Genes involved in synthesis of biogenic amines

• Dopa decarboxylase
• ebony (β-alanyl-dopamine synthase) Tyrosine decarboxylase 1 Tyrosine decarboxylase 2
• pale (Tyrosine hydroxylase)
• Tyramine β hydroxylase

• Allatostatin A
• Dopamine receptor
• Dopamine transporter
• ebony
• tan
• Serotonin receptor 1A
• Serotonin receptor 1B
• Serotonin receptor 2
• Stromal interaction molecule
• tan
• Tyramine receptor
• Tyramine receptor II
• Ubiquitin protein ligase E3A
• white

A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster

The modulation of an animal's behavior through external sensory stimuli, previous experience and its internal state is crucial to survive in a constantly changing environment. In most insects, octopamine (OA) and its precursor tyramine (TA) modulate a variety of physiological processes and behaviors by shifting the organism from a relaxed or dormant condition to a responsive, excited and alerted state. Even though OA/TA neurons of the central brain are described on single cell level in Drosophila melanogaster, the periphery was largely omitted from anatomical studies. Given that OA/TA is involved in behaviors like feeding, flying and locomotion, which highly depend on a variety of peripheral organs, it is necessary to study the peripheral connections of these neurons to get a complete picture of the OA/TA circuitry. This study describes the anatomy of this aminergic system in relation to peripheral tissues of the entire fly. OA/TA neurons arborize onto skeletal muscles all over the body and innervate reproductive organs, the heart, the corpora allata, and sensory organs in the antennae, legs, wings and halteres underlining their relevance in modulating complex behaviors (Pauls, 2018).

The adrenergic system of mammals influences various aspects of the animal's life. Its transmitters/hormones, adrenaline and noradrenaline, modulate a variety of physiological processes and behaviors. They are secreted into the bloodstream by the adrenal glands in response to stress. In addition, they are synthesized and released by axonal terminals in the central nervous system (CNS) as well as sympathetic fibers of the autonomic nervous system. Adrenaline and noradrenaline have been described as modulators to shift the organism from a relaxed or dormant state to a responsive, excited and alerted state. Stressful stimuli induce a metabolic and behavioral adaptation, leading to enhanced energy supply, increased muscle performance, increased sensory perception and a matched behavior. This so-called 'fight or flight' response can be seen in vertebrates and invertebrates. In insects, the stress response is mediated -- among others -- by octopamine (OA) and its precursor tyramine (TA). TA is synthesized from tyrosine by the action of a tyrosine decarboxylase enzyme (Tdc) and functions as an independent neurotransmitter/-modulator as well as the intermediate step in OA synthesis. For this, TA is catalyzed by the tyramine-β-hydroxylase (TΒH). Similar to the vertebrate adrenergic system, OA and TA act through specific G-protein coupled receptors. Besides structural similarities between OA/TA and adrenaline/noradrenaline and the corresponding receptors, functional similarities are illustrated by the action of these transmitters/hormones in the regulation of physiological processes and behaviors. OA and TA are known to modulate muscle performance, glycogenolysis, fat metabolism, heart rate, and respiration in insects (Pauls, 2018).

While the role of TA as an independent signaling molecule was underestimated for a long time, OA has been extensively studied and was shown to have effects on almost every organ, sensory modality and behavior in a great variety of insects. The most intensively studied peripheral organs regarding the modulatory role of OA are muscles. Here, OA is thought to not exclusively modulate muscle performance or motor activity. OA rather modulates muscle action according to metabolic and physiological processes, for example by promoting energy mobilization directly from the fat body, or indirectly by promoting the release of adipokinetic homones (AKH) from neuroendocrine cells in the corpora cardiaca (CC, a homolog of the vertebrate anterior pituitary gland and an analog of mammalian pancreatic alpha cells). In addition to the impact of OA/TA on muscles, fat body and AKH cells, OA is shown to modulate the heart, trachea and air sacs, gut, hemocytes, salivary glands, Malpighian tubules and ovaries in insects, mainly to induce a general stress or arousal state. However, in total OA seems to modulate a vast number of behaviors, which are not necessarily coupled to stress responses. The OA/TA system is shown to also act on inter alia (i.a.) learning and memory, sleep, feeding, flight, locomotion, and aggression (Pauls, 2018).

As mentioned above, OA and TA act as neurotransmitters and neuromodulators, allowing them to act in a paracrine, endocrine or autocrine fashion. In the fruit fly Drosophila, huge efforts were made to describe OA/TA neurons (OANs/TANs) in the brain and ventral nervous system (VNS) down to the single cell level. Nevertheless, although knowledge about physiological processes and behaviors modulated by the OA/TA system in the brain is rich, less is known about how OA and TA reach all its target organs and tissues in the periphery (exceptions: reproductive organs and muscles) (Pauls, 2018).

This study used the genetically tractable fruit fly Drosophila melanogaster to describe the arborizations of Tdc2-Gal4-positive, and therefore OANs and TANs in the periphery, as the Drosophila Tdc2 gene is expressed neurally. OANs/TANs were found to be widespread distributed throughout the fly's body with innervations in the skeletal muscles, reproductive organs, corpora allata, antennae, legs, wings, halteres and the heart. This diverse innervation pattern reflects the modulatory role of OA/TA in many different behaviors and physiological processes. These results provide, for the very first time, a complete and comprehensive map of the OA/TA circuitry in the entire insect body. This map allows assumptions about the type of OA/TA signaling (paracrine or endocrine) to a specific organ and, at the same time, it provides a deeper understanding to what extend the OA/TA-dependent activity of peripheral organs is altered, for example by genetically manipulating Tdc2-Gal4-positive neurons in the brain and VNS (Pauls, 2018).

This study used the Tdc2-Gal4 line, allowing Gal4 expression under the control of a regulatory sequence of the tyrosine decarboxylase enzyme. As this enzyme is essential for the synthesis of TA from tyrosine, the Tdc2-Gal4-line labels both TANs and OANs. Within the Drosophila brain, Tdc2-Gal4 labels in total about 137 cells, while additional 39 cells are located in the VNS. The small number of Tdc2Ns lead to arborizations in large parts of the central brain, optic lobes and the thoracic and abdominal ganglion. Based on the profound innervation of Tdc2Ns in the brain and VNS, the variety of behaviors modulated by the OA/TA system including learning and memory, feeding, vision, and sleep, are not surprising. Beyond the brain and VNS, OANs and TANs massively innervate regions within the periphery of the fly. This study described arborizations on most skeletal muscles, the antennae, wings, halteres and reproductive system and parts of the circulatory system and stomodaeal ganglion (Pauls, 2018).

The findings are in line with previous reports focusing on the expression of different OA and TA receptors in the fly. Accordingly, the OA receptor OAMB is expressed in reproductive organs (in both male and female flies) and muscles, which are directly innervated by Tdc2Ns. Additionally, the midgut and trachea contain OA and TA receptors, but do not seem to be innervated by Tdc2Ns, even though axons run in close vicinity to these organs. Likewise, the OA receptor Octβ2R is expressed in the fat body, salivary glands and Malpighian tubules, tissues that seem not to be innervated by Tdc2Ns, while the expression of Octβ1R and Octβ3R is more specific56,57. The three tyramine receptors TyrR, TyrRII and TyrRIII show a broad expression in the periphery, also in tissues not innervated by Tdc2Ns56. The lack of direct innervation of these peripheral tissues might argue for volume transmission or hemolymph released OA/TA from Tdc2Ns. Alternatively, Drosophila TDC1, the product of the non-neurally expressed Tdc gene, is expressed in the gut musculature, rectal papillae, Malpighian tubules and two small clusters in the thoracic nervous system and might be the source for peripheral TA. Interestingly, TyrR seems to be the only receptor expressed in the heart, suggesting that only TA modulates heart function. Contrary, OA has a modulatory effect on the heart of other insect species including honeybees, olive fruit flies and cockroaches. This is also in line with a previous report providing evidence that OA modulates the heart rate of the Drosophila fly and pupa, but not the larva (Pauls, 2018).

OA-dependent modulation of organs and tissues is mainly elicited through muscle action, especially in terms of its impact on the 'fight or flight' response. In line with this, it was observed Tdc2-Gal4-positive arborizations on nearly all skeletal muscles and many visceral muscles. In both Drosophila and desert locusts OA and TA is expressed in type II terminals of skeletal muscles50. OA has an excitatory effect on Drosophila flight muscles, while TA was shown to inhibit excitatory junction potentials, and thereby reduce muscle contractions and locomotion at least in the larva. In addition, flies lacking OA show severe deficits in flight initiation and maintenance. Interestingly, in an antagonistic effect to serotonin, OA reduces crop muscle activity presumably via Octβ1R, suggesting that OA has different effects on muscle activity dependent on the type of muscle. However, the data do not provide any convincing evidence of a direct innervation of Tdc2Ns of the crop, even though many fibers run in close vicinity, suggesting that OA might target the crop by volume transmission (Pauls, 2018).

Furthermore, OA modulates ovulation and fertilization in insects. Flies lacking OA display a severe egg-laying phenotype. Remarkably, within the female reproductive organ two different OA receptors, OAMB and Octβ2R, are necessary. Again, OA has a strong impact on muscle activity within the reproductive system. Octβ2R is expressed in the visceral oviduct muscle and elicits muscle relaxation through an increase of intracellular cAMP levels. Such an OA-dependent modulation appears to be conserved as OA is found in dorsal unpaired median neurons of locusts innervating oviduct muscles through the oviducal nerve. However, the data suggest that OA-positive fibers not only innervate oviduct muscles, but also enter the organs themselves. The OAMB receptor is expressed in epithelial cells inducing fluid secretion through increasing intracellular Ca2+ levels. Thus, OA affects different processes within the female reproductive organ due to the expression of different receptors and their coupled signaling pathways, which may be a general mechanism of the OA/TA system to fulfill an extensive modulatory function (Pauls, 2018).

OA does not exclusively modulate muscle activity, but also sensory neurons of external tissues like the antennae, halteres and wings. OA has also been shown to increase the spontaneous activity of olfactory receptor neurons (ORN). The modulation of ORNs allows OA to modulate the innate response to attractive stimuli like fruit odors or pheromones. Further, this modulation helps nestmate recognition in ants. In addition to Tdc2-Gal4-positive arborizations in the funiculus, Tdc2-Gal4-positive sensory neurons were found in the JO, a chordotonal organ sensitive to mechanosensory stimuli and thus important for hearing in insects. In mosquitos, OA modulates auditory frequency tuning and thereby affects mating behavior. In locusts, OA similarly modulates the response of chordotonal neurons in the legs to encode proprioceptive information. These data suggest that chordotonal neurons in the legs, wings, halteres and thorax are included in the Tdc2-Gal4 line suggesting a conserved modulatory role of OA/TA for insect proprioception (Pauls, 2018).

Taken together, this study suggests that the OA/TA system massively modulates various organs and tissues in the periphery of Drosophila. Through distinct receptors and coupled signaling pathways OANs/TANs mainly induce 'fight or flight' responses by modulating muscle activity, proprioception, and heart rate. As a result, the innervation pattern in the periphery supports the idea that the OA/TA system is crucial for insects to switch from a dormant to an excited state, by a positive modulation of muscle activity, heart rate and energy supply, and a simultaneous negative modulation of physiological processes like sleep (Pauls, 2018).

tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals

In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of β-alanine to dopamine or hydrolysis (tan) of the β-alanyl conjugate to liberate dopamine. β-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. When they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [³H]histamine into its β-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [³H]carcinine, the hydrolysis of which is blocked; ebony lacks [³H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas β-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [³H]carcinine and [³H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. These findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. It is suggested that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake (Borycz, 2002).

Shared mushroom body circuits underlie visual and olfactory memories in Drosophila

In nature, animals form memories associating reward or punishment with stimuli from different sensory modalities, such as smells and colors. It is unclear, however, how distinct sensory memories are processed in the brain. This study established appetitive and aversive visual learning assays for Drosophila that are comparable to the widely used olfactory learning assays. These assays share critical features, such as reinforcing stimuli (sugar reward and electric shock punishment), and allow direct comparison of the cellular requirements for visual and olfactory memories. It was found that the same subsets of dopamine neurons drive formation of both sensory memories. Furthermore, distinct yet partially overlapping subsets of mushroom body intrinsic neurons are required for visual and olfactory memories. Thus, these results suggest that distinct sensory memories are processed in a common brain center. Such centralization of related brain functions is an economical design that avoids the repetition of similar circuit motifs (Vogt, 2014).

Devising a transparent electric shock grid module made it possible to apply the same visual stimulation in aversive and appetitive conditioning assays. Also an integrated platform was developed for fully automated high-throughput data acquisition using customized software to control the presentation of electric shock and visual stimuli while making video recordings of behavior. In these assays, memory performance is based on altered visual preference in walking flies, a task likely to be less demanding than the constant flight required for flight simulator learning. These advantages facilitate behavioral examination of many genotypes (Vogt, 2014).

Circuits underlying olfactory and visual memory can be optimally compared when the sugar reward and electric shock punishment are matched between the two modalities. Visual and olfactory memories share the same subsets of dopamine neurons that convey reinforcing signals. This shared requirement of the transmitter system between visual and olfactory learning has been described in crickets. However, the pharmacological manipulation used in these studies does not allow further circuit dissection (Vogt, 2014).

For electric shock reinforcement, identified neurons in the PPL1 cluster, such as MB-MP1, MB-MV1 and MB-V1, drive aversive memories in both visual and olfactory learning, while the MB-M3 neurons in the PAM cluster seem to be involved specifically in aversive olfactory memory. Thus, overlapping sets of dopamine neurons appear to represent electric shock punishment in both visual and olfactory learning with olfactory aversive memory probably recruiting a larger set. Previous studies have shown that the MB-M3 neurons induce aversive olfactory memory that increases stability of other memory components. Olfactory memories last longer than visual memories potentially due to the recruitment of additional dopamine neurons (Vogt, 2014).

In appetitive conditioning, PAM cluster neurons play crucial roles in both olfactory and visual memories. Which cell types in these clusters are involved and whether there is a cellular distinction between olfactory and visual memory requires further analysis at the single cell level. Most importantly, all these neurons convey dopamine signals to restricted subdomains of the MB. The blockade of octopamine neurons did not impair appetitive visual memories with sucrose. The involvement of octopamine neurons may be more substantial when non-nutritious sweet taste rewards are used, as has been shown in olfactory learning (Vogt, 2014).

In addition to these shared reinforcement circuits in the MB, the necessity of MB output for visual memory acquisition and retrieval is also consistent with olfactory conditioning. Taken together, these results suggest that the MBs harbor associative plasticity for visual memories and support the conclusion that similar coincidence detection mechanisms are used to form memories within the MBs. Centralization of similar brain functions spares the cost of maintaining similar circuit motifs in different brain areas and may be an evolutionary conserved design of information processing. Such converging inputs of different stimuli into a multisensory area have even been described in humans (Vogt, 2014).

'Flight simulator' visual learning was shown to require the central complex but not the MBs. Although this appears to contradict the current study, it is noted that there are important differences between the behavioral paradigms employed. In the flight simulator, a single tethered flyingDrosophilais trained to associate a specific visual cue with a laser beam punishment, to later on avoid flying towards this cue in the test. Although this study controlled for visual context consistency and the 'operant component' of the flight simulator training, any other difference could account for the differential requirement of brain structures. Given that flies during flight show octopamine-mediated modulation of neurons in the optic lobe, similar state-dependent mechanisms might underlie different requirement of higher brain centers. Thus, it is critical to design comparable memory paradigms (Vogt, 2014).

This study together with previous results in associative taste learning highlights the fact that the role of the MB in associative learning is not restricted to one sensory modality or reinforcer. This study found that olfactory and visual memories recruit overlapping, yet partly distinct, sets of Kenyon cells. In contrast to the well-described olfactory projection neurons, visual inputs to the MB remain unidentified. No anatomical evidence has been reported in Drosophila for direct connections between optic lobes and MBs although such connections are found in other insects. Also afferents originating in the protocerebrum were found to provide multi-modal input to the MB lobes of cockroaches. Thus, Drosophila MBs may receive indirect visual input from optic lobes, and the identification of such a visual pathway would significantly contribute to understanding of the MB circuits (Vogt, 2014).

Given the general requirement of the γ lobe neurons, visual and olfactory cues may be both represented in the γ neurons. Consistently, the dopamine neurons that convey appetitive and aversive memories heavily project to the γ lobe. In olfactory conditioning, the γ lobe was shown to contribute mainly to short-term memory. This converging evidence from olfactory and visual memories suggests a general role for the γ lobe in short-lasting memories across different sensory modalities. Previous studies found that the MB is also involved in sensorimotor gating of visual stimuli or visual selective attention. Therefore, the MB circuits for visual associative memories might be required for sensorimotor gating and attention (Vogt, 2014).

Interestingly, the contribution of the α'/β' lobes is selective for olfactory memories. This Kenyon cell class is more specialized to odor representation, as the cells have the broadest odor tuning and the lowest response threshold among the three Kenyon cell types (Vogt, 2014).

The role of α/β neurons in visual memories is also limited. The α/β neurons might play more modulatory roles in specific visual memory tasks, such as context generalization, facilitation of operant learning and occasion setting. This modulatory role of the α/β neurons is corroborated in olfactory learning, where they are preferentially required for long-lasting memories (Vogt, 2014).

Differentiated but overlapping sensory representations by KCs may be conserved among insect species. In honeybees, different sensory modalities are represented in spatially segregated areas of the calyx, whereas the basal ring region receives visual and olfactory inputs. The MB might thus have evolved to represent the sensory space of those modalities that are subject to associative modulation (Vogt, 2014).

Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila

During olfactory learning in fruit flies, dopaminergic neurons assign value to odor representations in the mushroom body Kenyon cells. This study identified a class of downstream glutamatergic mushroom body output neurons (MBONs) called M4/6, or MBON-β2β'2a, MBON-β'2mp, and MBON-γ5β'2a, whose dendritic fields overlap with dopaminergic neuron projections in the tips of the β, β', and γ lobes. This anatomy and their odor tuning suggests that M4/6 neurons pool odor-driven Kenyon cell synaptic outputs. Like that of mushroom body neurons, M4/6 output is required for expression of appetitive and aversive memory performance. Moreover, appetitive and aversive olfactory conditioning bidirectionally alters the relative odor-drive of M4β' neurons (MBON-β'2mp). Direct block of M4/6 neurons in naive flies mimics appetitive conditioning, being sufficient to convert odor-driven avoidance into apprroach, while optogenetically activating these neurons induces avoidance behavior. It is therefore proposed that drive to the M4/6 neurons reflects odor-directed behavioral choice. See Three Pairs of Glutamatergic Output Neurons Innervate the Tips of the Horizontal Mushroom Body Lobes (Owald, 2015).

Many prior studies have concluded that mushroom body neurons are dispensable for naive odor-driven behavior and subsets are either required or are dispensable for particular memory functions. However, these experiments simultaneously blocked all the outputs from a given population of KCs using cell-wide expression of shi^ts1. The current results suggest that these models should be reconsidered. Blocking the specific M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a output from the mushroom body, as opposed to blocking all outputs, has a radical effect on naive odor-driven behavior. It is proposed that ordinarily, in naive flies, the multiple mushroom body output channels are ultimately pooled and contribute a net zero to odor-driven behavior. Therefore, if one uses a mushroom body neuron-driven UAS-shi^ts1 that simultaneously blocks all outputs, there is no apparent effect on naive behavior. If, however, one blocks only one channel, or alters its efficacy by learning, the odor-driven behavior can be changed. A similar logic could also account for why clear memory retrieval defects are seen when blocking M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neurons that presumably pool outputs from the tip of the γ and β' lobe, yet blocking all α'β' neuron outputs did not demonstrably disrupt later memory retrieval. Others have shown a role for α'β' neuron output to retrieve earlier forms of memory (Owald, 2015).

Both the physiological and behavioral results are consistent with a depression of the M4β'/MBON-β'2mp and M6/MBON-γ5β'2a output being sufficient to code learned approach. Learning-related plasticity has been reported at the β-lobe outputs in both bees and locusts, although the importance of these synaptic connections in the behavior of these insects is not known. At this stage it is not certain that the observed decrease in the relative odor drive reflects plasticity of the synapses between odor-specific KCs and the M4/6 neurons. However, it seems plausible, because this synaptic junction is addressed by the relevant rewarding dopaminergic neurons. Given that blocking M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neurons converts avoidance to approach, other mushroom body output channels, perhaps some of which lie on the vertical α-lobe projection, must drive the approach behavior. It is therefore conceivable that a similar plasticity of odor drive to these putative approach outputs could be critical for aversive conditioning. Such an idea is consistent with several prior reports of aversive memory traces that are specific to the vertical α-branch of the mushroom body. In addition, aversive learning has been reported to depress odor drive in the vertical lobe of downstream MB-V2α/MBON-α2sc and MB-V2α'/MBON-α'3 neurons and to potentiate odor drive of MB-V3/MBON-α3 output neurons. However, it is notable that blocking either the MB-V2α/MBON-α2sc and MB-V2α'/MBON-α'3 neurons or MB-V3/MBON-α3 neurons did not affect naive odor avoidance behavior in the current experiments or those of others. Therefore, although MB-V2α/MBON-α2sc, MB-V2α'/MBON-α'3, and MB-V3/MBON-α3 neurons are required for memory expression, it is not currently known which reinforcing neurons address MB-V2α/MBON-α2sc, MB-V2α'/MBON-α'3, and MB-V3/MBON-α3 connections and how these outputs specifically contribute to odor-guided behavior (Owald, 2015).

The physiological analyses suggest bidirectional plasticity of odor-evoked responses, with aversive learning increasing the relative conditioned odor drive to the M4β'/MBON-β'2mp neurons. This could account for why output from M4/6 neurons is also required for expression of aversive memory. Moreover, whereas blocking the M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a neurons converts odor avoidance into approach, activation of M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a neurons drives avoidance. It therefore seems likely that plasticity of the relative odor drive to M4β'/MBON-β'2mp neurons is also part of the aversive memory engram. Again, it is not known that the increased odor drive after training reflects synaptic potentiation between odor-specific KCs and the M4β'/MBON-β'2mp neurons. Increased odor drive to M4β'/MBON-β'2mp neurons could, for example, also result from plasticity elsewhere in the KCs that enhances signal propagation along the horizontal KC arbor. Nevertheless, the MB-M3 dopaminergic neurons that are required to reinforce aversive memory also innervate the tips of the β and β' lobe. In addition, a recent study reported that aversive learning specifically decreased unconditioned odor-evoked neurotransmission from the γ neurons, a result that presumably would mirror a relative increase in the response to the reinforced odor. Lastly, aversive conditioning using relative shock intensity utilizes the rewarding dopaminergic neurons that occupy the same zones on the mushroom body as the M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neuron dendrites. With the caveat that GRASP is only an indicator of proximity, the anatomical studies suggest that dendrites of rewarding dopaminergic neurons may connect to the M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neuron presynaptic terminals, forming a potential feedback or forward loop that could serve such a relative-judgment function (Owald, 2015).

It is perhaps noteworthy that KC outputs in the vertical lobe are onto excitatory cholinergic MB-V2α/MBON-α2sc and MB-V2α'/MBON-α'3 neurons, whereas the horizontal outputs are onto glutamatergic, potentially inhibitory, M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a neurons. This suggests that distinct signaling modes may be driven from the bifurcated collaterals of KCs. It will be crucial to understand how these outputs from the different branches, and those from discrete lobes, are ultimately pooled to guide appropriate behavior (Owald, 2015).

Dopaminergic neurons write and update memories with cell-type-specific rules

Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. The anatomy of the adult MB and 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments have been previously defined and described. This study compared the properties of memories formed by optogenetic activation of individual DAN cell types. Extensive differences were found in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. These results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences. The mechanisms that generate these distinct learning rules are unknown. They could arise from differences in the dopamine release properties of different DAN cell types or from local differences in the biochemical response to dopamine signaling in each MB compartment. For example, KCs express four distinct dopamine receptors, which might be deployed differently in each compartment. Or they could originate from circuit properties (Aso, 2016).

Circuit analysis of a Drosophila Dopamine type 2 receptor that supports anesthesia-resistant memory

Dopamine is central to reinforcement processing and exerts this function in species ranging from humans to fruit flies. It can do so via two different types of receptors (i.e., D1 or D2) that mediate either augmentation or abatement of cellular cAMP levels. Whereas D1 receptors are known to contribute to Drosophila aversive odor learning per se, this study shows that D2 receptors are specific for support of a consolidated form of odor memory known as anesthesia-resistant memory. By means of genetic mosaicism, this function was localized to Kenyon cells, the mushroom body intrinsic neurons, as well as GABAergic APL neurons and local interneurons of the antennal lobes, suggesting that consolidated anesthesia-resistant memory requires widespread dopaminergic modulation within the olfactory circuit. Additionally, dopaminergic neurons themselves require D2R, suggesting a critical role in dopamine release via its recognized autoreceptor function. Considering the dual role of dopamine in balancing memory acquisition (proactive function of dopamine) and its 'forgetting' (retroactive function of dopamine), this analysis suggests D2R as central player of either process (Scholz-Kornehl, 2016).

Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster

Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. This study describes the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. Endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) were used to identify neurons, based on cell class and transmitter content, that express each receptor. Specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs (5-HT2 type and 5-HT7), the 5-HT1 type receptors are generally inhibitory, and distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing (Sizemore, 2016).

Neuromodulators often act through diverse sets of receptors expressed by distinct network elements and in this manner, differentially affect specific features of network dynamics. Knowing which network elements express each receptor for a given neuromodulator provides a framework for making predictions about the mechanistic basis by which a neuromodulator alters network activity. This study provides an 'atlas' of 5-HTR expression within the AL of Drosophila, thus revealing network elements subject to the different effects of serotonergic modulation. In summary, different receptors are predominantly expressed by distinct neuronal populations. For example, the 5-HT2B is expressed by ORNs, while the 5-HT2A and 7 are expressed by cholinergic PNs. Additionally, each receptor was found to be expressed by diverse populations of LNs, with the exception the 5-HT1B. For instance, 5-HT1A is expressed by GABAergic and peptidergic (TKK and MIP) LNs, while 5-HT2A and 2B are not expressed by peptidergic LNs. However, the vPNs are the exception to the general observation that distinct neuronal classes differ from each other in the 5-HTRs and the implications of this are discussed below. Together, these results suggest that within the AL, 5-HT differentially modulates distinct populations of neurons that undertake specific tasks in olfactory processing (Sizemore, 2016).

A recurring theme of neuromodulation is that the expression of distinct receptor types by specific neural populations allows a single modulatory neuron to differentially affect individual coding features. For instance, GABAergic medium spiny neurons (MSNs) in the nucleus accumbens express either the D1 or D2 dopamine receptor allowing dopamine to have opposite effects on different MSNs via coupling to different Galpha subunits (reviewed in56). MSNs that differ in dopamine receptor expression also differ in their synaptic connectivity. Dopamine activates D1-expressing MSNs that directly inhibit dopaminergic neurons in the ventral tegmental area (VTA), and inhibits D2-expressing MSNs that inhibit GABAergic VTA interneurons thus inducing suppression of dopamine release. In this manner, a single neuromodulator differentially affects two populations of principal neurons via different receptors to generate coordinated network output. This principle also holds true for the effects of 5-HT within the olfactory bulb. For instance, 5-HT enhances presynaptic inhibition of olfactory sensory neurons by 5-HT2C-expressing juxtaglomerular cells57, while increasing excitatory drive to mitral/tufted cells and periglomerular cells via 5-HT2A-expressing external tufted cells. Similarly, distinct classes of AL neurons were observed to differ in their expression of 5-HTRs. For instance, ePNs express the 5-HT2A, 5-HT2B and 5-HT7 receptors, while peptidergic LNs predominantly express the 5-HT1A receptor. This suggests that the cumulative effect of 5-HT results from a combination of differential modulation across neuronal populations within the AL. Interestingly, although it was found that 5-HT2B is expressed by ORNs, previous reports found that 5-HT does not directly affect Drosophila ORNs. In this study, ORNs were stimulated using antennal nerve shock in which the antennae were removed in order to place the antennal nerve within a suction electrode. Thus, if 5-HT2B is localized to the ORN cell body, removal of the antennae would eliminate any effect of 5-HT on ORNs. In several insects, 5-HT within the antennal haemolymph modulates ORN odor-evoked responses. Therefore, it is plausible ORNs are modulated by a source of 5-HT other than the CSD neurons within the AL. Serotonergic modulation of LN activity has widespread, and sometimes odor specific, effects on olfactory processing. LNs allow ongoing activity across the AL to shape the activity of individual AL neurons, often in a glomerulus specific manner creating non-reciprocal relationships. It is fairly clear that 5-HT directly modulates LNs, although 5-HT almost certainly affects synaptic input to LNs. Serotonin modulates isolated Manduca sexta LNs in vitro and, consistent with the current results, a small population of GABAergic LNs in the AL of Manduca also express the 5-HT1A receptor. Furthermore, 5-HT has odor-dependent effects on PN odor-evoked activity, suggesting that odor specific sets of lateral interactions are modulated by 5-HT. Different populations of LNs were found to express different sets of 5-HT receptors, however LNs were categorized based on transmitter type, so it is possible that these categories could be even further sub-divided based on morphological type, synaptic connectivity or biophysical characteristics. Regardless, the results suggest that 5-HT modulates lateral interactions within the AL by selectively affecting LN populations that undertake different tasks. For instance, the TKKergic LNs that express the 5-HT1A receptor provide a form of gain control by presynaptically inhibiting ORNs32. The results suggest that 5-HT may affect TKK mediated gain control differently relative to processes undertaken by other LN populations. Furthermore, the expression of the TKK receptor by ORNs is regulated by hunger, allowing the effects of TKK to vary with behavioral state. It would be interesting to determine if the expression of 5-HTRs themselves also vary with behavioral state as a means of regulating neuromodulation within the olfactory system (Sizemore, 2016).

Although it was primarily found that individual populations of AL neurons chiefly expressed a single or perhaps two 5-HTR types, the vPNs appear to be an exception. As a population, the vPNs express all of the 5-HTRs and the vPNs that express each 5-HTR did not appear to differ in terms of the proportion of those neurons that were GABAergic or cholinergic (roughly 3:2). Unfortunately, the approach does not allow determination of the degree to which individual vPNs co-express 5-HTRs. However, it is estimated that there are ~51 vPNs and even if this is an underestimate, there is likely some overlap of receptor types as a large number of vPNs expressed the 5-HT1A, 1B, 2B and 7 receptors. It is possible that a single vPN expresses one 5-HTR in the AL and a different 5-HTR in the lateral horn. However, the current approach only allows identification of which neurons express a given 5-HTR, not where that receptor is expressed. The CSD neurons ramify throughout both ALs and both lateral horns, thus vPNs could have differential spatial expression of individual 5-HTRs. Individual neurons expressing multiple 5-HTRs has been demonstrated in several neural networks. For instance, pyramidal cells in prefrontal cortex express both the 5-HT1A and 5-HT2A7. This allows 5-HT to have opposing effects that differ in their time course in the same cell. In terms of the vPNs, the results suggest that the current understanding of the diversity of this neuron class is limited. The expression of receptors for different signaling molecules could potentially be a significant component to vPN diversity (Sizemore, 2016).

Neuromodulators are often released by a small number of neurons within a network, yet they can have extremely diverse effects depending upon patterns of receptor expression. For the most part, individual populations of AL neurons differed in the receptor types that they expressed. This suggests that 5-HT differentially acts on classes of neurons that undertake distinct tasks in olfactory processing. In the case of the vPNs, this differential modulation may be fairly complex due to the diversity within this neuronal class. The goal of this study was to establish a functional atlas of 5-HTR expression in the AL of Drosophila. This dataset therefore provides a mechanistic framework for the effects of 5-HT on olfactory processing in this network (Sizemore, 2016).

Pharmacological characterization of the fifth serotonin receptor subtype of Drosophila melanogaster

Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor's pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B's pharmacology, this study evaluated the receptor's response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo (Blenau, 2017).

Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila

Learned and adaptive behaviors rely on neural circuits that flexibly couple the same sensory input to alternative output pathways. This study shows that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, depending on the state and experience of the fly (see Compartmentalized Architecture of the Mushroom Body). Using functional synaptic imaging and electrophysiology, it was shown that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, the dopaminergic neurons function as an interconnected network, encoding information about both an animal's external context and internal state to coordinate synaptic plasticity throughout the mushroom body. These data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity (Cohn, 2015).

This study took advantage of the mushroom body's orderly architecture to gain insight into the circuit mechanisms through which neuromodulation mediates flexible sensory processing. Compartmentalized dopaminergic signaling permits independent tuning of synaptic transmission between an individual KC and its repertoire of postsynaptic MBON targets. As a consequence, the same KC odor representation can evoke different patterns of output activity, depending on the state of the animal and the dopaminergic network. Recent data indicate that the ensemble of MBONs acts in concert to bias an animal's behavioral response to an odor such that altering the balance of their activity can modify the olfactory preferences of both naive and trained animals. In accord with such a model, this study revealed how a distributed neuromodulatory network is poised to orchestrate plasticity across all 15 compartments of the mushroom body and reweight the net output of the MBONs, allowing for adaptive behavioral responses based on the immediate needs or past experience of the animal (Cohn, 2015).

Distinct subsets of DANs are sufficient to drive learned olfactory associations, leading to the suggestion they may act autonomously to encode the rewarding or punishing contextual stimuli that assign meaning to an odor. The current data, however, suggest a more complex circuit architecture, in which rich functional interconnectivity between compartments contributes to coordinated and bidirectional patterns of activity across the DAN population. This raises the possibility that reinforcement experiences may be represented by combinatorial patterns of DAN excitation and inhibition in different compartments, endowing the dopaminergic population with a greater capacity to instruct behavior via the limited repertoire of mushroom body outputs. Intriguingly, midbrain dopaminergic neurons responsive to punishment and reward also project to distinct targets in the mammalian brain and display a similar functional opponency as a consequence of reciprocal network interactions. Thus, the concerted and partially antagonistic action of neuromodulatory pathways may represent a general and conserved circuit principle for generating adaptive behavioral responses (Cohn, 2015).

Distinct DAN network activity states are evoked by electric shock and sugar ingestion, reinforcers classically used in associative olfactory conditioning paradigms because of their strong inherent valence. However, similarly distributed patterns of DAN activity are correlated with the fly's motor activity, implying that an animal's behavioral state might serve as a reinforcement stimulus that itself drives synaptic plasticity to shape odor processing. Metabolic states, such as thirst and hunger, have been shown to gate appetitive reinforcement by water and sugar rewards, permitting state-dependent formation of olfactory associations only in motivated animals. The current data highlight an additional facet of how an animal's internal state can regulate dopamine release to adjust the salience of contextual cues. Together, these observations indicate that the distributed DAN network integrates information about external context and internal state with MBON feedback to represent the moment-by-moment experience of an animal and dynamically regulate the flow of olfactory signals through the mushroom body (Cohn, 2015).

The independent regulation of synapses along an axon is thought to permit a single neuron to convey specialized information to different downstream targets, providing additional flexibility and computational power to neural circuits. In the mushroom body, synapse-specific plasticity is achieved through spatially restricted patterns of dopaminergic modulation that divide a KC axon into functionally distinct segments. Thus, the ensemble of synapses within a compartment, as the site of convergence for sensory and contextual signals, represents the elementary functional unit that underlies experience-dependent mushroom body output (Cohn, 2015).

Within a compartment, multiple neuromodulatory mechanisms appear to shape synaptic signaling. Broad potentiation of KC-MBON synapses is seen after DAN activation, but odor-specific depression is seen if DANs were coincidently activated with KCs, consistent with the synaptic changes previously proposed to occur after learning. Taken together, these findings indicate that neuromodulation in the mushroom body instructs opposing forms of synaptic plasticity, analogous to the bidirectional tuning of synaptic strength by dopamine in mammalian brain centers. The molecular mechanisms through which dopamine can direct diverse synaptic changes within a compartment remain to be elucidated, but they may depend on signaling through different dopamine receptors or downstream signaling cascades that function as coincidence detectors. Indeed, while DopR1 in KCs is essential to the formation of learned olfactory associations, this receptor was found to play only a subtle role in the context-dependent patterning of Ca2+ along their axons. Conversely, DopR2 strongly influences the topography of presynaptic Ca2+ along KC axons, in accord with evidence that tonic release of dopamine during ongoing behavior acts through this receptor to interfere with the maintenance of specific learned olfactory associations. Thus, distinct molecular pathways may transform the same dopaminergic reinforcement signals into synaptic changes of opposite polarity to shape olfactory processing based on both the present context and prior experiences of an individual (Cohn, 2015).

The mushroom body has been most extensively studied as a site for associative learning in which the temporal pairing of an odor with a reinforcement experience selectively alters subsequent behavioral responses to that odor. The current data suggest that the convergence of DAN network activity and KC olfactory representations within the mushroom body lobes may drive associative plasticity in each compartment, allowing the odor tuning of the MBON repertoire to reflect the unique experiences of an individual. However, these observations also provide insight into the mushroom body's broader role in the context-dependent regulation of innate behaviors. The ongoing activity of the distributed DAN network, encoding information about an animal's current environmental context and behavioral state, is poised to continuously reconfigure the activity patterns of the MBON population to allow for adaptive responses based on the acute needs of the animal. This context-dependent synaptic modulation could potentially erode odor-specific learned associations within the mushroom body, permitting the immediate circumstances of an animal to dominate over previously learned olfactory associations that may no longer be predictive or relevant. The axons of MBONs ultimately converge with output pathways from the lateral horn, a Drosophila brain center thought to mediate stereotyped responses to odors, providing a potential substrate for learned and context-dependent output from the mushroom body to influence inherent olfactory preferences (Cohn, 2015).

Thus, the dual role of neuromodulation in the mushroom body-to select among alternative circuit states that regulate both innate and learned behaviors-is reminiscent of its function in other higher integrative brain centers. In the basal ganglia, for example, different temporal patterns of dopamine release are thought to select the relevant circuit configurations that control inherently motivated behaviors as well as reinforcement learning. The generation of flexible behavioral responses based on experience, whether past or present, may therefore rely on common integrative brain structures in which neuromodulatory networks act with exquisite spatial precision to shape sensory processing (Cohn, 2015).

Trace conditioning in Drosophila induces associative plasticity in mushroom body kenyon cells and dopaminergic neurons

Dopaminergic neurons (DANs) signal punishment and reward during associative learning. In mammals, DANs show associative plasticity that correlates with the discrepancy between predicted and actual reinforcement (prediction error) during classical conditioning. Also in insects, such as Drosophila, DANs show associative plasticity that is, however, less understood. This study examined ssociative plasticity in DANs and their synaptic partners, the Kenyon cells (KCs) in the mushroom bodies (MBs), while training Drosophila to associate an odorant with a temporally separated electric shock (trace conditioning). In most MB compartments DANs strengthened their responses to the conditioned odorant relative to untrained animals. This response plasticity preserved the initial degree of similarity between the odorant- and the shock-induced spatial response patterns, which decreased in untrained animals. Contrary to DANs, KCs (α'/β'-type) decreased their responses to the conditioned odorant relative to untrained animals. No evidence was found for prediction error coding by DANs during conditioning. Rather, the data supports the hypothesis that DAN plasticity encodes conditioning-induced changes in the odorant's predictive power (Dylla, 2017).

Associative learning enables animals to anticipate negative or positive events. The neural mechanisms of associative learning are commonly studied in classical conditioning paradigms, in which animals are trained to associate a cue (conditioned stimulus; CS) with a punishment or reward (unconditioned stimulus; US). In the standard conditioning paradigm CS and US overlap in time, while in the trace conditioning paradigm there is a temporal gap between the CS and US. During both standard conditioning and trace conditioning, the US is mediated by dopaminergic neurons (DANs), in animals as diverse as monkeys and fruit flies (Dylla, 2017).

Genetic tools for monitoring and manipulating neuronal activity in the fruit fly Drosophila melanogaster promoted the understanding of the neural mechanisms of dopamine-mediated learning. Those mechanisms are well-described for standard 'odor-shock conditioning' in Drosophila, in which an olfactory CS is paired with a temporally overlapping electric shock US. During conditioning, an odor-shock association is formed in the mushroom body (MB) neuropil. The intrinsic neurons of the MB, the Kenyon cells (KCs), receive olfactory input in the MB-calyx and project to the vertical (α and α'), and the medial (β, β', and γ) MB-lobes. During odor-shock conditioning, the olfactory CS activates an odorant-specific KC population, and the electric shock US activates DANs that innervate the MB-lobes. In KCs, the CS-induced increase in intracellular calcium and the US-(dopamine)-induced second messengers synergistically activate an adenylyl cyclase, which alters the synaptic strength between KCs and MB output neurons (MBONs). This change in KC-to-MBON synapses is thought to encode the associative odor memory (Dylla, 2017).

The MB-lobes are divided into 15 compartments (α1-3, β1-2, α'1-3, β'1-2, and γ1-5), each of which is innervated by a distinct population of DANs and MBONs. These compartments constitute functional units, which are involved in different forms of associative learning. In compartments such as γ1, γ2, and β2, DANs mediate electric shock reinforcement. Besides mediating reinforcement during classical conditioning, Drosophila DANs are involved in long-term memory formation, forgetting, extinction learning and memory reconsolidation, and in integrating internal states with memory and sensory processing. A single DAN can even serve different functions, for example, PPL1-γ1pedc (also referred to as MB-MP1) signals reinforcement, and controls state-dependent memory retrieval (Dylla, 2017).

The functional complexity of Drosophila DANs is further increased by the fact that DANs show learning-induced associative plasticity: they increase their response to the CS during classical conditioning. Mammalian DANs also increase their CS-induced responses during classical conditioning. In addition, they decrease their response to the US, and when a predicted US does not occur, they decrease their activity below baseline level. This pattern of response plasticity in mammalian DANs is compatible with the hypothesis that animals only learn to associate a CS with a US, when the US occurs unpredictably. Thus, mammalian DANs appear to encode this prediction error. In Drosophila, however, DANs do not change their response to the US. Therefore, Drosophila DANs appear to encode the US prediction by the CS rather than encoding the US prediction error during classical conditioning. It is not clear, whether classical conditioning in insects is driven by US prediction error. There is evidence for prediction error-driven conditioning in crickets, but there is also a controversy about whether or not blocking (a failure to learn, when the US is already predicted by another CS) occurs (Dylla, 2017).

This study reassessed the hypothesis that Drosophila DANs do not encode the prediction error during classical conditioning (Riemensperger, 2005). Different from Riemensperger (2005) who pooled DAN activity across the mushroom body lobes, this study differentiated between DAN types that innervate different compartments of the MB lobes. Moreover, instead of using standard conditioning, trace conditioning with a 5 s gap between the CS and the US was used, allowing for precise distinguishing between responses to either the CS or the US (Dylla, 2017).

This study investigated associative plasticity in the responses of DANs and their synaptic partners, the KCs, across the compartments of the Drosophila MB. Using calcium imaging, CS- and US-induced responses of a subpopulation of DANs (labeled by TH-GAL4) and of KCs (labeled by OK107-GAL4) were recorded during odor-shock trace conditioning. Note, that most compartments are innervated by multiple TH-GAL4-labeled DANs. Therefore, the average activity that was recorded in most of the compartments might mask possible differences in the response properties and plasticity between individual DANs and KCs. Only DAN responses in the compartments γ2 and α'1 reflect the responses of a single neuron (Dylla, 2017).

Across MB compartments, DANs and KCs differed in their response strength to odorants and electric shock, and they differed in CS-US pairing-induced plasticity. Compared to the unpaired control groups, KCs decreased their responses to the CS in all compartments of the β'-lobe and in the junction, while DANs increased their responses to the CS in all compartments of the γ- and β'-lobe, and in the junction. The occurrence of associative plasticity in DANs in the compartments γ3-5 and β'1 is surprising, given that these DANs are not known to be involved in odor-shock conditioning, after training there was neither an associative change in US-induced DAN responses nor a change of activity during US-omission after CS presentation. It is therefore concluded that Drosophila DANs do not encode the US-prediction error during classical conditioning (Dylla, 2017).

Previous studies suggested that DANs in the MB lobes respond strongly to electric shock and weakly to odorants. The compartment-resolved analysis of the calcium imaging data refines this picture: It is confirmed that DANs of all imaged compartments respond to both electric shock and odorants, and it was shown that their relative response strength to odorants and electric shock differs across compartments. For example, DANs innervating γ1 responded stronger to electric shock than to odorants, while DANs innervating β'2 responded equally strong to odorants and electric shock. The strongest DAN responses to electric shock were shown to be in the compartments γ1 and γ2. These compartments receive input from PPL1-γ1pedc and PPL1-γ2α'1 DANs that mediate electric shock reinforcement. In all compartments, except in α1/α'1, the DAN response strength correlated positively with the current strength encountered by individual flies. Thus, DANs are capable of encoding the strength of the electric shock US, and this property may account for the positive dependence between electric shock strength and learning performance in flies (Dylla, 2017).

Calcium responses in KCs differ between MB lobes, and they differ between the compartments of a given lobe, possibly due to compartment-specific modulation by DANs and MBONs. KCs in γ2 and γ3 responded strongest to odorants, confirming the results of Cohn (2015). KCs generally responded only weakly to electric shocks. Previously published strong KC responses to electric shock may be because electric shocks were applied to the flies' abdomen rather than to their legs, which might have resulted in a stronger stimulation (Dylla, 2017).

The associative strengthening of DAN responses to the olfactory CS (as compared to the unpaired control group), confirms the previous report by Riemensperger (2005). Associative plasticity occurred in those DANs that innervate the MBs (PPL1 and PAM cluster DANs; note that the used TH-GAL4 driver line covers only a small subpopulation of PAM neurons but not in DANs that innervate the central complex (PPL1 and PPM3 cluster DANs). This is in line with the established role of MB innervating-DANs in associative memory formation, while central complex-innervating DANs are involved in behaviors such as locomotion, wakefulness, arousal, and aggression, and are therefore not expected to show odor-shock conditioning-induced plasticity (Dylla, 2017).

In contrast to previous studies, this study did not find an associative increase in KC calcium responses to the CS in the MB-lobes after odor-shock conditioning. This may indicate either a difference between trace conditioning and standard conditioning, or a difference in other experimental parameters that may also account for inconsistencies in the published effects of odor-shock conditioning (Dylla, 2017).

The associative decrease in KC responses in the β'-lobe compartments is in line with previous studies that showed conditioning-induced depression of KC-to-MBON synapses (Cohn, 2015; Hige, 2015). Therefore, it propose that the associative decrease in KC responses to the CS reflects a presynaptic depression at KC-to-MBON synapses in β'-lobe compartments (Dylla, 2017).

What is the site of neuronal plasticity that underlies the relative increase in DANs' responses to the olfactory CS? Riemensperger (2005) proposed that DANs get odorant-driven excitatory input via a MBON feedback loop that is strengthened during odor-shock conditioning. However, the DAN population is composed of different neuron types that do not share a common input either from MBONs or from other neurons that could explain the global associative plasticity across MB compartments. Because KCs presumably provide the only common odor-driven input to all MB-innervating DANs, it is suggested that the site of associative plasticity is located in a KC-to-DAN synapse. Indeed, KC-to-DAN synapses have recently been reported in Drosophila (Cervantes-Sandoval, 2017). Associative increase in CS-induced DAN responses occurred despite unaltered or decreased KC responses in the same compartment. This suggests that the associative plasticity occurs post-synaptic in DANs and is not inherited from KCs (Dylla, 2017).

What is the neuronal substrate of CS-US coincidence detection in DANs and KCs? Drosophila trace conditioning depends on dopamine receptor-triggered signaling in KCs, as is the case for standard conditioning. However, the CS-US coincidence detection mechanism in trace conditioning is unknown. In standard conditioning the CS-induced increase in KCs' calcium concentration coincides with the US-(dopamine)-induced second messengers, which is thought to synergistically activate the rutabaga adenylyl cyclase, and ultimately alters the strength of KC-to-MBON synapses. This mechanism would not work for trace conditioning, because (1) at the time the US occurs, CS-induced increase in KCs' calcium concentration is back to baseline levels, and (2) trace conditioning does not involve the rutabaga adenylyl cyclase. It is therefore hypothesized that a non-rutabaga adenylyl cyclase or a protein kinase C could serve as a molecular coincidence detector for the CS trace and the US. For example, the CS-induced calcium and dopamine signaling could lead to a sustained activation of an adenylyl cyclase or protein kinase C in KCs, which then would increase synergistically and drive synaptic plasticity during the US-induced dopamine signaling (Dylla, 2017).

DAN responses to odorants and associative strengthening of DAN responses to the CS-odorant are not included in current models of associative learning in the MB. However, associative plasticity is a common feature of US-mediating neurons, which occurs in mammalian and Drosophila DANs, and in an octopaminergic neuron in honey bees (Dylla, 2017).

What could be the function of odorant-induced responses and odor-shock conditioning-induced plasticity in DANs? MB-innervating DANs strengthened their response to the CS (as compared to the unpaired group) during odor-shock conditioning, in line with Riemensperger (2005). However, other than in monkey DANs, this study did not observe associative plasticity in DANs' response to the US. The data therefore support the idea that Drosophila DANs encode predictive power of the CS, e.g., US-prediction, but not the US-prediction error during classical conditioning (Dylla, 2017).

This study found shock-induced responses and associative plasticity in DANs that are not involved in odor-shock conditioning, for example in DANs innervating β'1, γ3, γ4, and γ5. This suggests that those DANs serve a function in aversive odor learning which is not captured by the commonly applied conditioning paradigms. For example, the relative strengthening of CS-induced responses could mediate reinforcement during second-order conditioning, in which a previously reinforced CS1 can act as US in subsequent conditioning of a second CS2. As Drosophila is capable of second-order learning, this theory can be tested in behavioral experiments: if associative strengthening of DAN responses to the CS underlies CS1-induced reinforcement in second-order conditioning, then preventing associative plasticity in DANs, or blocking their output during CS2-CS1 pairing should abolish second-order conditioning (Dylla, 2017).

The occurrence of CS-induced responses and associative plasticity in most of the MB-innervating DANs suggests that the separation between the CS- and US-pathway and between different US-pathways is less strict than suggested in current models of associative learning in the MB. Associative plasticity in the spatial pattern of CS-induced DAN responses makes them a potential neuronal substrate for encoding the US identity in CS-US memories and the predictive power of a CS (Dylla, 2017).

These data revealed similar response properties and plasticity rules across Drosophila DANs in the γ- and β'-lobe. This contrasts with their anatomical and functional heterogeneity, which indicates yet undiscovered mechanisms and functions of DAN plasticity. Note, that this study could not test whether the flies learned in the imaging setup, as currently no behavioral readout exists for odor-shock conditioning during physiological experiments. Nevertheless, since a conditioning protocol and stimulus application comparable to an established behavioral paradigm was used, it is believed that the associative plasticity in neuronal responses that was found underlies behavioral associative plasticity. Therewith the data lay the foundations for causal studies on the function of associative plasticity in DANs (Dylla, 2017).

Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster

Biogenic amines are important signaling molecules in the central nervous system of both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, biogenic amines take part in the regulation of various vital physiological processes such as feeding, learning/memory, locomotion, sexual behavior, and sleep/arousal. Consequently, several morphological studies have analyzed the distribution of aminergic neurons in the CNS. Previous descriptions, however, did not determine the exact spatial location of aminergic neurite arborizations within the neuropil. The release sites and pre-/postsynaptic compartments of aminergic neurons also remained largely unidentified. This study used gal4-driven marker gene expression and immunocytochemistry to map presumed serotonergic (5-HT), dopaminergic, and tyraminergic/octopaminergic neurons in the thoracic and abdominal neuromeres of the Drosophila larval ventral ganglion relying on Fasciclin2-immunoreactive tracts as three-dimensional landmarks. With tyrosine hydroxylase- (TH) or tyrosine decarboxylase 2 (TDC2)-specific gal4-drivers, the distribution of ectopically expressed neuronal compartment markers was examined in presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons, respectively. The results suggest that thoracic and abdominal 5-HT and TH neurons are exclusively interneurons whereas most TDC2 neurons are efferent. 5-HT and TH neurons are ideally positioned to integrate sensory information and to modulate neuronal transmission within the ventral ganglion, while most TDC2 neurons appear to act peripherally. In contrast to 5-HT neurons, TH and TDC2 neurons each comprise morphologically different neuron subsets with separated in- and output compartments in specific neuropil regions. The three-dimensional mapping of aminergic neurons now facilitates the identification of neuronal network contacts and co-localized signaling molecules, as exemplified for DOPA decarboxylase-synthesizing neurons that co-express crustacean cardioactive peptide and myoinhibiting peptides (Vömel, 2008).

This study used gal4-driven marker gene expression and immunocytochemistry to three-dimensionally map presumed serotonergic, dopaminergic and tyraminergic/octopaminergic neurons within the Fas2 landmark system of the larval VG. Furthermore, several ectopically expressed pre- and postsynaptic markers were employed to reveal the in- and output compartments of presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons. The results allow comparison of the segmental distribution patterns of aminergic neurons and to trace aminergic projections to defined neuropil areas within the VG. In the following, the morphology of aminergic neurons are related to known biogenic amine (BA) functions and describes putative neuronal network interactions with other VG neurons. This work also exemplifies how Fas2-based mapping can simplify the identification of co-localized signaling molecules, and allocate all neurons within the complex Ddc-gal4 expression pattern to distinct neuron subsets (Vömel, 2008).

Throughout the insects, similar neuron groups synthesize BAs. These groups typically comprise only few neurons with large branching patterns. In agreement with previous studies, 5-HT neurons in t1-a8 of the Drosophila larval VG represent interneurons with intrasegmental neurites. The 5-HT neurons of a8, however, appear to supply only the neuropil of a7, but not that of a8 and the adjacent 'terminal plexus'. Like 5-HT neurons, the presumptive dopaminergic TH neurons lack peripheral projections and appear to exclusively represent interneurons. In contrast, presumptive tyraminergic/octopaminergic TDC2 neurons mostly represent efferent vumTDC2 neurons. The vumTDC2 neurons obviously project to larval body wall muscles including M1 and M2 since these muscles showed TA- and OA-immunoreactive type II boutons. In a8, dorsally located dmTDC2 neurons send axons through the associated segmental nerves, and hence are efferent neurons as well. These dmTDC2 neurons probably innervate the reproductive tract in the adult female fly. Besides the dmTDC2 neurons of a8, typically two additional dmTDC2 neurons reside in the dorsal cortex between the last subesophageal neuromere and t1. These dmTDC2 neurons were not described in previous morphological studies on TA- and OA-/TβH-immunoreactive neurons. Nevertheless, all dmTDC2 neurons in the VG consistently showed strong Tdc2-gal4-driven mCD8GFP expression as well as TßH immunoreactivity. Thus, they likely synthesize both TA and OA. Although their neurites could not be traced, the dmTDC2 neurons resemble a pair of anterior medial neurons in locusts and crickets that localize to t1 and innervate the anterior connectives. Alternatively, dmTDC2 neurons may correspond to a single dorsal unpaired median neuron which resides in t1 of the locust and supplies the subesophageal nerves. Like dmTDC2 neurons, pmTDC2 neurons are probably interneurons as well. The soma position of pmTDC2 neurons highly resembles that of descending OA-immunoreactive interneurons detected in the subesophageal and thoracic neuromeres of bees, crickets, cockroaches, locusts, and moths (Vömel, 2008).

Within the larval VG of Drosophila, aminergic neurons typically show a segmentally reiterated distribution. The number of aminergic modules, however, often varies between different neuromeres. 5-HT neurons, for instance, typically occur as two bilateral pairs per neuromere. Yet, t1 comprises three 5-HT neuron pairs and a8 only one pair. The presumptive dopaminergic TH neurons also lack a strict serial homology since three ventral median TH (vmTH) neurons are present in t1, but only one in t2-a7. Furthermore, dlTH neurons locate to a1-7, but appear to be missing in t1-3. The neuromere a8 lacks TH neurons. The number of presumptive tyraminergic/octopaminergic TDC2 neurons differs between various neuromeres as well. Whereas t1 comprises one or two dmTDC2 neurons, comparable neurons are absent in t2-a7. Putative descending pmTDC2 interneurons localize to t1-a1, but appear to be missing in the remaining abdominal neuromeres. Taken together, the number of aminergic modules in t1 and a8 often deviated from that of t2-a7. This difference may-at least partially-reflect unique neuronal circuits in t1 and a8. While t1 specific physiological functions in larvae are unknown, a8 and the adjacent 'terminal plexus' are associated with the tail region, and hence contain a specific set of sensory neurons and motoneurons. The terminal neuromeres also supply several unique structures such as the spiracles or the anal pads (Vömel, 2008).

Besides the segmental differences in neuron number, the density of aminergic innervation and the amount of immunolabeling/marker gene expression varies between neuromeres as well. In particular, presumptive dopaminergic TH neurons show a striking neuromere-specific labeling pattern. Whereas a1-5 contain only few labeled TH projections, t1-3 and a6-7 comprise a comparably dense network of TH neurites. Similar to TH neurons, 5-HT neurons most densely innervate the neuropil of a7. Since a high extracellular concentration of 5-HT decreases the density of 5-HT-immunoreactive arborizations within the neuropil, a7 may represent a minor 5-HT release site. In contrast to a7, the neuropil of a8 and the adjacent 'terminal plexus' (which receive prominent peptidergic innervation) typically lack aminergic neurite arborizations. Consequently, larval aminergic neurons may play a subordinate role in tail-related physiological processes (Vömel, 2008).

To reveal putative synaptic in- and output zones of aminergic neurons, the neuronal compartment markers neuronal synaptobrevin-GFP, synaptotagmin 1-GFP, and Drosophila Down syndrome adhesion molecule [17.1]-GFP were employed. Neuronal synaptobrevin is a vesicle associated membrane protein that plays a role in the SNARE complex during vesicle transport and fusion with the plasma membrane. In accordance with this function, ectopically expressed neuronal synaptobrevin-GFP (SybGFP) accumulates at nerve terminals. SybGFP therefore served to define the presynaptic compartments of several Drosophila neurons, e.g. in the visual system. However, neuronal synaptobrevin is not restricted to small synaptic vesicles, but also locates to the membrane of large dense core vesicles, which contain BAs or neuropeptides. Consequently, in a7, SybGFP localized to putative release sites of presumptive serotonergic DDC neurons. SybGFP was also used to identify non-synaptic release sites in several peptidergic neurons. In aminergic neurons, the distribution of gal4-driven SybGFP highly resembled the corresponding mCD8GFP expression pattern. SybGFP localized in dotted patterns to aminergic neuron somata and associated neurites. It is therefore suggested that SybGFP does not exclusively label the presynaptic compartments of aminergic neurons. This fits to the assumption that ectopically expressed synaptic proteins can either localize to transport vesicles or non-synaptic compartments in peptidergic neurons. On the other hand, the ubiquitous distribution of SybGFP in aminergic neurites may suggest a widespread BA release/recycling from non-synaptic active sites. In mammals, BA release/recycling is not restricted to synapses. Vesicular monoamine transporters, which transport BAs into secretory vesicles, reside within neuron somata, axons, and dendrites. In Drosophila, the vesicular monoamine transporter DVMAT-A localizes to somata as well as neurites of several aminergic neurons both in the larval. Thus, the widespread distribution of SybGFP and DVMAT-A in aminergic neurons suggests that a considerable amount of aminergic vesicles resides at non-synaptic sites. Non-synaptic BA release/recycling might therefore play a major role for aminergic neuronal network signaling (Vömel, 2008).

Like neuronal synaptobrevin, synaptotagmins also represent integral membrane proteins of both small synaptic and large dense core vesicles. In Drosophila, the products of seven synaptotagmin genes localize to distinct neuronal compartments including the postsynaptic site. At the presynaptic site, synaptotagmin 1 does not participate in the SNARE complex, but acts as a Ca²⁺-sensor for synaptic vesicle fusion. Furthermore, synaptotagmin 1 appears to be the only crucial isoform for synaptic vesicle release. Consequently, a synaptotagmin 1-GFP fusion construct (SytGFP) was developed as a synaptic vesicle marker that specifically labels presynaptic sites. In aminergic neurons, the distribution pattern of SytGFP strikingly differed from the observed mCD8GFP and SybGFP labeling. Primary neurites of aminergic neurons always completely lacked SytGFP. Varicose neurite structures which were less evident in the mCD8GFP and SybGFP expression patterns showed strong SytGFP labeling. In agreement with the SytGFP distribution in other Drosophila neuron types, SytGFP hence appears to exclusively accumulate at the presynaptic sites of aminergic neurons. Thus, SytGFP represents a valuable marker to separate synapses from other neuronal compartments in aminergic neurons. However, since BA release is not restricted to synapses, SytGFP may not label all BA release sites of aminergic neurons. The sparse co-localization of SytGFP and SybGFP in aminergic neurites in fact suggests that aminergic vesicles-which are located distal to presynaptic sites-generally lack SytGFP. Consequently, non-synaptic BA release appears to be independent of synaptotagmin 1, but may depend on other synaptotagmin isoforms such as synaptotagmin α or β. The differing distribution of SytGFP and SybGFP also suggests that aminergic neurons contain several types of aminergic vesicles which are either associated with presynaptic or non-synaptic BA release. Alternatively, aminergic neurons may synthesize additional non-aminergic neurotransmitters like acetylcholine, GABA, or glutamate. Presumed octopaminergic efferent neurons, for instance, appear to release glutamate from type II terminals at the neuromuscular junction. In such neurons, SytGFP likely labels presynaptically located transmitter vesicles and may not reveal BA release sites (Vömel, 2008).

In contrast to SybGFP and SytGFP, ectopically expressed Drosophila Down syndrome adhesion molecule [17.1]-GFP (DscamGFP) localized to postsynaptic compartments and not to axons or presynaptic sites. Consequently, DscamGFP has served as dendrite marker in mushroom body lobe neurons. Aminergic neurons showed only weak DscamGFP labeling. DscamGFP primarily localized to neurites that lacked SytGFP labeling. Since SytGFP accumulates at presynaptic sites, DscamGFP appears to represent a valuable marker to define dendritic compartments in aminergic neurons (Vömel, 2008).

In 5-HT neurons, the distribution of ectopically expressed neuronal compartment markers was not examined since specific gal4 drivers are not available. The Ddc-gal4 driver induces marker gene expression not only in presumed serotonergic, but also in dopaminergic and additional peptidergic neurons. Consequently, neurites of different DDC neuron subsets overlap in specific neuropil areas. Presumptive serotonergic as well as dopaminergic DDC neurites, for instance, localize to the VG neuropil above the CI tracts. These conditions prevent an accurate description and interpretation of the compartment marker distribution in presumptive serotonergic DDC neurons. Thus, appropriate gal4 drivers (e.g. Dtph-gal4) are needed to further analyze 5-HT neuron morphology (Vömel, 2008).

5-HT neurons bifurcate strongly in the whole neuropil of t1-a7, and hence may influence various VG neurons including sensory, inter- as well as motoneurons. However, putative neuronal network contacts of 5-HT neurons were not examined since previous morphological studies on Drosophila 5-HT receptors did not describe the exact spatial location of the respective receptors in the larval VG (Vömel, 2008).

In TH neurons, the distribution of ectopically expressed mCD8GFP, SybGFP, SytGFP and DscamGFP differed only slightly. This might relate to the fact that the VG contains two different TH neuron groups, the vmTH and dlTH neurons, whose neurites contact each other at longitudinal projections. Consequently, pre- and postsynaptic compartments of both TH neuron groups appeared to overlap, e.g. at longitudinal projections next to the VL tracts. Since additional TH neurons located in the brain or subesophageal ganglia also innervate the VG, it was not possible to clarify which TH neuron group attributes to a particular neuronal projection. Several morphological findings, however, suggest that TH neurons possess distinct in- and output sites: Most strikingly, a1-5 contained less TH neurites labeled with mCD8GFP, SybGFP and DscamGFP, as compared to t1-3 and a6-7. In t1-a7, high amounts of SybGFP and SytGFP located to lateral longitudinal projections next to the VL tracts. These longitudinal TH neurites also contained a comparably high amount of DscamGFP, and hence likely represent synaptic in- as well as output compartments of different TH neuron groups. Besides lateral longitudinal TH projections, SybGFP and SytGFP also co-localized to the median neuropil between the DM/VM tracts. At least in a1-5, this neuropil area lacked DscamGFP, and hence probably represents a presynaptic output site of TH neurons. In a6-7, a comparably strong SybGFP and SytGFP labeling was observed in arborizations around transversal TH neurites. Whereas SybGFP mainly located to the dorsal branches of the transversal TH neurite loops, SytGFP and DscamGFP primarily labeled the ventral branches. Thus, the dorsal branches of the transversal TH neurite loops may represent non-synaptic DA release sites, while the ventral branches seem to comprise overlapping synaptic in- and output compartments of different TH neuron groups (Vömel, 2008).

Both vmTH and dorso-lateral TH (dlTH) neurons innervate distinct neuropil areas within the VG. The vmTH neurons send their primary neurites dorsally and then project through the dorsal part of the neuropil above Transversal projection (TP) 3. Since the dorsal neuropil comprises the dendritic compartments of most motoneurons, vmTH neurites are ideally located to modulate locomotor activity. This fits to the finding that DA application onto intact larval CNS-segmental preparations rapidly decreased the rhythmicity of CNS motor activity and synaptic vesicle release at the neuromuscular junction. Unlike vmTH neurons, dlTH neurons exclusively innervate the ventral part of the VG neuropil beneath TP 3. There, putative dendritic compartments of TH neurons mainly localize to lateral longitudinal and to transversal projections adjacent to the main output site of several afferent sensory neurons, e.g. tactile and proprioreceptive neurons. Thus, some TH neurons may receive synaptic input from specific sensory neurons. In contrast, TH neurons also seem to have output sites in the ventral part of the neuropil, and hence may influence the signal transmission between sensory neurons and interneurons. This fits to the finding that peptidergic apterous neurons, which appear to transmit sensory input from the VG to the brain, express DA receptors. Concomitantly, dendritic compartments of apterous neurons seem to reside adjacent to the putative DA release sites of TH neurons at the CI tracts. Besides the overlap between transversal TH neurites and sensory/interneuron projections in the ventral neuropil, TH neurons may influence several neuron groups at other locations within the VG. For instance, the putative synaptic output sites of TH neurons in the median neuropil between the DM/VM tracts overlap with presumptive input compartments of both interneurons and efferent neurons expressing peptides such as CCAP, corazonin, FMRFa, or MIP. Furthermore, the putative output sites at longitudinal TH projections next to the VL tracts lay adjacent to presumptive input compartments of e.g. efferent leucokininergic neurons (Vömel, 2008).

In the VG, most TDC2 neurons are efferent vumTDC2 neurons and showed a differential distribution of ectopically expressed SybGFP, SytGFP, and DscamGFP. The primary neurites and transversal projections of vumTDC2 neurons were labeled with DscamGFP, but lacked SytGFP. Therefore, these neurites likely represent dendritic input sites. This fits to the finding that vumTDC2 neurons possess output sites at larval body wall muscles. However, vumTDC2 neurites within the VG also contained high amounts of SybGFP, and hence may release TA/OA from non-synaptic sites. Besides vumTDC2 neurites, SybGFP strongly labeled longitudinal TDC2 neurites and associated arborizations in the dorso-lateral neuropil between TP 1 and 3. These TDC2 projections showed prominent SytGFP labeling and TßH immunoreactivity, but largely lacked DscamGFP. Thus, the dorsal part of the VG neuropil likely contains output compartments of TDC2 neurons. Since the larval brain seems to contain only tyramine- and no octopamine-immunoreactive neurons, these output sites likely derive from descending interneurons located in the subesophageal ganglia, dmTDC2 or pmTDC2 neurons. Noteworthy, the strong SybGFP and SytGFP labeling in TDC2 neurites projecting through the dorso-lateral neuropil of the VG overlapped with DscamGFP in transverse vumTDC2 neurites. Thus, descending TDC2 neurons may interact with vumTDC2 neurons (Vömel, 2008).

The VG comprises efferent vumTDC2 neurons as well as several putative TDC2 interneuron groups. Since all vumTDC2 neurons appear to have synapses at peripheral targets and dendrites in the dorsal neuropil, they show the typical motoneuron morphology. This corresponds to the finding that OA inhibited synaptic transmission at the neuromuscular junction by affecting both pre- and postsynaptic mechanisms. In addition, T?H mutant larvae, with altered levels of TA and OA, showed severe locomotion defects, which seemed to be linked to an imbalance between TA and OA signaling. Hence, vumTDC2 neurons likely regulate peripheral processes such as body wall muscle activity, whereas TDC2 interneurons centrally modulate the neuronal activity of motoneurons and interneurons involved in locomotor control. Interestingly, presumptive presynaptic compartments of descending TDC2 interneurons reside adjacent to transversal vumTDC2 dendrites. Thus, both TDC2 neuron groups may interact to modulate larval locomotor activity. Besides their function for locomotion, descending TDC2 neurons may also influence other neurons which project into the dorsal neuropil between TP 1 and 3. The putative output sites of TDC2 interneurons, for instance, lay adjacent to several peptidergic projections showing allatostatin-A, FMRFa, MIP or tachykinin immunoreactivity. However, nothing is known about TA/OA receptor distribution in the larval VG (Vömel, 2008).

During the morphological analysis of DDC neurons in the L3 larval VG, two DDC neuron groups were identified that obviously synthesize neither 5-HT nor DA. This corresponds to the previous finding that Ddc-gal4-driven marker gene expression is not restricted to presumptive serotonergic 5-HT and dopaminergic TH neurons. However, it cannot be excluded that the putative non-aminergic DDC neurons transiently synthesize BAs during other developmental stages. Ddc-gal4-driven mCD8GFP expression never revealed the dlTH neurons. This may relate to the fact that the onset of Ddc expression varies between different DDC neuron groups, and high DDC and TH levels do not temporally coincide. Taken together, these results suggest that-at least in the L3 larval VG-the Ddc-gal4 expression pattern 1) contains additional non-aminergic neurons, and 2) typically comprises most, but not all 5-HT and TH neurons. These particular characteristics of the Ddc-gal4 driver line should be carefully considered for the interpretation of studies that employed Ddc-gal4-driven expression to genetically manipulate serotonergic or dopaminergic neurons. Nevertheless, since all Ddc-gal4 expressing neurons within the VG showed at least faint DDC immunoreactivity, the Ddc-gal4 driver appears to restrict ectopical gene expression to DDC neurons. Noteworthy, the DDC neurons which lacked 5-HT and TH immunoreactivity showed corazonin and CCAP/MIP immunoreactivity respectively. In the moth Manduca sexta, these peptides play vital roles during ecdysis. At least the CCAP/MIP neurons are also necessary for the proper timing and execution of ecdysis behavior in Drosophila. Since dopaminergic DDC neurons regulate the titers of the molting hormones 20-hydroxyecdyson and juvenile hormone, both aminergic and peptidergic DDC neurons may interact to control ecdysis-related events. Recent findings indeed suggest that CCAP/MIP neurons modulate TH activity after eclosion to control the precise onset of tanning (Vömel, 2008).

Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). This study shows that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulated ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. The study proposes that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant (Ohhara, 2015).

In many animal species, the developmental transition is a well-known biological process in which the organism alters its body morphology and physiology to proceed from the juvenile growth stage to the adult reproductive stage. For example, in mammals, puberty causes a drastic change from adolescent to adulthood, whereas in insects, metamorphosis initiates alteration of body structures to produce sexually mature adults, a process accompanied by changes in habitat and behavior. These developmental transitions are primarily regulated by steroid hormones, production of which is regulated coordinately by developmental timing and nutritional conditions. How these processes are precisely regulated in response to developmental and environ mental cues is a longstanding question in biology (Ohhara, 2015).

In holometabolous insects, the steroid hormone ecdysone plays a pivotal role in metamorphosis. In Drosophila, metamorphic development from the third-instar larva into the adult, through the prepupa and pupa, initiates 90-96 h after hatching (hAH) at 25°C under a standard culture condition. At the onset of the larval-prepupal transition, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the peripheral organs. The activities of 20E terminate larval development and growth and initiates metamorphosis. Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during development. The best-known stimulator of ecdysone biosynthesis is prothoracico-tropic hormone (PTTH), which is produced by neurons in the CNS. PTTH activates the receptor tyrosine kinase Torso in the PG to stimulate expression of ecdysone biosynthetic genes through the Ras85D/Raf/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Insulin-like peptides (Ilps), members of another class of neuron-derived factors, activate PI3K in the PG, resulting in production of ecdysone biosynthetic proteins. The Activin/transforming growth factor-β (TGF-β) signaling pathway is also required in the PG for the expression of PTTH and Ilps receptors, although to date it remains unclear which organ produces the ligand that acts on the PG (Ohhara, 2015).

In addition to these neuropeptides, the larval-prepupal transition is modulated by environmental cues such as nutritional conditions that influence larval body size. For example, at 56 hAH, early third-instar larvae attain the minimal viable weight (MVW), at which sufficient nutrition is stored in larvae to ensure their survival through metamorphosis. After attaining MVW, larvae pass another checkpoint, critical weight (CW), defined as the minimum larval size at which starvation no longer delays the larval-prepupal transition. In Drosophila, both checkpoints occur almost simultaneously, making it difficult to distinguish them. However, CW is regarded as a body size checkpoint that initiates metamorphosis and is therefore believed to ultimately modulate ecdysone production in the PG. However, its downstream effectors and signaling pathway remain elusive (Ohhara, 2015).

Based on data obtained in Manduca and Bombyx, a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. This study shows that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval-prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. It is proposed that this autocrine system acts downstream of the CW checkpoint to allow the larval-prepupal transition. It is speculated that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions (Ohhara, 2015).

Previously studies have shown that the GPCR Octβ3R is expressed in multiple larval tissues, including the PG. To determine whether Octβ3R is involved in ecdysone biosynthesis and metamorphosis, RNAi was used to knock down Octβ3R function specifically in the PG, using the Gal4-upstream activation sequence (UAS) system. Two different UAS-Octβ3RRNAi constructs targeting distinct regions of the Octβ3R mRNA (Octβ3R^RNAi-1 and Octβ3R^RNAi-2) were used to knock down Octβ3R in the PG with the help of a phantom (phm)-22-Gal4 driver. Strikingly, larvae expressing Octβ3RRNAi in the PG never developed into adult flies, and 96% of phm>Octβ3R^RNAi-1 animals and 34% of phm>Octβ3R^RNAi-2 animals arrested at the larval stage. When UAS-dicer2 was introduced into phm>Octβ3R^RNAi-2 larvae (phm>Octβ3R^RNAi-2+dicer2) to increase RNAi activity, all of these animals arrested at the larval stage. Using in situ hybridization, a significant reduction was confirmed in the Octβ3R mRNA levels in the PG of knockdown animals relative to control larvae. These data suggest that Octβ3R expression in the PG is essential for executing the larval-prepupal transition in metamorphosis (Ohhara, 2015).

Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3R^RNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

Octβ3R is thought to be activated by octopamine and tyramine binding. Octopamine is synthesized from tyramine by tyramine β-hydroxylase (Tbh), and tyramine is synthesized from tyrosine by tyrosine decarboxylase (Tdc). In Drosophila, two Tdc genes (Tdc1 and Tdc2) and one Tbh gene have been identified, and all of them are expressed in the larval CNS. Tdc1, Tdc2, and Tbh are also expressed in the PG. Furthermore, octopamine and tyramine were detected in the PG by immunostaining. Thus, octopamine and/or tyramine synthesized in the PG may activate Octβ3R in an autocrine manner to induce ecdysone production (Ohhara, 2015).

To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2^RNAi-1 and Tdc2^RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2^RNAi-1+dicer2 and phm > Tdc2^RNAi-2+dicer2). All phm > Tdc2^RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2^RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2^RNAi-1+ dicer2 and phm > Tdc2^RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2^RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2^RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

To determine which Octβ3R ligand is responsible for this autocrine signaling, Tbh was knocked down in the PG to prevent conversion of tyramine into octopamine. To knock down Tbh, two constructs targeting distinct regions of the Tbh transcript (Tbh^RNAi-1 and Tbh^RNAi-2) were expressed along with dicer2 under the control of phm-22-Gal4 (phm > Tbh^RNAi-1+ dicer2 and phm > Tbh^RNAi-2+dicer2). Although the Tbh knockdown caused a reduction in octopamine production in the PG, these larvae did not exhibit any obvious defects in the larval-prepupal transition or subsequent metamorphosi. These data suggest that tyramine, rather than octopamine, is the Octβ3R ligand that activates ecdysone production in the PG (Ohhara, 2015).

Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3R^RNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3R^RNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3R^RNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3R^RNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3R^RNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

The observations described above demonstrate that phm>Octβ3RRNAi affects Ilps and PTTH signaling in the PG as early as 60 hAH, raising the question of when Octβ3R function is required in the PG for execution of the larval-prepupal transition. To address this issue, the Gal80ts and Gal4/UAS system, which restricts expression of Octβ3R dsRNA in the PG at 18^oC, but allows its expression at 28^oC, was used. The results of temperature upshift and downshift experiments revealed that the larval-prepupal transition was impaired only when Octβ3R dsRNA was expressed in the PG at around 60 hAH. Notably, 60 hAH is the critical period during which larvae attain CW under nutrient-rich conditions. As noted above, when larvae are starved before attainment of CW, they are unable to transit into the prepupal stage. By contrast, starved larvae can successfully transit to prepupal/pupal stage without developmental delay once they have attained CW by growing beyond the critical period (~56 hAH) under nutrient-rich conditions in standard Drosophila medium. Thus, it is hypothesized that Octβ3R signaling acts downstream of the body-size checkpoint, or attainment of CW, to allow the larval-prepupal transition (Ohhara, 2015).

Several lines of evidence support this hypothesis. First, Octβ3R function is required for activation of Ilps and PTTH signaling detected in the PG at 60 hAH. By contrast, at 48 hAH, before the attainment of CW, neither signaling pathway is active in the PG. Second, Ilps and PTTH signaling was not activated in the PG when the larvae were starved from 48 hAH onward (early starvation), whereas these signaling pathways were active when the larvae were starved after 60 hAH (late starvation). Finally, a ligand for Octβ3R, tyramine, was detectable in the PG at 60 hAH, but decreases after this stage under a nutrient-rich condition. This decrease in tyramine was abrogated by early starvation but not by late starvation. Assuming that this decrease in tyramine in the PG is due to its secretion from PG cells, it is reasonable to propose that attainment of CW causes tyramine secretion from the PG at around 60 hAH, which in turn activates Octβ3R to regulate the Ilps and PTTH pathways, leading to the larval-prepupal transition (Ohhara, 2015).

This study demonstrates that monoaminergic regulation plays a pivotal role in ecdysone biosynthesis to induce metamorphosis and that Octβ3R acts as an upstream regulator essential for the Ilps and PTTH signaling. In addition, the data indicate that Octβ3R ligands are produced in the PG to stimulate ecdysone biosynthesis in an autocrine manner. Autocrine signaling has been proposed to mediate the community effect, in which identical neighboring cells are coordinated in their stimulation and maintenance of cell type-specific gene expression and their differentiation, as observed in muscle development of amphibian embryos. Thus, it is proposed that monoaminergic autocrine signaling among PG cells acts to increase their responsiveness to Ilps and PTTH, thereby allowing coordinated ex- pression of ecdysone biosynthetic genes within a time window following exposure to neuropeptides (Ohhara, 2015).

These findings raise the larger question of whether monoamine acts as part of an evolutionarily conserved mechanism of steroid hormone production. In vertebrates, there is limited evidence of monoaminergic regulation of steroid hormone biosynthesis. For example, in cultured adrenal glands, catecholamine stimulates the biosynthesis of the steroid hormone cortisol in a paracrine manner to elicit a stress reaction. Another example is the Leydig cells of the mammalian testes, in which the steroid hormone testosterone is produced mainly in response to pituitary gonadotropin. However, catecholamine signaling through β-adrenergic receptors, the orthologs of Octβ3R, also promotes the production of testosterone from cultured fetal Leydig cells, which may be the site of catecholamine synthesis in the fetal and mature human testes. Thus, monoamines may play a conserved role in modulating and/or stimulating steroid hormone production during physiological and developmental transitions (Ohhara, 2015).

Limited distal organelles and synaptic function in extensive monoaminergic innervation

Organelles such as neuropeptide-containing dense-core vesicles (DCVs) and mitochondria travel down axons to supply synaptic boutons. DCV distribution among en passant boutons in small axonal arbors is mediated by circulation with bidirectional capture. However, it is not known how organelles are distributed in extensive arbors associated with volume transmission and neuromodulation by monoamines and neuropeptides and mammalian dopamine neuron vulnerability. Therefore, this study examined presynaptic organelle distribution in Drosophila octopamine neurons that innervate approximately 20 muscles with approximately 1500 boutons. Unlike in smaller arbors, distal boutons in these arbors contain fewer DCVs and mitochondria, although active zones are present. Absence of vesicle circulation is evident by proximal nascent DCV delivery, limited impact of retrograde transport and older distal DCVs. Traffic studies show that DCV axonal transport and synaptic capture are not scaled for extensive innervation, thus limiting distal delivery. Activity-induced synaptic endocytosis and synaptic neuropeptide release are also reduced distally. It is proposed that limits in organelle transport and synaptic capture compromise distal synapse maintenance and function in extensive axonal arbors, thereby affecting development, plasticity and vulnerability to neurodegenerative disease (Tao, 2017).

Octopamine mediates starvation-induced hyperactivity in adult Drosophila

Starved animals often exhibit elevated locomotion, which has been speculated to partly resemble foraging behavior and facilitate food acquisition and energy intake. Despite its importance, the neural mechanism underlying this behavior remains unknown in any species. This study confirmed and extended previous findings that starvation induced locomotor activity in adult fruit flies Drosophila melanogaster. It was also shown that starvation-induced hyperactivity was directed toward the localization and acquisition of food sources, because it could be suppressed upon the detection of food cues via both central nutrient-sensing and peripheral sweet-sensing mechanisms, via induction of food ingestion. It was further found that octopamine, the insect counterpart of vertebrate norepinephrine, as well as the neurons expressing octopamine, were both necessary and sufficient for starvation-induced hyperactivity. Octopamine was not required for starvation-induced changes in feeding behaviors, suggesting independent regulations of energy intake behaviors upon starvation. Taken together, the current results establish a quantitative behavioral paradigm to investigate the regulation of energy homeostasis by the CNS and identify a conserved neural substrate that links organismal metabolic state to a specific behavioral output (Yang, 2015).

Upon starvation, animals exhibit increased feeding and foraging behaviors, which in turn increases energy intake and restores energy homeostasis. The neural mechanism of feeding behavior, particularly how feeding is regulated by metabolic signals, has been extensively studied in both rodents and insect species. In contrast, how organismal metabolism influences foraging remains largely unclear (Yang, 2015).

This study aimed to establish a behavioral paradigm for foraging behavior and to study how it is regulated by starvation. It has been reported that starvation induces hyperactivity in both rodents and fruit flies, but its relationship with foraging was unclear. It was first confirmed that starvation induced a robust and sustained increase in locomotion in adult fruit flies. Furthermore, it was found that starvation-induced hyperactivity could be suppressed upon the detection of nutritious substrate via an internal energy sensor SLC5A11, as well as the detection of food palatability via sweet-sensing gustatory neurons expressing Gr5a and Gr64a. It was also shown that although food intake per se was not sufficient to drive the suppressive effect on locomotion, nutrient/sweetness-induced fluid ingestion was indeed required. In addition, the data showed that mutant flies with a deficit in starvation-induced hyperactivity took longer time to locate and occupy desired food sources. Collectively, it is concluded that starvation-induced hyperactivity in adult flies resembles foraging behavior in laboratory conditions, because it is directed toward and facilitates the localization and acquisition of food. This set of behavioral assays therefore offers a platform and an entry point to further dissect the neural basis of this evolutionarily conserved and critical behavior (Yang, 2015).

By using the foraging assay described above, octopamine, the insect counterpart of vertebrate norepinephrine, was found to be both necessary and sufficient for starvation-induced hyperactivity. In fruit flies, octopamine is only synthesized and released in a small number of CNS neurons (~100-150 neurons per fly). Therefore, the findings presented in this study offer a clear entry point to further dissect the neural circuitry that underlies foraging behavior in fruit flies. In addition, it is of obvious interest to examine whether norepinephrine is also involved in locomotor responses to starvation in rodents, such as FAA (Yang, 2015).

Tyramine is the precursor of octopamine synthesis and itself can also function as a neurotransmitter in insects. In the present study, the possibility was exclued that tyramine alone was either necessary or sufficient for starvation-induced hyperactivity. However, the possibility was not excluded that octopamine and tyramine work in a synergistic way to regulate this behavior. Fruit flies express several receptors that are sensitive to both octopamine and tyramine in vitro, which serves as a potential 'hub' to integrate octopamine and tyramine signaling in vivo. Future research is needed to clarify the role of octopamine and tyramine in starvation-induced hyperactivity (Yang, 2015).

Octopamine plays an important role in the regulation of a variety of fly behaviors, such as sleep, learning, and aggression. It is of interest to investigate whether and how different subsets of octopaminergic neurons modulate different behaviors in flies. Meanwhile, it is noteworthy that many of these behaviors regulated by octopamine signaling have a locomotor component. It is therefore plausible that the octopamine system may function as a general 'arousal' center, modulating physical activity of flies in response to external/internal cues such as circadian rhythm, conspecific chemosensory stimuli, and metabolic signals (Yang, 2015).

Despite its importance in starvation-induced hyperactivity, this study found that octopamine was not required for starvation-induced changes in feeding behavior. These findings argue for independent regulations of multiple starvation-induced behavioral responses. Consistent with this idea, rodent studies have shown that several aspects of foraging behavior (e.g., FAA and food hoarding) do not require the genes and brain regions that are important for feeding control. In contrast, activating hypothalamic neurons expressing neuropeptide Y and agouti-related peptide promotes both feeding and locomotion, suggesting that feeding and foraging pathways converge at some point. It is therefore important to study whether there exists a common 'hunger' center in the CNS that coordinates various starvation-induced behaviors or whether different neural mechanisms independently sense changes in the metabolic state and modulate energy homeostasis (Yang, 2015).

Octopamine drives endurance exercise adaptations in Drosophila

Endurance exercise is an effective therapeutic intervention with substantial pro-healthspan effects. Male Drosophila respond to a ramped daily program of exercise by inducing conserved physiological responses similar to those seen in mice and humans. Female flies respond to an exercise stimulus but do not experience the adaptive training response seen in males. This study used female flies as a model to demonstrate that differences in exercise response are mediated by differences in neuronal activity. The activity of octopaminergic neurons is specifically required to induce the conserved cellular and physiological changes seen following endurance training. Furthermore, either intermittent, scheduled activation of octopaminergic neurons or octopamine feeding is able to fully substitute for exercise, conferring a suite of pro-healthspan benefits to sedentary Drosophila. These experiments indicate that octopamine is a critical mediator of adaptation to endurance exercise in Drosophila (Sujkowski, 2017).

Octopamine controls starvation resistance, life span and metabolic traits in Drosophila

The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. This study shows that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. It was inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes (Li, 2016).

Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila

Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. This study investigated how nutritional states modulate starvation-induced hyperactivity in adult Drosophila. The receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, is required for starvation-induced hyperactivity. AKHR is expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminates starvation-induced hyperactivity, whereas activation of these neurons accelerates the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons are involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposes suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila (Yu, 2016).

Food seeking and food consumption are essential for the acquisition of food sources, and hence survival, growth, and reproduction of animal species. Starvation influences food-seeking behavior via both modulating the perception of food cues as well as enhancing flies' locomotor activity. Accumulated evidence has suggested that starvation modulates the activity of ORNs via multiple neural and hormonal cues, which in turn facilitates odor driven food search and food consumption. Similarly, starvation also modulates the perception of food taste via the relative sensitivity of appetitive sweet-sensing and aversive bitter-sensing GRNs,which may in turn increase the attractiveness of food taste. However, how starvation increases the locomotor activity of flies remains largely uncharacterized (Yu, 2016).

Consistent with previous reports, this study has shown that starved fruit flies exhibit sustained increase in their locomotor activity, which can be suppressed by food consumption induced by both nutritive and non-nutritive food cues. The present study has shown that a small group of neurons located in the subesophageal zone (SEZ) region of the fly brain are both necessary and sufficient for starvation induced hyperactivity. These neurons sense the changes in flies' internal nutritional states by directly responding to two sets of hormones, AKH and DILPs, and modulate locomotor activity in response. Single cell analysis has identified that these AKHR+dInR+ neurons are octopaminergic, which offers an entry point to trace the downstream neural circuitry that regulates starvation-induced hyperactivity. For example, there are seven candidate octopamine receptors in fruit flies and it would be of interest to investigate whether any of these receptors and the receptor-expressing neurons are involved in locomotor regulation upon starvation (Yu, 2016).

AKH and DILPs are two sets of functionally counteracting hormones in fruit flies. As its mammalian analog glucagon, the reduction in circulating sugars induces the release of AKH, which in turn mobilizes fat storage and provides energy supply for flies. In contrast, DILPs, the insect analog of mammalian insulin, function as satiety hormones. Dietary nutrient induces the release of DILPs into the hemolymph, which in turn promotes protein synthesis, body growth, and other anabolic processes. This study has shown that these two hormonal signaling systems exert opposite effects on starvation-induced hyperactivity via a small group of AKHR+InR+ octopaminergic neurons. These results suggest that these AKHR+dInR+ neurons can integrate the inputs from the two hormonal signaling systems representing hunger and satiety at the same time, and modulate flies' locomotor activity. This elegant yet concise design allows these neurons to be responsive to rapid changes in the internal nutritional states as well as food availability. Furthermore, it is possible that besides hunger and satiety, other physiological states such as wakefulness, stress, and emotions also influence flies' locomotor activity. Notably, single cell analysis has shown that these AKHR+dInR+ neurons also sparsely express other neuropeptide receptors, suggesting that at least small portions of these neurons may also receive input from other neuropeptidergic systems (Yu, 2016).

Starved animals exhibited increased locomotion and food consumption, the transition of which relies on the detection of food cues. But whether these two behaviors are interdependently or independently regulated remains unclear. This study has shown that these two behaviors are dissociable from each other in fruit flies. On the one hand, although AKHR+ neurons exert robust modulatory effect on starvation-induced hyperactivity, these neurons are neither necessary nor sufficient for starvation-induced food consumption. On the other hand, the regulation of food consumption is independent of starvation-induced hyperactivity as well. Previous studies have shown that a small subset of GABAergic neurons in the fly brain regulates food consumption but exerts no effect on 10 starvation-induced hyperactivity (Pool, 2014). In addition, several neuropeptides are known to regulate food consumption, such as Hugin, NPF, sNPF, Leucokinin, and AstA. However this study found in an RNAi screen that the receptors of these neuropeptides were not involved in the regulation of starvation-induced hyperactivity. Taken together, it is likely that starvation-induced hyperactivity and food consumption are independently regulated by different sets of hormonal cues, and that AKHR+ neurons are only involved in the former but not the latter. These results may shed light on the regulation of food intake in mammals, especially whether starvation-induced hyperactivity and food consumption are also independently regulated by different sets of hormones and distinct neural circuitry in mammals (Yu, 2016).

PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine

Male sexual desire typically declines with ageing. However, understanding of the neurobiological basis for this phenomenon is limited by knowledge of the brain circuitry and neuronal pathways controlling male sexual desire. A number of studies across species suggest that dopamine (DA) affects sexual desire. This study used genetic tools and behavioural assays to identify a novel subset of DA neurons that regulate age-associated male courtship activity in Drosophila. Increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies. These results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila. These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain (Kuo, 2015).

Novel Drosophila model for parkinsonism by targeting phosphoglycerate kinase

Patients with Parkinson's disease (PD) show a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, called parkinsonism. Several lines of recent evidence support the hypothesis that mutations in the gene encoding phosphoglycerate kinase (PGK) play an important role in the PD mechanism. PGK is a key enzyme in the glycolytic pathway that catalyzes the reaction from 1,3-diphosphoglycerate to 3-phosphoglycerate. This study established a parkinsonism model targeting Drosophila Pgk. Dopaminergic (DA) neuron-specific Pgk knockdown lead to locomotive defects in both young and aged adult flies and was accompanied by progressive DA neuron loss with aging. Pgk knockdown in DA neurons decreased dopamine levels in the central nervous system (CNS) of both young and aged adult flies. These phenotypes are similar to the defects observed in human PD patients, suggesting that the Pgk knockdown flies established herein are a promising model for parkinsonism. Furthermore, pan-neuron-specific Pgk knockdown induced low ATP levels and the accumulation of reactive oxygen species (ROS) in the CNS of third instar larvae. Collectively, these results indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons (Shimizu, 2020).

Calycosin Alleviates Paraquat-Induced Neurodegeneration by Improving Mitochondrial Functions and Regulating Autophagy in a Drosophila Model of Parkinson's Disease

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder with limited clinical treatments. The occurrence of PD includes both genetic and environmental toxins, such as the pesticides paraquat (PQ), as major contributors to PD pathology in both invertebrate and mammalian models. Calycosin, an isoflavone phytoestrogen, has multiple pharmacological properties, including neuroprotective activity. However, the paucity of information regarding the neuroprotective potential of calycosin on PQ-induced neurodegeneration led to an exploration of whether calycosin can mitigate PD-like phenotypes and the underlying molecular mechanisms. A PQ-induced PD model in Drosophila was used as a cost-effective in vivo screening platform to investigate the neuroprotective efficacy of natural compounds on PD. Calycosin showed a protective role in preventing dopaminergic (DA) neuronal cell death in PQ-exposed Canton S flies. Calycosin-fed PQ-exposed flies exhibit significant resistance against PQ-induced mortality and locomotor deficits in terms of reduced oxidative stress, loss of DA neurons, the depletion of dopamine content, and phosphorylated JNK-caspase-3 levels. Additionally, mechanistic studies show that calycosin administration improves PQ-induced mitochondrial dysfunction and stimulates mitophagy and general autophagy with reduced pS6K and p4EBP1 levels, suggestive of a maintained energy balance between anabolic and catabolic processes, resulting in the inhibition of neuronal cell death. Collectively, this study substantiates the protective effect of calycosin against PQ-induced neurodegeneration by improving DA neurons' survival and reducing apoptosis, likely via autophagy induction, and it is implicated as a novel therapeutic application against toxin-induced PD pathogenesis (Chaouhan, 2022).

The role of tyrosine hydroxylase-dopamine pathway in Parkinson's disease pathogenesis

Parkinson's disease (PD) is characterized by selective and progressive dopamine (DA) neuron loss in the substantia nigra and other brain regions, with the presence of Lewy body formation. Most PD cases are sporadic, whereas monogenic forms of PD have been linked to multiple genes, including Leucine kinase repeat 2 (LRRK2) and PTEN-induced kinase 1 (PINK1), two protein kinase genes involved in multiple signaling pathways. There is increasing evidence to suggest that endogenous DA and DA-dependent neurodegeneration have a pathophysiologic role in sporadic and familial PD. This study generated patient-derived dopaminergic neurons and human midbrain-like organoids (hMLOs), transgenic (TG) mouse and Drosophila models, expressing both mutant and wild-type (WT) LRRK2 and PINK1. Using these models,the effect of LRRK2 and PINK1 on tyrosine hydroxylase (TH)-DA pathway was studied. PD-linked LRRK2 mutations were able to modulate TH-DA pathway, resulting in up-regulation of DA early in the disease which subsequently led to neurodegeneration. The LRRK2-induced DA toxicity and degeneration were abrogated by wild-type (WT) PINK1 (but not PINK1 mutations), and early treatment with a clinical-grade drug, α-methyl-L-tyrosine (α-MT), a TH inhibitor, was able to reverse the pathologies in human neurons and TG Drosophila models. Opposing effects between LRRK2 and PINK1 on TH expression were also identified, suggesting that functional balance between these two genes may regulate the TH-DA pathway. These findings highlight the vital role of the TH-DA pathway in PD pathogenesis. LRRK2 and PINK1 have opposing effects on the TH-DA pathway, and its balance affects DA neuron survival. LRRK2 or PINK1 mutations can disrupt this balance, promoting DA neuron demise. These findings provide support for potential clinical trials using TH-DA pathway inhibitors in early or prodromic PD (Zhou, 2022).

Impact of age on the rotenone-induced sporadic Parkinson's disease model using Drosophila melanogaster

Rotenone, a naturally occurring toxin, has been used to induce sporadic Parkinson's disease (PD) in Drosophila melanogaster for decades. However, the age of flies varies considerably between studies in this model. To investigate the impact of age on the rotenone-induced PD model, male flies were collected at the age of 1, 5, 7, and 10 days post-eclosion, respectively. Then, flies were immediately exposed to a feeding medium supplemented with 250 μM rotenone for seven days. The motor ability of Drosophila was detected by negative geotaxis assay, and the number of dopamine (DA) neurons and tyrosine hydroxylase (TH) expression levels were evaluated. The results showed that both the motor deficits and mortality increased with age. The flies older than five days showed typical PD features, including the loss of DA neurons, decreased TH expression levels, and decreased locomotive ability. However, 1-day-old flies displayed an unstable motor deficit and little TH expression changes after seven days of rotenone exposure. Lastly, after 7 days of exposure to rotenone, the death rate of flies rapidly increased with increasing starting age. The death rates of 1-, 5-, 7-, and 10-days old flies were 10.0%, 22.8%, 41.5%, and 50.4%, respectively. The findings of this study suggest that age is a crucial factor impacting the Drosophila PD model. This information provides a reference for the age selection to use this model for future studies (Li, 2023).

Identification of a New Allosteric Binding Site for Cocaine in Dopamine Transporter

Dopamine (DA) transporter (DAT) is a major target for psychostimulant drugs of abuse such as cocaine that competitively binds to DAT, inhibits DA reuptake, and consequently increases synaptic DA levels. In addition to the central binding site inside DAT, the available experimental evidence suggests the existence of alternative binding sites on DAT, but detection and characterization of these sites are challenging by experiments alone. This study integrated multiple computational approaches to probe the potential binding sites on the wild-type Drosophila melanogaster DAT and identified a new allosteric site that displays high affinity for cocaine. This site is located on the surface of DAT, and binding of cocaine is primarily dominated by interactions with hydrophobic residues surrounding the site. Cocaine binding to this new site allosterically reduces the binding of DA/cocaine to the central binding pocket, and simultaneous binding of two cocaine molecules to a single DAT seems infeasible. Furthermore, binding of cocaine to this site was found to stabilize the conformation of DAT but alters the conformational population and thereby reduces the accessibility by DA, providing molecular insights into the inhibitory mechanism of cocaine. In addition, the results indicate that the conformations induced by cocaine binding to this site may be relevant to the oligomerization of DAT, highlighting a potential role of this new site in modulating the function of DAT (Xu, 2020).

Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons

Inherited mutations in the LRRK2 protein are the common causes of Parkinson's disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern ('sleep'), and courtship memory determined in aged flies. Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. It is concluded that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson's disease (Fellgett, 2021).

Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster

Dopamine (DA) is required for movement, sleep, and reward, and DA signaling is tightly controlled by the presynaptic DA transporter (DAT). Therapeutic and addictive psychostimulants, including methylphenidate (Ritalin; MPH), cocaine, and amphetamine (AMPH), markedly elevate extracellular DA via their actions as competitive DAT inhibitors (MPH, cocaine) and substrates (AMPH). DAT silencing in mice and invertebrates results in hyperactivity, reduced sleep, and blunted psychostimulant responses, highlighting DAT's essential role in DA-dependent behaviors. DAT surface expression is not static; rather it is dynamically regulated by endocytic trafficking. PKC-stimulated DAT endocytosis requires the neuronal GTPase, Rit2, and Rit2 silencing in mouse DA neurons impacts psychostimulant sensitivity. However, it is unknown whether or not Rit2-mediated changes in psychostimulant sensitivity are DAT-dependent. This study leveraged Drosophila melanogaster to test whether the Drosophila Rit2 ortholog, Ric, impacts dDAT function, trafficking, and DA-dependent behaviors. Orthologous to hDAT and Rit2, dDAT and Ric directly interact, and the constitutively active Ric mutant Q117L increased dDAT surface levels and function in cell lines and ex vivo Drosophila brains. Moreover, DAergic RicQ117L expression caused sleep fragmentation in a DAT-dependent manner but had no effect on total sleep and daily locomotor activity. Importantly, this study found that Rit2 is required for AMPH-stimulated DAT internalization in mouse striatum, and that DAergic RicQ117L expression significantly increased Drosophila AMPH sensitivity in a DAT-dependent manner, suggesting a conserved impact of Ric-dependent DAT trafficking on AMPH sensitivity. These studies support that the DAT/Rit2 interaction impacts both baseline behaviors and AMPH sensitivity, potentially by regulating DAT trafficking (Fagan, 2021).

Context-dependent representations of movement in Drosophila dopaminergic reinforcement pathways

Dopamine plays a central role in motivating and modifying behavior, serving to invigorate current behavioral performance and guide future actions through learning. This study examined how this single neuromodulator can contribute to such diverse forms of behavioral modulation. By recording from the dopaminergic reinforcement pathways of the Drosophila mushroom body during active odor navigation, this study reveals how their ongoing motor-associated activity relates to goal-directed behavior. Dopaminergic neurons were found to correlate with different behavioral variables depending on the specific navigational strategy of an animal, such that the activity of these neurons preferentially reflects the actions most relevant to odor pursuit. Furthermore, this study shows that these motor correlates are translated to ongoing dopamine release, and acutely perturbing dopaminergic signaling alters the strength of odor tracking. Context-dependent representations of movement and reinforcement cues are thus multiplexed within the mushroom body dopaminergic pathways, enabling them to coordinately influence both ongoing and future behavior (Zolin, 2021).

Fast-Scan Cyclic Voltammetry (FSCV) Reveals Behaviorally Evoked Dopamine Release by Sugar Feeding in the Adult Drosophila Mushroom Body

Drosophila melanogaster, the fruit fly, is an excellent model organism for studying dopaminergic mechanisms and simple behaviors, but methods to measure dopamine during behavior are needed. This study developed fast-scan cyclic voltammetry (FSCV) to track in vivo dopamine during sugar feeding. First, acetylcholine stimulation was used to evaluate the feasibility of in vivo measurements in an awake fly. Next, sugar feeding was tested by placing sucrose solution near the fly proboscis. In the mushroom body medial tip, 1 pmol acetylcholine and sugar feeding released 0.49±0.04 μM and 0.31#177;0.06 μM dopamine, respectively but sugar-evoked release lasted longer than with acetylcholine. Administering the dopamine transporter inhibitor nisoxetine or D2 receptor antagonist flupentixol significantly increased sugar-evoked dopamine. This study develops FSCV to measure behaviorally evoked release in fly, enabling Drosophila studies of neurochemical control of reward, learning, and memory behaviors (Shin, 2022).

Serotonin and dopamine modulate aging in response to food odor and availability

An organism's ability to perceive and respond to changes in its environment is crucial for its health and survival. This study reveals how the most well-studied longevity intervention, dietary restriction, acts in-part through a cell non-autonomous signaling pathway that is inhibited by the presence of attractive smells. Using an intestinal reporter for a key gene induced by dietary restriction but suppressed by attractive smells, this study identified three compounds that block food odor effects in C. elegans, thereby increasing longevity as dietary restriction mimetics. These compounds clearly implicate serotonin and dopamine in limiting lifespan in response to food odor. A chemosensory neuron that likely perceives food odor, an enteric neuron that signals through the serotonin receptor 5-HT1A/SER-4, and a dopaminergic neuron that signals through the dopamine receptor DRD2/DOP-3. Aspects of this pathway are conserved in D. melanogaster. Thus, blocking food odor signaling through antagonism of serotonin or dopamine receptors is a plausible approach to mimic the benefits of dietary restriction (Miller, 2022).

An internal expectation guides Drosophila egg-laying decisions

To better understand how animals make ethologically relevant decisions, egg-laying substrate choice was studied in Drosophila. Flies were found to dynamically increase or decrease their egg-laying rates while exploring substrates so as to target eggs to the best, recently visited option. Visiting the best option typically yielded inhibition of egg laying on other substrates for many minutes. The data support a model in which flies compare the current substrate's value with an internally constructed expectation on the value of available options to regulate the likelihood of laying an egg. Dopamine neuron activity is critical for learning and/or expressing this expectation, similar to its role in certain tasks in vertebrates. Integrating sensory experiences over minutes to generate an estimate of the quality of available options allows flies to use a dynamic reference point for judging the current substrate and might be a general way in which decisions are made (Vijayan, 2022).

Dopamine promotes head direction plasticity during orienting movements

In neural networks that store information in their connection weights, there is a tradeoff between sensitivity and stability. Connections must be plastic to incorporate new information, but if they are too plastic, stored information can be corrupted. A potential solution is to allow plasticity only during epochs when task-specific information is rich, on the basis of a 'when-to-learn' signal. It was reasoned that dopamine provides a when-to-learn signal that allows the brain's spatial maps to update when new spatial information is available-that is, when an animal is moving. This study shows that the dopamine neurons innervating the Drosophila head direction network are specifically active when the fly turns to change its head direction. Moreover, their activity scales with moment-to-moment fluctuations in rotational speed. Pairing dopamine release with a visual cue persistently strengthens the cue's influence on head direction cells. Conversely, inhibiting these dopamine neurons decreases the influence of the cue. This mechanism should accelerate learning during moments when orienting movements are providing a rich stream of head direction information, allowing learning rates to be low at other times to protect stored information. These results show how spatial learning in the brain can be compressed into discrete epochs in which high learning rates are matched to high rates of information intake (Fisher, 2022).

Locomotor and olfactory responses in dopamine neurons of the Drosophila superior-lateral brain

The Drosophila brain contains about 50 distinct morphological types of dopamine neurons. Physiological studies of Drosophila dopamine neurons have been largely limited to one brain region, the mushroom body, where they are implicated in learning. By comparison, little is known about the physiology of other Drosophila dopamine neurons. Interestingly, a recent whole-brain imaging study found that dopamine neuron activity in several fly brain regions is correlated with locomotion. This is notable because many dopamine neurons in the rodent brain are also correlated with locomotion or other movements; however, most rodent studies have focused on learned and rewarded behaviors, and few have investigated dopamine neuron activity during spontaneous (self-timed) movements. This study monitored dopamine neurons in the Drosophila brain during self-timed locomotor movements, focusing on several previously uncharacterized cell types that arborize in the superior-lateral brain, specifically the lateral horn and superior-lateral protocerebrum. It was found that activity of all of these dopamine neurons correlated with spontaneous fluctuations in walking speed, with different cell types showing different speed correlations. Some dopamine neurons also responded to odors, but these responses were suppressed by repeated odor encounters. Finally, the same identifiable dopamine neuron encode different combinations of locomotion and odor in different individuals. If these dopamine neurons promote synaptic plasticity-like the dopamine neurons of the mushroom body-then, their tuning profiles would imply that plasticity depends on a flexible integration of sensory signals, motor signals, and recent experience (Marquis, 2022).

Differential coding of absolute and relative aversive value in the Drosophila brain

Animals use prior experience to assign absolute (good or bad) and relative (better or worse) value to new experience. These learned values guide appropriate later decision making. Even though understanding of how the valuation system computes absolute value is relatively advanced, the mechanistic underpinnings of relative valuation are unclear. This study uncovered mechanisms of absolute and relative aversive valuation in Drosophila. Three types of punishment-sensitive dopaminergic neurons (DANs) respond differently to electric shock intensity. During learning, these punishment-sensitive DANs drive intensity-scaled plasticity at their respective mushroom body output neuron (MBON) connections to code absolute aversive value. In contrast, by comparing the absolute value of current and previous aversive experiences, the MBON-DAN network can code relative aversive value by using specific punishment-sensitive DANs and recruiting a specific subtype of reward-coding DANs. Behavioral and physiological experiments revealed that a specific subtype of reward-coding DAN assigns a "better than" value to the lesser of the two aversive experiences. This study therefore highlights how appetitive-aversive system interactions within the MB network can code and compare sequential aversive experiences to learn relative aversive value (Villar, 2022)

Value-based decisions require animals to make choices between several options based on a prediction of their relative subjective value learned through prior experience. Associative learning provides a means to assign absolute (good or bad) values to experience that can be used to guide future approach or avoidance behaviors. During learning, animals can also compare the value of their current experience with that of prior knowledge and assign a relative value (better or worse) between these experiences to promote more accurate economic-based choices. Notwithstanding that a substantial body of research has investigated mechanisms for relative reward-value coding, less is known about how relative aversive value is computed during learning to guide appropriate value-based decisions. Reinforcement learning models propose that learning occurs when actual outcome value differs from predicted value. This process and the error computed between actual and predicted value are driven by a valuation circuit that includes dopaminergic and GABAergic neurons (Villar, 2022)

However, it is unclear whether similar circuitry compares current and previous experience to assign relative aversive value to sensory stimuli during learning. Anatomically discrete dopaminergic neurons (DANs) in Drosophila and mice provide either positive or negative teaching signals]. In flies, these different DANs project to unique compartments of the mushroom body (MB), a central brain structure essential for olfactory learning and memory as well as several goal-directed behaviors. DANs from the protocerebral posterior lateral 1 (PPL1) cluster projecting to the vertical and proximal horizontal lobes of the MB relay punishment and signal negative value during learning. Many DANs from the protocerebral anterior medial (PAM) cluster projecting to the horizontal lobes of the MB assign positive reward value during learning (Villar, 2022)

Sparse activation within the ~4,000 MB intrinsic Kenyon cells (KCs), which indirectly receive odorant information from sensory neurons in the periphery, provides the specificity of olfactory memories. KCs synapse onto mushroom body output neurons (MBONs), which project into downstream structures to drive (for most of them) approach or avoidance behavior. Some MBONs are synaptically interconnected, providing cross-excitation or -inhibition between MB compartments. Lastly, many MBONs make feedback or feedforward synapses outside the MB onto DAN dendrites (Villar, 2022)

During olfactory associative learning, specific DANs releasing dopamine in individual MB compartments depress synaptic strengths between sparse odor-activated KCs and the MBONs whose dendrites reside within the relevant compartments. As a result, learning-induced plasticity within different MB compartments reconfigures the MBON ensemble output signal to promote either learned approach or avoidance behavior. Olfactory aversive learning reduces odor drive of approach-directing MBONs, hence tilting the MBON network toward promoting odor avoidance. By contrast, appetitive learning reduces odor drive of avoidance-directing MBONs, leaving the network in a configuration promoting odor approach (Villar, 2022)

Flies can perceive, learn, and compare differences in the intensity of punishment and adapt their behavior accordingly. This study combined genetic interventions with behavioral analyses, anatomical characterization, and in vivo two-photon calcium imaging to investigate the detailed circuit requirements that allow flies to write and compare olfactory aversive memories of different intensities during learning to promote appropriate value-based choices. This study found that aversive PPL1 DANs show differential responses to electric shock punishment of varying intensity. As a result, the intensity of shock reinforcement correlates with the magnitude of learning-driven plasticity at the corresponding KC to MBON junctions. Using a specific behavioral paradigm in which flies associate three odors with 0, 60, and 30 V punishment, respectively, the circuits involved in coding relative aversive value were identified. Loss-of-function screening revealed a role for specific aversive DANs, in addition to the rewarding PAM-β'2aγ5n DANs, to learn relative aversive value. Recording from PAM-β'2aγ5n DANs during learning revealed these neurons to signal relative aversive value by increasing their responsiveness when the odor-low shock association is better than a previous odor-high shock association. Recording from three MBONs presynaptic to PAM-β'2aγ5n DANs revealed a positive difference in the odor responses of MBON-γ2α'1 between current and previous aversive experiences. This increased responsiveness of cholinergic MBON-γ2α'1 likely provides excitatory input necessary to drive the PAM-β'2aγ5n DANs 'better than' value signal for the less aversive experience, and they thereby learn relative aversive value. In support of this model, optogenetic activation of PAM-β'2aγ5n reward DANs, during learning, assigns a relative 'better than' value to one of two identical odor-punishment associations (Villar, 2022)

This study addresses how animals assign absolute aversive value during learning and how they compare and ascribe relative aversive value information to consecutive negative experiences for them to make appropriate value-based decisions afterwards. Using the fruit fly Drosophila permitted a cellular resolution view of how the interaction between the appetitive and aversive DAN systems, within the MB network, is at the heart of the mechanistic underpinnings that compute a relative aversive value teaching signal. This work also indicates that coding of a relative 'worse than' aversive value likely involves different circuit mechanisms to those for 'better than' but that there may be some overlap (Villar, 2022)

PPL1 DANs reinforce a range of aversive memories with differing strength and persistence. The data provide new insight into the functional diversity of these anatomically discrete DANs. It was found that individual aversively reinforcing PPL1- γ1pedc, PPL1- γ2 α'1, and PPL1- α2 α'2 DANs exhibit different intensity response profiles when flies were exposed to a series of shock voltages. Importantly, the strength of their responses to electric shocks strongly correlated with the magnitude of plasticity of the odor-evoked responsiveness of their corresponding MBONs after differential conditioning. These results indicate that absolute aversive value is assigned to odors in different ways in the γ1pedc, γ2 α'1, and α2 α'2 MB compartments, consistent with the conclusion of a prior study that artificially activated individual DANs (Villar, 2022)

Of note, no significant shock responses was observed in PPL1- α2 α'2 DANs. These results are also in accordance with the absence of odor-evoked changes in the corresponding MBON- α2sc immediately after training and a lack of reinforcing properties when pairing artificial activation of PPL1- α2 α'2 DANs with an odor. In addition, learning-dependent depression of odor responses in MBON- α2sc has been reported to be most relevant for expression of later forms of memory (Villar, 2022)

This study observed that the stronger the aversive experience, the greater the PPL1- γ1pedc DAN-driven depression of the CS+-evoked response of MBON- γ1ped> αβ. Feedforward GABAergic inhibition from MBON- γ1ped> αβ to the primary axon of MBON- γ5β'2a is therefore reduced in a graded manner by aversive conditioning. MBON- γ5β'2a should therefore display a proportional increase in its CS+-evoked response to drive learned avoidance behavior (Deng, 2022).

These experiments uncovered a very different effect of absolute aversive conditioning at the MBON- γ2 α'1 junction. Although the PPL1- γ2 α'1 DANs were significantly triggered by shocks ≥30 V, their responses were comparable at all voltages between 30 and 90 V. Moreover, aversive conditioning did not significantly depress the CS+ responses of MBON- γ2 α'1. Instead, this study observed that the responses to the CS- odor were specifically increased, and the CS- CS+ differential responses were correlated with the intensity of the shocks applied. The data therefore suggest that any odor that follows the CS+ with ≥45 V presentation during training gains the capacity to drive more activity in the cholinergic MBON- γ2 α'1. In addition, recordings indicate that the more aversive the first experience is, the stronger the cholinergic MBON- γ2 α'1 activity will be to the subsequent 'better than' experience. These data reveal a key role for MBON- γ2 α'1 in coding relative aversive value (Deng, 2022).

MBON- γ2 α'1 input to PAM-β'2a γ5n DANs provides a 'better than' reward signal during relative aversive training. Output from PAM-β'2a γ5n DANs was critical during the odor Z + 30 V presentation for relative aversive learning. These DANs receive direct excitatory cholinergic input from MBON- γ2 α'1, and it is proposes that the strength of this excitation is key for the flies to assign a 'better than' reward value to the lesser of the two aversive experiences. As mentioned above, when odor Y is paired with 60 V shock in a differential conditioning assay, the CS- responses of MBON- γ2 α'1 become elevated. This means that when Y + 60 V is followed by Z + 30 V, the Z odor will more strongly drive MBON- γ2 α'1 and as a result will activate the PAM-β'2a γ5n DANs. In effect, it is hypothesized that any odor that follows a Y + 60 V experience is predisposed to be judged as 'better than,' unless it is itself accompanied by 60 V or a greater voltage. These analyses that subtracted MBON- γ2 α'1 odor-evoked responses are entirely consistent with this model. Odor-driven activity of MBON- γ2 α'1 is greater during the first period of the following Z + 30 V experience than during the same period (just after the first shock) of the prior Y + 60 V experience is observed. Critically, this is also the time period during which an elevation of PAM-β'2a γ5n DAN activity. It is speculated that the first of the 30 V shocks somehow further releases the PAM-β'2a γ5n DAN activity to be fully driven by MBON- γ2 α'1, perhaps as a release of feedforward inhibition in the MBON- γ1ped> αβ to MBON- γ5β'2a to PAM-β'2a γ5n DAN pathway. The results and proposed models of PAM-β'2a γ5n DANs providing a 'better than' reward signal are in accordance with previous reports that PAM-β'2a γ5n activation provides appetitive reinforcement (Villar, 2022).

Are there limits to comparable aversive memories? Individual PPL1-DAN subtypes have different thresholds for activation, and intensity-dependent plasticity in their corresponding MBON junctions have similar thresholds. It was noted that these thresholds seem reflected in the range of comparisons that flies can make in a relative choice between different aversive memories, which point toward a threshold and a difference between voltages of 30 V as being optimal to efficiently estimate a relative difference. In the recordings of this study, 30 V was the threshold for observing shock-evoked responses in PPL1- γ1pedc and PPL1- γ2 α'1, but it did not trigger PPL1- α2 α'2. In addition, 30 V produced significant plasticity of MBON- γ1ped> αβ odor responses, but plasticity was not evident in MBON- γ2 α'1 responses until 45 V. Thus, perhaps every odor paired with a voltage of ≥30 V is considered to be 'not so bad,' because it only depresses the GABAergic MBON- γ1ped> αβ responses and not the cholinergic MBON- γ2 α'1 responses, thereby leaving CS+ odor-driven excitation of PAM-β'2a γ5n DANs from these MBONs. Although flies can differentiate between stronger aversive memories such as 90 versus 60 V, their relative choice performances are less good than 60 versus 30 V.8 While significant shock responses were not observed for PPL1- α2 α'2 DANs, this study found a role for these neurons during learning of relative aversive value. MBON- γ1ped> αβ is GABAergic and is connected to PPL1- α2 α'2 DANs (Villar, 2022)

It is therefore possible that repeated pairing of odor Y + 60 V electric shocks (or anything above their threshold) during relative training induces enough CS+-evoked depression at MBON- γ1ped> αβ to release inhibition in PPL1- α2 α'2 DANs while pairing the odor Z with 30 V shocks. The resulting plasticity in MBON- α2sc could explain the requirement of αβ surface and αβ core KCs during a relative choice between Y60 versus Z (Villar, 2022)

The results of this study show that learning a relative 'better than' aversive value requires an interplay between aversively reinforcing PPL1 DANs modulating KC-MBON connections, which provide feedforward and recurrent feedback input that determines the activity of specific subtypes of rewarding PAM DANs. These results support long-held and recent models in both vertebrates and invertebrates, suggesting that learning requires critical interactions between appetitive and aversive reinforcement systems. In the fly, and likely also in mammals, this process relies on opposing populations of DANs providing predictive signals needed to compare current and previous experience to assign (and update) both absolute and relative value to stimuli during learning. For instance, aversive memory extinction and reversal learning require the reward system in both vertebrates and invertebrates (Villar, 2022)

In all these cases, stimuli that represent the absence of a punishment are rewarded. In humans, the ventral striatum, targeted by numerous DA inputs from the ventral tegmental area (VTA) providing rewarding information, is essential to compare aversive experiences of different intensities. In the orbitofrontal cortex, relative coding of aversive (but also appetitive) experiences seem to require overlapping neuronal ensembles to select a preferred option and promote appropriate economical decisions in a specific spatial and temporal context. In the dopaminergic system, reward is also computed in a relative manner to broadcast value signals in different brain regions. These DANs from the VTA and substantia nigra compute a prediction error to signal positive, but also negative, value. A similar value prediction error calculation has not yet been demonstrated experimentally in the fly. Instead, results from several studies in the fly suggest that errors are registered in the MB network by the action of DANs that signal the opposing value. The current experiments suggest that a similar interplay between opposing populations of DANs, and plasticity at different MBON junctions in the MB network, permits computation of relative aversive value (or difference) between a prior and a new aversive experience. Combined with previous work and current computational models, these data provide key features of how the appetitive-aversive system interactions in the MB network using heterogeneous DANs can compare previous and current experience to 'pre-compute' a relative value during learning that facilitates future value-based decisions (Villar, 2022)

A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila

Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. This study has identified a subset of dopaminergic neurons (THD') that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding and growing, whereas expression of STIM in THD' neurons rescues feeding, growth and viability of STIM null mutants to a significant extent. Moreover STIM is essential for maintaining excitability and release of dopamine from THD' neurons. Optogenetic stimulation of THD' neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD' cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary this study has identified a novel STIM-dependent function of dopamine neurons that modulates developmental changes in larval feeding behaviour and growth (Kasturacharya, 2023).

In Drosophila, as in other holometabolous insects, growth is restricted to the larval stages. In early stages of larval development cells exit mitotic quiescence and re-enter mitosis resulting in organismal growth. This change is accompanied by an increase in the feeding rate of the organism so as to provide sufficient nutrition for the accompanying growth in organismal size. In STIM^KO larvae a loss of this ability to feed persistently was observed starting from early second instar larvae. The focus of this feeding deficit lies in a subset of central dopaminergic neurons that require STIM function to maintain excitability. Importantly, these dopaminergic neurons communicate with multiple neuropeptidergic cells in the brain to regulate appropriate changes in larval feeding behaviour. The identified dopaminergic cells also communicate with ilp producing neuropeptidergic cells, the MNSc, through which they appear to impact larval growth (Kasturacharya, 2023).

The THD' cells were identified as critical for larval feeding from their inability to function in the absence of the store-operated Ca2+ entry (SOCE) regulator STIM. Loss of excitability and the absence of dopamine release from THD' cells in STIM^KO larvae suggests that voltage-dependent receptor activity is required to maintain growth in early 2nd instar larvae. Changes in expression of ion channels and presynaptic components have been observed earlier upon knockdown of STIM in Drosophila and mammalian neurons. Moreover, loss of STIM-dependent SOCE in Drosophila neurons effects their synaptic release properties. Partial rescue of viability in STIM^KO organisms by over-expression of a bacterial sodium channel NaChBac and restoration of dopamine release upon rescue by STIM+ supports the idea that STIM-dependant SOCE maybe required for appropriate function and/or expression of ion channels and synaptic components in THD' neurons. Changes in ER-Ca2+ suggest that STIM is also required to maintain neuronal Ca2+ homeostasis (Kasturacharya, 2023).

While mechanisms that regulate developmental progression of Drosophila larvae have been extensively studied, neural control of essential changes in feeding behaviour that need to accompany each larval developmental stage have not been identified previously. Artificial manipulation of activity in the central dopaminergic neuron subset examined in this study (THD'), either by expression of an inward rectifying potassium channel (Kir2.1) or the bacterial sodium channel NaChBac, suggests an important role for THD' neurons during larval development. This idea is supported by the altered dynamics of muscarinic acetylcholine receptor (mAChR) stimulated Ca2+ release observed in THD' neurons between early, mid and late third instar larvae when larval feeding slows down and ultimately stops and re-iterates that signaling in and from these neurons drives larval feeding whereas lower carbachol-induced Ca2+ responses signal cessation of feeding. A weaker rescue of STIM^KO larvae is also obtained from STIM+ expression in the THC' neuron subset. Taken together these observations suggest a neuromodulatory role for dopamine, where DA release from THD' neurons has a greater influence on feeding than the DA release from THC' neurons, possibly due to the DL1 and DL2 cluster (among THD' marked neurons) receiving more feeding and metabolic inputs. A role for cells other than THD', in maintaining kinetics of dopamine release required for feeding behaviour are also indicated because expression of STIM+ in THD' neurons did not revert kinetics of dopamine release to wild type levels. The prolonged dopamine release observed in wild-type THD' neurons may arise from synaptic/modulatory inputs to THD' neurons from other neurons that require STIM function (Kasturacharya, 2023).

Though the cells that provide cholinergic inputs to THD' cells have not been identified it is possible that such neurons sense the nutritional state. In this context, two pairs of cells in the THD' subset also motivate the search for food in hungry adult Drosophila. Starved flies with knock down of the mAChR on THD' neurons exhibit a decrease in food seeking behaviour. Cholinergic inputs to THD' neurons for sensing nutritional state/hunger may thus be preserved between larval and adult stages (Kasturacharya, 2023).

Interestingly, dopamine is also required for reward-based feeding, initiation, and reinforcement of feeding behaviour in adult mice. These findings parallel past studies where prenatal mice genetically deficient for dopamine (DA-/-), were unable to feed and died from starvation. Feeding could however be initiated upon either enforced supplementation or injection with L-DOPA allowing them to survive. More recent findings show that dopaminergic neurons in the ventral tegmental area (VTA), and not the substantia nigra, drive motivational behaviour and facilitate action initiation for feeding in adult mice (Kasturacharya, 2023).

Both activation and inhibition of specific classes of neuropeptidergic cells by optogenetic activation of THD' cells suggests a dual role for dopamine possibly due to the presence of different classes of DA receptors. The Drosophila genome encodes four DA receptors referred to as Dop1R1, Dop1R2, DD2R and a non-canonical DopEcR [66]. Dop1R1, Dop1R2 and DopEcR activate adenylate cyclase and stimulate cAMP signaling whereas DD2R is inhibitory. Cell specific differences among dopamine receptors have been observed in adults. Down regulation of Dop1R1 on AstA and NPF cells shifted preference towards sweet food whereas down regulation of DopEcR in DH44 cells shifted preference towards bitter food. In third instar larvae a dopaminergic-NPF circuit, arising from central dopaminergic DL2 neurons, two cells of which are marked by THD'GAL4, motivates feeding in presence of appetitive odours. The dopamine-neuropeptide axis identified in this study demonstrates a broader role for dopamine in regulating neuropeptide release and/or synthesis, in the context of larval feeding behaviour, perhaps similar to the mammalian circuit described above (Kasturacharya, 2023).

Of specific interest is the untimely upregulation of ilp3 transcripts in STIM^KO larvae. Rescue of lethality in STIM^KO larvae either by bringing back activity to THD' neurons or by reducing ilp3 levels suggests an interdependence of Dopamine-Insulin signaling that is likely conserved across organisms. Thr data suggest that ilp3 expression is suppressed during the feeding and growth stages of larvae, and once enough nutrition accumulates expression of ilp3 is up-regulated, concurrent with a reduction in carbachol-induced Ca2+ signals in THD' neurons, possibly followed by upregulation and release of ilp3. The idea of ilp3 as a metabolic signal whose expression is antagonistic to larval growth is supported by the observation that knock-down of ilp3 in the MNSc leads to larger pupae in wild type animals and larger larvae in STIM^KO. This is the first report of ilp3 as a larval signal that is antagonistic to growth. Given that Drosophila encode a single Insulin receptor for ilp2, ilp3 and ilp5 the cellular mechanism of ilp3 action remains to be elucidated. Possibly, ilps with different affinity for the insulin receptor stimulate different cellular subsets and/or different intracellular signaling mechanisms, including ecdysone signaling that is essential for larval transition to pupae. Interestingly, in STIM^KO larval brains there is a significant increase in expression of the Insulin Receptor. Further studies are needed to fully understand ilp3 function in larvae (Kasturacharya, 2023).

Expression of other neuropeptides did not show significant changes in STIM^KO larval brains, suggesting that for neuropeptidergic cells in the LNC and SEZ, dopamine signals alter release properties rather than synthesis. However, it was not possible to to identify specific neuropeptides for cells in the LNC and the SEZ that responded upon activation of THD' (Kasturacharya, 2023).

The importance of dopamine for multiple aspects of feeding behaviour is well documented in juvenile and adult mice. Of interest are more recent findings linking dysregulation of dopamine-insulin signaling with the regulation of energy metabolism and the induction of binge eating. The identification of a simple neuronal circuit where dopamine-insulin signaling regulates feeding and growth could serve as a useful model for investigating new therapeutic strategies targeted towards the treatment of psychological disorders for obesity and metabolic syndrome (Kasturacharya, 2023).

Huntingtin Plays a Role in the Physiological Response to Ethanol in Drosophila

Huntingtin (htt) protein is an essential regulator of nervous system function through its various neuroprotective and pro-survival functions, and loss of wild-type htt function is implicated in the etiology of Huntington's disease. While its pathological role is typically understood as a toxic gain-of-function, some neuronal phenotypes also result from htt loss. Therefore, it is important to understand possible roles for htt in other physiological circumstances. To elucidate the role of htt in the context of ethanol exposure, this study investigated how loss of htt impacts behavioral and physiological responses to ethanol in Drosophila. Flies lacking htt were tested for ethanol sensitivity and tolerance, preference for ethanol using capillary feeder assays, and recovery of mobility after intoxication. Levels of dopamine neurotransmitter and numbers of dopaminergic cells in brains lacking dhtt were also measured. dhtt-null flies were found to be both less sensitive and more tolerant to ethanol exposure in adulthood. Moreover, flies lacking dhtt are more averse to alcohol than controls, and they recover mobility faster following acute ethanol intoxication. dhtt was shown to mediate these effects at least in part through the dopaminergic system, as dhtt is required to maintain normal levels of dopamine in the brain and normal numbers of dopaminergic cells in the adult protocerebrum. These results demonstrate that htt regulates the physiological response to ethanol and indicate a novel neuroprotective role for htt in the dopaminergic system, raising the possibility that it may be involved more generally in the response to toxic stimuli (Clabough, 2023).

Dopaminergic systems create reward seeking despite adverse consequences

Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking, the underlying reasons for this behaviour are unclear. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals (Jovanoski, 2023).

Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations

Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration (Vujnovic, 2023).

Contribution of DA signaling to appetitive odor perception in a Drosophila model

Understanding cognitive processes that translate chemically diverse olfactory stimuli to specific appetitive drives remains challenging. Food-related odors arouse impulsive-like feeding of food media that are palatable and readily accessible in well-nourished Drosophila larvae. This study provides evidence that two assemblies of four dopamine (DA) neurons, one per brain hemisphere, contribute to perceptual processing of the qualitative and quantitative attributes of food scents. These DA neurons receive neural representations of chemically diverse food-related odors, and their combined neuronal activities become increasingly important as the chemical complexity of an appetizing odor stimulus increases. Furthermore, in each assembly of DA neurons, integrated odor signals are transformed to one-dimensional DA outputs that have no intrinsic reward values. Finally, a genetic analysis has revealed a D1-type DA receptor (Dop1R1)-gated mechanism in neuropeptide Y-like neurons that assigns appetitive significance to selected DA outputs. These findings suggest that fly larvae provide a useful platform for elucidation of molecular and circuit mechanisms underlying cognitive processing of olfactory and possibly other sensory cues (Pu, 2018).

Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila

Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. This study shows that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. Specific fan-shaped body neurons were identified that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, this study provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation (Dag, 2019).

The activity of DAN-aSP13s, which is essential for courtship memory acquisition, is also necessary during a discrete post-learning time window for LTM consolidation. Because neuronal reactivation occurs during sleep in rodents, it was hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism. Using behavioral analysis and neuronal activity monitoring and perturbation approaches, thus study shows that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience. This sleep is necessary for LTM consolidation, and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the fan shaped body (vFB). These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s. Thus, these data provide a causal link between sleep promoting neurons in the vFB, post-learning activation of dopaminergic neurons, and LTM consolidation (Dag, 2019).

Based on these data, the following model is proposed for sleep-dependent consolidation of courtship LTM in Drosophila (see Post-learning activation of DAN-aSP13 neurons mediates LTM consolidation). During a prolonged learning experience, γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female, respectively. Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB, it is hypothesized that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB. In turn, the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s. It is thought that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation and sleep regulation. Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence (Dag, 2019).

Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus, worth remembering. Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area (VTA) has been implicated in the consolidation of fear memory in rodents. Interestingly, this activity is critical during a discrete time window after learning. Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience. This study shows that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila. It is proposed that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories. Specifically, reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6, which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition. This study considers two hypotheses to account for this selectivity. During sleep, vFB neurons might selectively reactivate only the relevant DANs, or alternatively, they might activate all DANs but only the relevant subset is able to consolidate LTM. The selective-reactivation model would require some marker to distinguish which DANs were activated, whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition, for example translational regulator Orb2, which regulates translation upon neuronal activity during LTM consolidation (Dag, 2019).

Activity of dopaminergic neurons regulates sleep-wake states in animals, including flies. Artificial activation of DAN-aSP13s has been shown to increase wakefulness. In contrast, the data presented in this study imply that activation of vFB neurons, although they activate DAN-aSP13s, do not promote wakefulness. These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies. One potential explanation is that post-learning sleep involves activation of the vFB circuit, which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons. Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness (Dag, 2019).

Thia study has identified a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB, which regulate sleep homeostasis. Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation, whereas dFB neurons are neither necessary nor sufficient for LTM consolidation, it is hypothesized that dFB and vFB neurons promote distinct components of sleep that have different functions. Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness. In contrast, the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition. Thus, the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and, if appropriate, incorporating it into long-term storage (Dag, 2019).

Recent studies have implied that sleep in flies, as in humans and rodents, exhibits sleep stages characterized by distinct electrophysiological signatures. Interestingly, the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern, recorded by local field potentials, than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons. Thus, these data support the hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions (Dag, 2019).

It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention. Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior. Accordingly, studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension. Hence, it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila, such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency (Dag, 2019).

This work has established a functional link between a novel class of sleep-promoting neurons in the FB, post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM. Moreover, the data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons. Thus, this study uncovered a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila. In addition, courtship LTM was establish in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep, neuronal reactivation and LTM consolidation (Dag, 2019).

Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning

The GABAergic system serves as a vital negative modulator in cognitive functions, such as learning and memory, while the mechanisms governing this inhibitory system remain to be elucidated. In Drosophila, the GABAergic anterior paired lateral (APL) neurons mediate a negative feedback essential for odor discrimination; however, their activity is suppressed by learning via unknown mechanisms. In aversive olfactory learning, a group of dopaminergic (DA) neurons is activated on electric shock (ES) and modulates the Kenyon cells (KCs) in the mushroom body, the center of olfactory learning. This study found that the same group of DA neurons also form functional synaptic connections with the APL neurons, thereby emitting a suppressive signal to the latter through Drosophila dopamine 2-like receptor (DD2R). Knockdown of either DD2R or its downstream molecules in the APL neurons results in impaired olfactory learning at the behavioral level. Results obtained from in vivo functional imaging experiments indicate that this DD2R-dependent DA-to-APL suppression occurs during odor-ES conditioning and discharges the GABAergic inhibition on the KCs specific to the conditioned odor. Moreover, the decrease in odor response of the APL neurons persists to the postconditioning phase, and this change is also absent in DD2R knockdown flies. Taken together, these findings show that DA-to-GABA suppression is essential for restraining the GABAergic inhibition during conditioning, as well as for inducing synaptic modification in this learning circuit. Such circuit mechanisms may play conserved roles in associative learning across species (Zhou, 2019).

Independent contributions of discrete dopaminergic circuits to cellular plasticity, memory strength, and valence in Drosophila

Dopaminergic neurons play a key role in encoding associative memories, but little is known about how these circuits modulate memory strength. This study reports that different sets of dopaminergic neurons projecting to the Drosophila mushroom body (MB) differentially regulate valence and memory strength. PPL2 neurons increase odor-evoked calcium responses to a paired odor in the MB and enhance behavioral memory strength when activated during olfactory classical conditioning. When paired with odor alone, they increase MB responses to the paired odor but do not drive behavioral approach or avoidance, suggesting that they increase the salience of the odor without encoding strong valence. This contrasts with the role of dopaminergic PPL1 neurons, which drive behavioral reinforcement but do not alter odor-evoked calcium responses in the MB when stimulated. These data suggest that different sets of dopaminergic neurons modulate olfactory valence and memory strength via independent actions on a memory-encoding brain region (Boto, 2019).

Dopaminergic neurons are involved in associative learning across taxa. In Drosophila, activation of certain dopaminergic neurons during associative learning tasks drives conditioned approach or avoidance, suggesting that they function as part of the reinforcement pathway and may encode stimulus valence (positive or negative). There are eight clusters of dopaminergic neurons in the fly brain. Neurons in three clusters project to the mushroom body (MB), a region that receives olfactory information and is required for olfactory learning. PAM dopaminergic neurons project to the horizontal lobes (β, β', and γ); PPL1 neurons project to the vertical lobes (α and α'), heel, and peduncle; and PPL2ab neurons project to the calyx. Different subsets of PPL1 and PAM neurons modulate reinforcement during learning (Boto, 2019).

Different sets of dopaminergic neurons play discrete roles in reinforcement during learning. Activating PPL1 dopaminergic neurons in lieu of reinforcement induces behavioral aversion to a paired odor. Conversely, activation of PAM neurons is sufficient to generate appetitive memories. These dopaminergic neurons respond strongly to the unconditioned stimulus during conditioning, releasing dopamine into the MB that integrates with odor-evoked spiking activity to drive learning-induced, cyclic AMP (cAMP)-dependent plasticity in the MB. Little is known about the third MB-innervating cluster, PPL2ab. These neurons innervate the ipsilateral MB calyx, as well as the lateral horn, lobula, optical track and esophagus, and medial and posterior protocerebrum. They have been implicated in the regulation of courtship behaviors but have no known role in learning and memory. How the multiple dopaminergic circuits that converge on the MB regulate learning is a major question (Boto, 2019).

This study has investigated the distinct roles of MB-innervating dopaminergic circuits in neuronal plasticity and behavioral memory. PPL2 neurons were found to play a role in learning, modulating neuronal gain and memory strength without imparting a strong valence. Thus, different subsets of dopaminergic neurons converging on memory-encoding neurons (the MB neurons) play roles in modulation of memory strength and valence (Boto, 2019).

This study provides insight into how PPL2 dopaminergic neurons regulate neuronal plasticity in the MB and behavioral learning. PPL2 neurons project to the MB calyx, where intrinsic MB neurons receive input from olfactory projection neurons. This places them in a position to exert strong influence over MB olfactory responses. The present data suggest that they both act as a gain control that modulates the MB olfactory responses and increase the strength of aversive short-term memory. Activation of PPL2 neurons in a differential conditioning protocol increased the relative responsivity to the paired odor in γ neurons. Behaviorally, this did not drive memory on its own but increased the strength of memory if paired with odor-shock conditioning. Therefore, PPL2 neurons appear to modulate the strength of aversive memory, rather than dictating its content. One mechanism underlying this effect could be that PPL2 neurons enhance MB responses to the odor during training, facilitating the generation of synaptic plasticity that has been observed at the MB output synapses. The memory enhancement effect that this study observed in flies may reflect a more general role that dopaminergic circuits play in other species. For instance, in mice, dopaminergic projections to the medial prefrontal cortex are not sufficient to induce memory, but they improve learning via effects on stimulus discrimination (Popescu et al., 2016) (Boto, 2019).

Previous studies have suggested that PPL2 neurons could regulate motivation and arousal. Increased responses in the MB do not likely represent the valence of a memory directly, but they may reflect a salience or motivational component of memory. This is supported by PPL1 stimulation failing to induce changes in the odor representation in the MB but inducing conditioned aversion that drives heterosynaptic depression at certain MB-mushroom body output neuron (MBON) synapses. In contrast, PPL2 neurons drive strong Ca2+ response plasticity in the MB but do not encode strong valence on their own. The effect was limited to aversive memory, possibly because the starvation necessary for appetitive protocols had already maximized the animals' arousal state and/or salience of the sensory cues (though other possibilities are discussed later) (Boto, 2019).

One function of PPL2 dopaminergic neurons may be to regulate the net responsivity of MB γ neurons to odorants and thereby alter the potential for stimuli to drive memory strength. Alternatively, the plasticity could regulate the balance of excitation across downstream MBONs that innervate spatially discrete zones of the MB and drive approach or avoidance behavior. For instance, increasing responses of MB γ neurons alone could increase the net excitatory drive to aversive MBONs relative to appetitive MBONs. In a previous study, appetitive conditioning was found to robustly increase Ca2+ responses to CS+ across the MB lobes (including both γ and α/β) (Louis, 2018). This could be interpreted to indicate either that the motivational component of appetitive conditioning differentially engages MB circuitry relative to aversive conditioning or that the appetitive valence is encoded as a bona fide cellular-level memory trace, comprising an increase in Ca2+ responses across all MB lobes. If the latter is true, perhaps a selective increase in Ca2+ responses in γ reflects a more aversive signature. Previous studies have demonstrated a critical role of γ neurons in short-term memory. Rescue of Rutabaga in the γ lobe of rut mutants is sufficient to restore performance in short-term memory, and rescue of the D1-like DopR receptor in the γ lobe is sufficient to rescue both short- and long-term memory. In addition, aversive learning induces plasticity in synaptic vesicle release from the MB γ lobes (Boto, 2019).

Several caveats in experimental interpretations should be noted. First, it is not known whether the MB plasticity forms in parallel to memory enhancement or directly drives it. Contributions of polysynaptic circuit elements to the physiological effects (MB plasticity) and/or behavioral effects (enhanced memory) are possible. Future mapping studies may identify additional circuit elements contributing to the memory networks underlying these phenomena. Nonetheless, anatomical innervation of the MB calyx by PPL2 neurons positions them to provide strong modulatory input to the MB dendrites and associated neuronal circuitry. Thus, while valence is layered at the MB output synapses, the data suggest that PPL2 neurons may be a control mechanism that influences how responsive the MB is to odors, potentially altering the propensity for synaptic plasticity downstream (Boto, 2019).

Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells

Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells, evidence is provided that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, it is argued that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. This study further shows that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. These data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization (Lyutova, 2019).

Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility. This study shows that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. These results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems (Aso, 2019).

An animal's survival in a dynamically changing world depends on storing distinct sensory information about their environment as well as the temporal and probabilistic relationship between those cues and punishment or reward. Thus it is not surprising that multiple distributed neuronal circuits in the mammalian brain have been shown to process and store distinct facets of information acquired during learning. Even a simple form of associative learning such as fear conditioning induces enduring changes, referred to as memory engrams, in circuits distributed across different brain areas. Do these multiple engrams serve different mnemonic functions, what molecular and circuit mechanisms underlie these differences, and how are they integrated to control behavior? Localizing these distributed engrams, understanding what information is stored in each individual memory unit and how units interact to function as one network are important but highly challenging problems. The Drosophila mushroom body (MB) provides a well-characterized and experimentally tractable system to study parallel memory circuits. Olfactory memory formation and retrieval in insects requires the MB. In associative olfactory learning, exposure to an odor paired with a reward or punishment results in formation of a positive- or negative-valence memory, respectively. In the MB, sensory stimuli are represented by the sparse activity of ~2,000 Kenyon cells (KCs). Each of 20 types of dopaminergic neurons (DANs) innervates compartmental regions along the parallel axonal fibers of the KCs. Similarly, types of mushroom body output neurons (MBONs) arborize their dendrites in specific axonal segments of the KCs; together, the arbors of the DANs and MBONs define the compartmental units of the MB. Activation of individual MBONs can cause behavioral attraction or repulsion, depending on the compartment in which their dendrites arborize, and MBONs appear to use a population code to govern behavior (Aso, 2019).

A large body of evidence indicates that these anatomically defined compartments of the MB are also the units of associative learning. Despite the long history of behavioral genetics in fly learning and memory, many aspects of the signaling pathways governing plasticity -- especially whether they differ between compartments -- remain poorly understood. Nevertheless, dopaminergic neurons and signaling play a key role in all MB compartments, and flies can be trained to form associative memories by pairing the presentation of an odor with stimulation of a single dopaminergic neuron. Punishment or reward activates distinct sets of DANs that innervate specific compartments of the MB. Activation of the DAN innervating a MB compartment induces enduring depression of KC-MBONs synapses in those specific KCs that were active in that compartment at the time of dopamine release. Thus, which compartment receives dopamine during training appears to determine the valence of the memory, while which KCs were active during training determines the sensory specificity of the memory (Aso, 2019).

Compartments operate with distinct learning rules. Selective activation of DANs innervating specific compartments has revealed that they can differ extensively in their rates of memory formation, decay dynamics, storage capacity, and flexibility to learn new associations. For instance, the dopaminergic neuron PAM-α1 can induce a 24h memory with a single 1-minute training session, whereas PPL1-α3 requires ten repetitions of the same training to induce a 24h memory. PPL1-γ1pedc (aka MB-MP1) can induce a robust short-lasting memory with a single 10-second training, but cannot induce long-term memories even after 10 repetitions of a 1-minute training. PAM-α1 can write a new memory without compromising an existing memory, whereas PPL1-γ1pedc extinguishes the existing memory when writing a new memory. What molecularand cellular differences are responsible for the functional diversity of these compartments? Some differences might arise from differences among KC cell types, but memory dynamics are different even between compartments that lie along the axon bundles of the same Kenyon cells (for example, α1 and α3). This paper shows that differences in memory dynamics between MB compartments can arise from the deployment of distinct cotransmitters by the DAN cell types that innervate them (Aso, 2019).

Evidence from a wide range of organisms establishes that dopaminergic neurons often release a second neurotransmitter, but the role of such cotransmitters in diversifying neuronal signaling is much less clear. In rodents, subsets of dopaminergic neurons co-release glutamate or GABA. In mice and Drosophila, single-cell expression profiling reveals expression of diverse neuropeptides in dopaminergic neurons. EM connectome studies of the mushroom body in adult and larval Drosophila reveal the co-existence of small-clear-core and large- dense-core synaptic vesicles in individual terminals of dopaminergic neurons; moreover, the size of the observed large-dense-core 02 vesicles differs between DAN cell types (Aso, 2019).

This study found that NOS, the enzyme that synthesizes NO, was located in the terminals of a subset of DAN cell types. NOS catalyzes the production of nitric oxide (NO) from L-arginine. Drosophila NOS is regulated by Ca2+/calmodulin, raising the possibility that NO synthesis might be activity dependent. Furthermore, the localization of the NOS1 protein in the axonal terminals of DANs is consistent with NO serving as a cotransmitter. The conclusion that NO acts as a neurotransmitter is supported by the observation that NO signaling requires the presence of a putative receptor, soluble guanylate cyclase, in the postsynaptic Kenyon cells. This role contrasts with the proposed cell-autonomous action of NOS in the ellipsoid body, in which NO appears to target proteins within the NOS-expressing ring neurons themselves, rather than conveying a signal to neighboring cells. The valence-inversion phenotype observed when PPL1-γ1pedc was optogenetically activated in a dopamine-deficient background can be most easily explained if NO induces synaptic potentiation between odor-activated KCs and their target MBONs. Modeling work is consistent with this idea, but testing this idea and 19 other possible mechanisms for NO action will require physiological experiments (Aso, 2019).

During olfactory learning, the concentration of Ca2+ in KC axons represents olfactory information. The coincidence of a Ca2+ rise in spiking KCs and activation of the G-protein-coupled Dop1R1 dopamine receptor increases adenylyl cyclase activity. The resultant cAMP in turn activates protein kinase A, a signaling cascade that is important for synaptic plasticity and memory formation throughout the animal phyla. In contrast, when DANs are activated without KC activity, and thus during low intracellular Ca2+ in the KCs, molecular pathways involving the Dop1R2 receptor, Rac1 and Scribble facilitate decay of memory (Aso, 2019).

This study found that NOS in PPL1-γ1pedc shortens memory retention, while facilitating fast updating of memories in response to new experiences. These observations could be interpreted as indicating that NO regulates forgetting. Indeed, NO-dependent effect requires scribble in KCs, a gene previously reported as a component of active forgetting. However, it is an open question whether the signaling pathways for forgetting, which presumably induce recovery from synaptic depression, are related to signaling cascades downstream of NO and guanylate cyclase, which appear to be able to induce memory without prior induction ofsynaptic depression by dopamine. Lack of detectable 1-day memory formation after spaced training with PPL1-γ1pedc can be viewed as a balance between two distinct, parallel biochemical signals, one induced by dopamine and the other by NO, rather than the loss of information (that is, forgetting). Confirming this interpretation will require better understanding of the signaling pathways downstream of dopamine and NO. The search for such pathways will be informed by the prediction from modeling that dopamine and NO may alter two independent parameters that define synaptic weights with a multiplicative interaction (Aso, 2019).

In the vertebrate cerebellum, which has many architectural similarities to the MB, long-term-depression at parallel fiber-Purkinje cell synapses (equivalent to KC-MBON synapses) induced by climbing fibers (equivalent to DANs) can coexist with long-term-potentiation by NO. In this case, the unaltered net synaptic weight results from a balance between coexisting LTD and LTP rather than recovery from LTD. This balance was suggested to play an important role in preventing memory saturation in the cerebellum 59 and allowing reversal of motor learning. In the Drosophila MB, a similar facilitation was observed of reversal learning by NO. The antagonistic roles of NO and synaptic depression may be a yet another common feature of the mushroom body and the cerebellum (Aso, 2019).

Opposing cotransmitters have been observed widely in both invertebrate and vertebrate neurons. A common feature in these cases is that the transmitters have distinct time courses of action. For instance, hypothalamic hypocretin-dynorphin neurons that are critical for sleep and arousal synthesize excitatory hypocretin and inhibitory dynorphin. When they are released together repeatedly, the distinct kinetics of their receptors result in an initial outward current, then little current, and then an inward current in the postsynaptic cells. In line with these observations, this study found that dopamine and NO show distinct temporal dynamics: NO-dependent memory requires repetitive training and takes longer to develop than dopamine-dependent memory. What molecular mechanisms underlie these differences? Activation of NOS may require stronger or more prolonged DAN activation than does dopamine release. Alternatively, efficient induction of the signaling cascade in the postsynaptic KCs might require repetitive waves of NO input. Direct measurements of release of dopamine and NO, and downstream signaling events by novel sensors will be needed to address these open questions (Aso, 2019).

Decades of behavioral genetic studies have identified more than one hundred genes underlying olfactory conditioning in Drosophila. Mutant andtargeted rescue studies have been used to map the function of many memory-related genes encoding synaptic or intracellular signaling proteins (for example, rutabaga, DopR1/dumb, DopR2/DAMB, PKA-C1/DC0, Synapsin, Bruchpilot, Orb2 and Rac1) to specific subsets of Kenyon cells. However, it is largely unknown if these proteins physically colocalize at the same KC synapses to form intracellular signaling cascades. Some of 97 these proteins might preferentially localize to specific MB compartments. Alternatively, they may distribute uniformly along the axon of Kenyon cells, but be activated in only specific compartments. Identification of cell-type-specific cotransmitters in DANs enabled a beginning to the exploration of this question (Aso, 2019).

Optogenetic activation of specific DANs was used to induce memory in specific MB compartments, while manipulating genes in specific types of KCs. This approach allowed mapping and characterization of the function of memory-related genes at a subcellular level. For example, the Gycbeta100B gene, which encodes a subunit of sGC, has been identified as 'memory suppressor gene' that enhances memory retention when pan-neuronally knocked down, but the site of its action was unknown. Gycbeta100B appears to be broadly dispersed throughout KC axons, based on the observed distribution of a Gycbeta100B-EGFP fusion protein. The experiments ectopically expressing NOS in PPL1-α3 DANs that do not normally signal with NO is most easily explained if sGC is available for activation in all MB compartments. What are the molecular pathways downstream to cGMP? How do dopamine and NO signaling pathways interact in regulation of KC synapses? Previous studies and RNA-Seq data suggest several points of possible crosstalk. In cultured KCs from cricket brains, cGMP-dependent protein kinase (PKG) mediates NO-induced augmentation of a Ca2+ channel current. However, no expression of either of the genes encoding Drosophila PKGs (foraging and Pkg21D) was detected in KCs in RNA-Seq studies. On the other hand, cyclic nucleotide-gated channels and the cGMP-specific phosphodiesterase Pde9 are expressed in KCs. Biochemical studies have shown that the activity of sGC is calcium dependent and that PKA can enhance the NO-induced activity of sGC by phosphorylating sGC; sGC isolated from flies mutant for adenylate cyclase, rutabaga, show lower activity than sGC from wild-type brains, suggesting crosstalk between the cAMP and cGMP pathways (Aso, 2019).

All memory systems must contend with a tension between the strength and longevity of the memories they form. The formation of a strong immediate memory interferes with and shortens the lifetimes of previously formed memories, and reducing this interference requires a reduction in initial memory strength that can only be overcome through repeated exposure. Theoretical studies have argued that this tension can be resolved by memory systems that exhibit a heterogeneity of timescales, balancing the need for both fast, labile memory and slow consolidation of reliable memories. The mechanisms responsible for this heterogeneity, and whether they arise from complex signaling within synapses themselves), heterogeneity across brain areas, or both, have not been identified (Aso, 2019).

NO acts antagonistically to dopamine and reduces memory retention while facilitating the rapid updating of memory following a new experience. Viewed in isolation, the NO-dependent reduction in memory retention within a single compartment may seem disadvantageous, but in the presence of parallel learning pathways, this shortened retention may represent a key mechanism for the generation of multiple memory timescales that are crucial for effective learning. During shock conditioning, for example, multiple DANs respond to the aversive stimulus, including PPL1-γ1pedc, PPL1-γ2α'1, PPL1-α3. This study has shown that optogenetic activation of these DAN cell types individually induces negative-valence olfactory memory with distinct learning rates. The NOS-expressing PPL1-γ1pedc induces memory with the fastest learning rate in a wild-type background, and this study shows that it induces an NO-dependent memory trace when dopamine synthesis is blocked, with a much slower learning rate and opposite valence (Aso, 2019).

Robust and stable NO-dependent effects were only observed when training was repeated 10 times. Under such repeated training, compartments with slower learning rates, such as α3, form memory traces in parallel to those formed in γ1pedc. Thus, flies may benefit from the fast and labile memory formed in γ1pedc without suffering the potential disadvantages of 58 shortened memory retention, as long-term memories are formed in parallel in other compartments. The Drosophila MB provides a tractable experimental system to study the mechanisms and benefits of diversifying learning rate, retention, and flexibility in parallel memory units, as well as exploring how the outputs from such unitsare integrated to drive behavior (Aso, 2019).

Molecular docking and biochemical validation of (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1->2)-β-D-glucopyranoside binding to an allosteric site in monoamine transporters

Albizia julibrissin Durazz is one of the most common herbs used for depression and anxiety treatment, but its mechanism of action as an antidepressant or anxiolytic drug have not been fully understood. Previous work has isolated and identified one lignan glycoside compound from Albizia Julibrissin Durazz, (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1->2)-β-D-glucopyranoside (SAG), that inhibited all three monoamine transporters with a mechanism of action different from that of the conventional antidepressants. This study, generated homology models for human dopamine transporter and human norepinephrine transporter, based on the X-ray structure of Drosophila dopamine transporter, and conducted the molecular docking of SAG to all three human monoamine transporters. The computational results indicated that SAG binds to an allosteric site (S2) that has been demonstrated to be formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule connected to the central site in these monoamine transporters. In addition, it wae demonstrated that SAG stabilizes a conformation of serotonin transporter with both the extracellular and cytoplasmic pathways closed. Furthermore, mutagenesis of the residues in both the allosteric and orthosteric sites was performed to biochemically validate SAG binding in all three monoamine transporters. These results are consistent with the molecular docking calculation and support the association of SAG with the allosteric site. It is expect that this herbal molecule could become a lead compound for the development of new therapeutic agents with a novel mechanism of action (Liu, 2022).

A neural mechanism for deprivation state-specific expression of relevant memories in Drosophila

Motivational states modulate how animals value sensory stimuli and engage in goal-directed behaviors. The motivational states of thirst and hunger are represented in the brain by shared and unique neuromodulatory systems. However, it is unclear how such systems interact to coordinate the expression of appropriate state-specific behavior. The activity of two brain neurons expressing leucokinin neuropeptide is elevated in thirsty and hungry flies, and leucokinin release is necessary for state-dependent expression of water- and sugar-seeking memories. Leucokinin inhibits two types of mushroom-body-innervating dopaminergic neurons (DANs) to promote thirst-specific water memory expression, whereas it activates other mushroom-body-innervating DANs to facilitate hunger-dependent sugar memory expression. Selection of hunger- or thirst-appropriate memory emerges from competition between leucokinin and other neuromodulatory hunger signals at the level of the DANs. Therefore, coordinated modulation of the dopaminergic system allows flies to prioritize the expression of the relevant state-dependent motivated behavior (Senapati, 2019).

Dopaminergic neurons can influence heat-box place learning in Drosophila

Dopamine provides crucial neuromodulatory functions in several insect and rodent learning and memory paradigms. However, an early study suggested that dopamine may be dispensable for aversive place memory in Drosophila. This study tested the involvement of particular dopaminergic neurons in place learning and memory. The thermogenetic tool Gr28bD was used to activate protocerebral anterior medial (PAM) cluster and non-PAM dopaminergic neurons in an operant way in heat-box place learning. Activation of PAM neurons influences performance during place learning, but not during memory testing. These findings provide a gateway to explore how dopamine influences place learning (Mishra, 2020).

Circuits that encode and guide alcohol-associated preference

A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. This study shows that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naive and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory (Scaplen, 2020).

A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. This study shows that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. This study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female (Ishimoto, 2020).

Overall, the present findings provide evidence for a neural relation that regulates the action selection of pre-mating behaviors in female Drosophila. The PPM3, a subset of DA neurons, forms a circuit with the R neurons R2/R4m and R4d in the EB. These different types of R neurons require different types of DA receptors, Dop1R1/D2R and Dop1R2. The knockdown of each DA receptor type indicates that all of these receptors are required to activate the expressing neurons, although the D2R conventionally inhibits D2R-expressing neurons. R2/R4m and R4d are GABAergic and cholinergic, respectively. Synaptic GRASP analysis revealed a neuronal connection from R2/R4m to R4d. R4d inhibition via the GABAA receptor is required for the proper reduction of pre-mating rejection. In addition to DA regulation of R2/R4m, the potential retrograde regulation may facilitate the GABA transmission of R2/R4m, depending on the R4d activity, with the production of NO via NMDAR/NOS signaling. This potentiation-like regulation provides the activation order of each R neuron with flexibility for the neural circuit output, and therefore to the rejection response for controlling the pre-mating behavioral kinetics (Ishimoto, 2020).

The pre-mating rejection should continue if the encounter does not match the female's criteria. Pheromones are important sexual cues provided by the male. Fruit flies produce cuticular hydrocarbons as pheromonal substances. cVA is a male-specific pheromonal cue that elicits female sexual arousal via the olfactory sensory system. This cVA signal activates pCd via a third-order olfactory interneuron, aSP-g. The SMP region contains aSP-g, pCd, and PPM3, although the direct connection between them has not been demonstrated. This aggregation of important components for female mating behavior suggests that pCd potentially integrates and verifies male information to execute the final decision for initiating the copulation. The female must resist the encounter until the evaluation process is complete-that is, the pre-mating rejection response controlled by the circuit found in the present study. These considerations lead to an assumption that the pCd and PPM3/R neuron circuits execute pre-mating computation in parallel. Many sexually dimorphic behaviors are reportedly mediated by sex-specific neural circuits (e.g., pCd, pC1). PPM3 and R neurons do not express fruitless or dsx, and no morphological sexual dimorphism has been detected. Thus, a non-sexually dimorphic circuit modulating sex-specific pre-mating behavior is proposed(Ishimoto, 2020).

Genetic manipulations of R neurons altered the pre-mating kinetics in virgin female flies, leading to the question of whether the modulation of R neurons also affects post-mating behavior, which is induced by injection of the seminal fluid sex peptide from the male fly and sustained for several days. One of the remarkable characteristics of post-mating rejection is ovipositor extrusion controlled by a distinct class of neurons in post-mating females, but either suppression of R2/R4m neurons or activation of R4d neurons, both of which reduced receptivity, rarely induced ovipositor extrusion. Mated females with R4d suppression, which makes virgin females highly receptive, exhibited a large rejection response similar to that in mated control females, and no copulation was observed during the 1-h observation time. In addition to these observations, the virgin females with Rdl knockdown in R4d reduced their walking speed with the progression of the male courtship; however, the walking speed of the mated females increases with the progression of the male courtship. Although further studies are required to elucidate the whole picture of rejection control in females, neuronal regulation of the post-mating response is presumably parallel to the pre-mating rejection response modulated by R neurons in the EB (Ishimoto, 2020).

This study found circuit functions that contain PPM3 DA subcluster neurons, and R neurons R2/R4m and R4d, were involved in the regulation of the kinetics of pre-mating behavior in virgin females. Pre-mating rejection is acutely elicited and then gradually decreased. A circuital feature of PPM3/R neurons may provide a theoretical action for controlling these pre-mating kinetics. PPM3 sends DA signals to both R2/R4m and R4d, and this recurrent circuit forms a feedforward motif with a repressor, the so-called incoherent type 1 feedforward loop (I1-FFL). I1-FFL is one of the most common network motifs implicated in gene and protein regulation networks, metabolic pathways, and neural networks from bacteria to humans. In the I1-FFL circuit, an activator X (e.g., PPM3) activates a target Z (e.g., R4d) and simultaneously activates another target Y (e.g., R2/R4m) that inhibits the target Z. Previous studies demonstrated that the I1-FFL accelerates the response of target Z, with a shorter response time. Moreover, upon input from X, Z activity increases and then, depending on the Y activity, it decreases toward basal levels. The I1-FFL circuit comprising PPM3, R2/R4m, and R4d would thus theoretically generate the acute activation of R4d, which promotes the pre-mating rejection behavior of females that would later be gradually attenuated by GABAergic signals from R2/R4m. This schema is consistent with the current findings, in which the circuit of PPM3/R neurons plays an important role in the action selection of pre-mating behavior (Ishimoto, 2020).

Genetic analysis indicates that retrograde signals from R4d to R2/R4m, mediated by NO and NMDAR, would add some flexibility to the Y-to-Z regulation in the I1-FFL circuit, contributing to progressive attenuation of the rejection response in virgin females. Depolarization of R4d activates NMDAR by the coincident input of glutamate from R2/R4m. The retrograde signals induced by NMDAR/NOS/sGC probably facilitate the GABA transmission of R2/R4m, which progressively suppresses R4d activity via the GABAA receptors. Because NMDARs require simultaneous activation by glutamate and depolarization, the NMDAR pore opening may be insufficient until the activity of R4d neurons will reach a sufficient level. This may lead to keeping the GABAergic R2/R4m function at a lower level that is under a certain threshold required for GABA release, and it may induce a continuous pre-mating rejection response. Recently, new subdivision of R neurons (R5 and R6) were classified by clonal morphological analysis and cell lineage analysis. These new subclasses of R neurons may provide a novel function of the putative I1-FFL. To investigate this further, the physiological properties of each R neuron should be analyzed (Ishimoto, 2020).

The distribution pattern of neurotransmitters in EB neurons has been documented. In the R2/R4m axonal ring structure, the glutamatergic population seems to be smaller than the GABAergic population, suggesting that the neuronal population in the R2/R4m is heterologous and contains few, if any, neurons that co-express both glutamate and GABA. In the vertebrate system, heterosynaptic regulation of GABAergic transmission is incorporated into inhibitory long-term potentiation. Inhibitory long-term potentiation of GABAergic neurons is induced at heterosynaptic sites containing glutamatergic and GABAergic neurons as presynaptic cells. In GABAergic inhibitory long-term potentiation, NO induced by glutamatergic activation of the NMDAR/NOS pathway retrogradely activates sGC, which augments cGMP levels to enhance GABA release. These intriguing analogies between vertebrate findings and the current results suggest that a similar molecular machinery potentiates GABAergic subsets of the R2/R4m neurons for attenuating the pre-mating rejection response, and hence for the action selection of pre-mating kinetics. Regarding the action selection for adaptive behaviors, others have proposed a correspondence of functions and neural architectures between vertebrate basal ganglia and the insect central complex, which contains EB neurons. In the basal ganglia, DA activates the neurons of the nucleus accumbens with modulation of glutamate and GABA release. The neurochemicals in the nucleus accumbens released by sexual interaction regulate female action selection for the affiliation of monogamous prairie voles (Microtus ochrogaster). The current genetic manipulations of particular DA, cholinergic, glutamatergic, and GABAergic systems in the central complex significantly affected the action selection for female fruit fly pre-mating behaviors, implying that the mechanisms are similar to those in the vertebrate system, which sheds light on the evolutionary parallels and diversities across the animal kingdom (Ishimoto, 2020).

Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning

Animals rely on the relative timing of events in their environment to form and update predictive associations, but the molecular and circuit mechanisms for this temporal sensitivity remain incompletely understood. This study shows that olfactory associations in Drosophila can be written and reversed on a trial-by-trial basis depending on the temporal relationship between an odor cue and dopaminergic reinforcement. Through the synchronous recording of neural activity and behavior, this study shows that reversals in learned odor attraction correlate with bidirectional neural plasticity in the mushroom body, the associative olfactory center of the fly. Two dopamine receptors, DopR1 and DopR2, contribute to this temporal sensitivity by coupling to distinct second messengers and directing either synaptic depression or potentiation. These results reveal how dopamine-receptor signaling pathways can detect the order of events to instruct opposing forms of synaptic and behavioral plasticity, allowing animals to flexibly update their associations in a dynamic environment (Handler, 2019).

While memories are often thought of as windows into the past, their adaptive value lies in the ability to predict the future. This study, took advantage of the concise circuitry of the Drosophila mushroom body to investigate how the precise timing of dopaminergic reinforcement allows animals to form and maintain predictive associations between cues and outcomes. While studies of associative learning have often focused on sensory cues that anticipate punishment or reward, equally informative are cues associated with their termination. This study demonstrates that shifting the relative timing of an odor and reinforcement by <1 s can switch the valence of an olfactory memory, underscoring the exquisite temporal sensitivity of this circuit. As a consequence, flies can form equivalent appetitive associations with odors that anticipate rewards or follow punishments, or aversive associations with odors that predict punishments or follow rewards. The symmetry of this behavioral modulation permits Drosophila to take advantage of all the temporally correlated features of their environment that can be used to infer causal relationships. Together, this work suggests a model in which the steep temporal sensitivity of associative learning arises from the concerted action of two dopamine receptor-signaling pathways that work in opposition to bidirectionally regulate the strength of KC-MBON signaling (see DopR1 and DopR2 Are Required for Behavioral Flexibility), allowing animals to maintain an accurate model of a complex and changing world (Handler, 2019).

In a dynamic environment, memories must be continually retouched and rewritten to maintain their relevance and predictive value. By monitoring how individual flies adapt their odor preferences over 50 conditioning trials, this study has revealed that Drosophila can form and reverse learned associations on a trial-by-trial basis, pointing to the fundamental flexibility of memory updating mechanisms (Handler, 2019).

Prior work in both Drosophila and mammals has suggested memory retention is regulated by multiple mechanisms at different timescales. If not reinforced, memories may passively fade over time, reflecting the natural turnover of molecular and neural hardware. Alternatively, memories can be actively eroded either by re-exposure to the learned odor in the absence of the anticipated dopaminergic reinforcement or the reinforcement in the absence of the odor, violating the expected contingency between these two events. In contrast, the brief episodes of odor and dopaminergic reinforcement (1-2 s) used in the current study are insufficient to overwrite an olfactory association when presented independently but can immediately reverse a prior association when paired together in time. The convergence of olfactory and DAN input to the mushroom body thus conveys information about their causal relationship, offering a mechanism to rapidly update memories to reflect the changing temporal structure of the environment (Handler, 2019).

While memory updating could rely on plasticity at various sites within this circuit, this study demonstrated that the bidirectional modulation of behavior is highly correlated with bidirectional changes in the strength of the same KC-MBON synapses within the mushroom body. Such bidirectional synaptic plasticity has been proposed to confer reversibility to learning circuits. For example, spike-timing dependent plasticity (STDP) can bidirectionally tune the strength of synaptic connections between neurons depending on the relative timing of spikes in pre- and post-synaptic neurons, mirroring the sensitivity to temporal order observed in associative learning. However, STDP requires nearly coincident firing patterns on a millisecond timescale, far more rapid than the temporal relationships between stimuli typically required for associative learning. In this study, by examining neural and behavioral modulation over the same timescales and even concurrently within the same individuals, the modulation of synaptic signaling within the mushroom body is linked to reversible changes in behavior (Handler, 2019).

Within the mushroom body, each compartment serves as a site of convergence between odor signaling conveyed by KCs and dopaminergic reinforcement, allowing dopamine-receptor pathways within KC axons to detect the temporal order of these inputs. The spatial patterns of dopamine release and dopamine receptor second-messenger cascades are found to adhere to the compartmentalized architecture of the lobes, permitting different synapses along the same KC axon to be independently regulated. These observations suggest that within a compartment, multiple neuromodulatory mechanisms tune neurotransmission depending on the temporal structure of conditioning. As a consequence, the distinct complement of KC-MBON synapses activated by odors that precede or follow a reinforcement are differentially regulated, allowing a single dopaminergic reinforcement to drive the synchronous formation of multiple odor associations, effectively enhancing the coding capacity of a compartment (Handler, 2019).

Dopamine shapes circuit function in diverse ways by engaging distinct classes of receptors that couple to different signaling cascades. In Drosophila, DopR1 and DopR2 have been proposed to play opposing roles in olfactory memory regulation at the behavioral level, with DopR1 essential to memory formation and DopR2 necessary for memory erosion. Yet the contribution of these receptors to synaptic modulation within the mushroom body has remained unclear. The current work reveals that the opposing behavioral roles of DopR1 and DopR2 are mirrored by their antagonistic regulation of KC-MBON signaling, with DopR1 required for the depression ensuing from forward pairing, while DopR2 is essential for the potentiation that follows backward pairing. Although DANs selectively innervating the mushroom body are sufficient to instruct bidirectional behavioral modulation, the broader expression of DopR1 and DopR2 leaves open the possibility that these receptors may also act at other sites within the nervous system to shape the temporal sensitivity of associative learning (Handler, 2019).

Using fluorescent sensors of DopR1 and DopR2 second messengers allowed the gaining insight into the spatial and temporal patterning of these intracellular signaling pathways during conditioning. While a potential limitation of optical reporters is their restricted sensitivity and dynamic range, these sensors nevertheless reveal that the selective recruitment of dopamine-receptor signaling cascades is sufficient to account for the temporal dependence of neural and behavioral modulation. Monitoring cAMP production during conditioning reveals that, while the DopR1 pathway serves as a coincidence detector, in accord with the coordinate regulation of adenylate cyclases by Gαs and calcium, it cannot autonomously encode the temporal order of events. In contrast, DopR2 signaling through Gαq strictly depends on the temporal sequence of KC and DAN activation (Handler, 2019).

Which component of the DopR2-signaling cascade is sensitive to the temporal ordering of odor and reinforcement? IP3 receptors that gate calcium release from the ER lumen represent an intriguing candidate, as their complex regulation by both IP3 and cytosolic calcium renders them inherently sensitive to the sequence of agonist binding: IP3 binding unmasks a calcium regulatory site required for channel opening, while high calcium in the absence of IP3 inhibits channel activity. Indeed, this study observed that ER calcium release is time locked to KC stimulation suggesting that the precise order dependence of this pathway relies on calcium entry subsequent to IP3 production. In the cerebellum, bidirectional plasticity at parallel fiber-Purkinje neuron synapses has been proposed to similarly rely on calcium release from the ER lumen via IP3 receptors. While the analogous circuit organization of the mushroom body and cerebellum has been well described, the current observations suggest they may share conserved molecular mechanisms for temporally precise synaptic modulation (Handler, 2019).

Together, this work points to dopamine receptor signaling pathways in KC axons as a key site of temporal coincidence and order detection during associative learning. While this study focused on the role of dopaminergic signaling within the γ4 compartment, timing-dependent bidirectional plasticity was observed to be shared characteristic of KC-MBON synapses in multiple compartments of the γ lobe. Therefore, the reversible modulation of behavior instructed by both the PAM or PPL DANs likely reflects bidirectional plasticity driven synchronously in the multiple compartments innervated by these DAN drivers. Aversive electric shock and sugar rewards evoke distributed patterns of activity across the DAN population, implying that these naturalistic reinforcers likewise instruct coordinated bidirectional plasticity across different compartments to rapidly shape the net output of the mushroom body. Similar patterns of DAN activity are also elicited by a fly's locomotion, raising the possibility that, in the context of an odor plume, an animal's behavior may serve as a reinforcement stimulus that itself drives bidirectional synaptic plasticity to regulate odor processing (Handler, 2019).

The ability to form or overwrite associations on a trial-by-trial basis allows for adaptive behavior in a noisy and uncertain environment where the temporal relationships between events may quickly change. However, animals must also have the capacity to store relevant memories persistently, even for a lifetime. Therefore, the reversible plasticity observed must co-exist with additional molecular and circuit mechanisms that underlie the formation and retention of longer-term associations. Indeed, recent work has described intrinsic differences between mushroom body compartments in their susceptibility to memory erosion as well as differences in second-messenger signaling in distinct KC subpopulations. Together, these results suggest that the differential expression or coupling of dopamine-receptor signaling pathways in different KC classes may tune synaptic plasticity rules to regulate the persistence of information storage. While this work connects molecular pathways within a sub-population of KCs to the emergence of short-term associations, functional dissection of these signaling cascades across the different lobes of the mushroom body will provide insight into the distinct timescales of memory formation and erosion (Handler, 2019).

Input Connectivity Reveals Additional Heterogeneity of Dopaminergic Reinforcement in Drosophila

Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions. This study used a recent electron microscope volume of the fly brain to reconstruct the fine anatomy of individual DANs within three MB compartments. The 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype γ5(fb) receives direct recurrent feedback from γ5β'2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from MBONs. The single aversively reinforcing PPL1-γ1pedc DAN was similarly reconstructed. The γ1pedc DAN inputs mostly differ from those of γ5 DANs and they cluster onto distinct dendritic branches, presumably separating its established roles in aversive reinforcement and appetitive motivation. Tracing also identified neurons that provide broad input to γ5, β'2a, and γ1pedc DANs, suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal further complexity of dopaminergic reinforcement circuits between and within MB compartments (Otto, 2020).

Carbon monoxide, a retrograde messenger generated in postsynaptic mushroom body neurons, evokes noncanonical dopamine release

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. This study found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca(2+) efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. It is proposed that DA neurons use two distinct modes of transmission to produce global and local DA signaling (Ueno, 2020).

Dopamine (DA) is required for various brain functions, including the regulation of global brain states, such as arousal and moods. To perform these functions, individual DA neurons innervate extensive areas of the brain, and release DA onto a wide range of target neurons through a processes known as volume transmission. However, this extensive innervation is not suitable for precise, localized release of DA, and it has been unclear how widely innervating dopaminergic neurons can also direct DA release onto specific target neurons (Ueno, 2020).

In Drosophila, olfactory associative memories are formed and stored in the mushroom bodies (MBs) where Kenyon cells, MB intrinsic neurons which are activated by different odors, form synaptic connections with various MB output neurons, which regulate approach and avoidance behaviors. Dopaminergic neurons (DA neurons) modulate plasticity of synapses between Kenyon cells and MB output neurons. However, while there are ~2000-2500 Kenyon cells that form thousands of synapses with MB output neurons, plasticity at these synapses is regulated by relatively few DA neurons. This indicates that canonical action potential-dependent release cannot fully explain DA release and plasticity. It was recently determined that in Drosophila, synaptic vesicular (SV) exocytosis from DA terminals is restricted to MB neurons that have been activated by coincident inputs from odor-activated cholinergic pathways, and glutamatergic pathways, which convey somatosensory (pain) information. Odor information is transmitted to the MBs by projection neurons from the antennal lobe (AL), while somatosensory information is transmitted to the brain via ascending fibers of the ventral nerve cord (AFV). AL stimulation evokes Ca2+ responses in the MB by activating nAChRs, and AFV stimulation evokes Ca2+ responses in the MBs by activating N-Methyl-D-aspartate receptors (NMDARs) in the MBs. Significantly, when the AL and AFV are stimulated simultaneously (AL 1 AFV) or the AL and NMDARs are stimulated simultaneously (AL 1 NMDA), plasticity occurs such that subsequent AL stimulations causes increased Ca2+ responses in the a3/a93 compartments. This plasticity is known as long-term enhancement (LTE) of MB responses and requires activation of D1 type DA receptors (D1Rs) in the MBs. Furthermore, while activation of D1Rs alone is sufficient to produce LTE, neither AL nor AFV stimulation alone is able to cause SV exocytosis from presynaptic DA terminals projecting onto the a3/a93 compartments of the vertical MB lobes. Instead, exocytosis from DA terminals occurs only when postsynaptic Kenyon cells are activated by coincident AL 1 AFV or AL 1 NMDA stimulation. Strikingly, while MBs are bilateral structures and DA neurons project terminals onto both sides of MBs, SV exocytosis occurs specifically in MB areas that have been coincidently activated. Based on these results, it is proposed that coincident inputs specify the location where DA is released, whereas DA induces plastic changes needed to encode associations. However, it has been unclear how activated Kenyon cells send a retrograde 'demand' signal to presynaptic DA terminals to induce SV release (Ueno, 2020).

This study used a Drosophila dissected brain system to examine synaptic plasticity and DA release, and found that coincidentally activated postsynaptic Kenyon cells generate the retrograde messenger, carbon monoxide (CO). CO is generated by heme oxygenase (HO) in postsynaptic MB neurons, and induces DA release from presynaptic terminals by evoking Ca2+ release from internal stores via ryanodine receptors (RyRs). Thus, while individual DA neurons extensively innervate the MBs, on-demand SV exocytosis allows DA neurons to induce plasticity in specific target neurons (Ueno, 2020).

CO functions as a retrograde on-demand messenger for SV exocytosis in presynaptic DA terminals A central tenet of neurobiology is that action potentials, propagating from the cell bodies, induce Ca2+ influx in presynaptic terminals to evoke SV exocytosis. However, recent mammalian studies have shown that only a certain fraction of a large number of presynaptic DA release sites is involved in canonical SV exocytosis. In this study, a novel mechanism of SV exocytosis was identified in which activity in postsynaptic neurons evokes presynaptic release to induce plastic changes. This mechanism allows the timing and location of DA release to be strictly defined by activity of postsynaptic neurons (Ueno, 2020).

On-demand SV exocytosis uses CO as a retrograde signal from postsynaptic MB neurons to presynaptic DA terminals. CO fulfills the criteria that have been proposed for a retrograde messenger: (1) it was demonstrated that HO, which catalyzes CO production, is highly expressed in postsynaptic MB neurons, indicating that MB neurons have the capacity to synthesize the messenger; (2) it was shown that pharmacological and genetic suppression of HO activity in the MBs inhibits CO production, presynaptic DA release, and LTE, and (3) using a CO fluorescent probe, COP-1, it was demonstrated that CO is generated in the MBs following coincident stimulation of the MBs, and CO generation is restricted to lobes of MB neurons that receive coincident stimulation. It was further shown that direct application of CO, or a CO donor, induces DA release from presynaptic terminals, whereas addition of a CO scavenger, HemoCD, suppresses release. Fourth, it was demonstrated that CO activates RyR in presynaptic terminals to induce SV exocytosis. Strikingly, CO-dependent SV exocytosis does not depend on influx of extracellular Ca2+ but instead requires efflux of Ca2+ from internal stores via RyR. Finally, it was shown that pharmacological inhibition and genetic suppression of RyR in DA neurons impair DA release after coincident stimulation and CO application (Ueno, 2020).

Other retrograde signals, such as NO and endocannabinoids, enhance or suppress canonical SV exocytosis, but this study finds that CO-dependent DA release occurs even in conditions that block neuronal activity and Ca2+ influx in presynaptic DA terminals. This suggests that CO does not function to modulate canonical SV exocytosis, but may instead evoke exocytosis through a novel mechanism. Several previous studies have indicated that CO and RyR-dependent DA release also occurs in mammals. A microdialysis study has shown that CO increases the extracellular DA concentration in the rat striatum and hippocampus, either through increased DA release or inhibition of DA reuptake. Also, pharmacological stimulation of RyRs has been reported to induce DA release in the mice striatum. This release is attenuated in RyR3-deficient mice, while KCl-induced DA release, which requires influx of extracellular Ca2+, is unaffected, suggesting that RyR-dependent release is distinct from canonical DA release. However, it has been unknown whether and how CO is generated endogenously. Physiologic conditions that activate RyR to evoke DA release have also been unclear (Ueno, 2020).

While most neurotransmitters are stored in synaptic vesicles and released on neuronal depolarization, the release of gaseous retrograde messengers, such as NO and CO, is likely coupled to activation of their biosynthetic enzymes, NOS and HO. In mammals, an HO isoform, HO-2, is selectively enriched in neurons, and HO-2-derived CO is reported to function in plasticity. HO-2 is activated by Ca2+/calmodulin (CaM) binding, and by casein kinase II (CKII) phosphorylation. Previously, it was shown that coincident AL 1 NMDA stimulation induces a robust Ca2+ increase in the MBs that is greater than the increase from either stimulation alone (Ueno, 2017). It is proposed that this increase may activate Drosophila HO in the MB to generate CO during associative stimulation. While Drosophila has a single isoform of RyR, mammals have three isoforms, RyR1-RyR3. Skeletal muscle and cardiac muscle primarily express RyR1 and RyR2, and the brain, including the striatum, hippocampus, and cortex, expresses all three isoforms. RyRs are known to be activated by Ca2+ to mediate Ca2+ induced Ca2+ release. However, CO-evoked DA release occurs even in the presence of Ca2+-free extracellular solutions containing TTX and EGTA, suggesting that CO activates RyR through a different mechanism. In addition to Ca2+, RyR can be activated by calmodulin, ATP, PKA, PKG, cADP-ribose, and NO. NO can directly stimulate RyR1 nonenzymatically by S-nitrosylating a histidine residue to induce Ca2+ efflux. CO has been reported to activate Ca2+-activated potassium channels (KCa) through a nonenzymatic reaction in rat artery smooth muscle, raising the possibility that it may activate RyR through a similar mechanism. Alternatively, both NO and CO can bind to the heme moiety of soluble guanlylate cyclase leading to its activation. Activated soluble guanlylate cyclase produces cGMP, and cGMP-dependent protein kinase (PKG) rapidly phosphorylates and activates RyRs. Interestingly, NO increases DA in the mammalian striatum in a neural activity-independent manner. Since activation of RyRs also increases extracellular DA in the striatum, hippocampus, and cortex, NO may play a pivotal role in RyRs activation and DA release in mammals. However, NOS expression has not been detected in the MBs, suggesting that, in Drosophila, CO rather than NO may function in this process (Ueno, 2020).

Previous studies have shown that electrical activity from the AL and AFV is transmitted to the MBs by cholinergic and glutamatergic neurons acting on nAChRs and NMDARs, respectively. Although the cholinergic inputs from the AL are known to be delivered by projection neurons, the glutamate inputs are still unclear. Previous work identified glutamatergic neurons that innervate a3/a93 compartments of the MBs and show SV release on electrical stimulation of the AFV (Ueno, 2017). It is proposed that these neurons may transmit information regarding AFV stimuli to the MBs. Alternatively, while NMDARs are localized throughout MB lobes, vesicular glutamate transporter-positive terminals are found only sparsely on the MBs. This suggests that neurons expressing a currently uncharacterized vesicular glutamate transporter may convey information from the AFV to MBs (Ueno, 2020).

DA plays a critical role in associative learning and synaptic plasticity. In flies, neutral odors induce MB responses by activating sparse subsets of MB neurons. After being paired with electrical shocks during aversive olfactory conditioning, odors induce larger MB responses in certain areas of the MBs. This plastic change was modeled in ex vivo brains as LTE, and it was shown that DA application alone is sufficient to induce this larger response (Ueno, 2017). However, in the Drosophila brain, only a small number of DA neurons (~12 for aversive and ~100 for appetitive) regulate plasticity in ~2000 MB Kenyon cells. Thus, to form odor-specific associations, there should be a mechanism regulating release at individual synapses. CO-dependent on-demand DA release provides this type of control. If on-demand release is involved in plasticity and associative learning, knockdown of genes associated with release should affect learning. Indeed, this study shows that knocking down either dHO in the MBs or RyR in DA neurons impairs olfactory conditioning. While these knockdowns did not completely abolish olfactory conditioning, this may be due to inefficiency of the knockdown lines. Alternatively, on-demand release may not be the only mechanism responsible for memory formation, but may instead be required for a specific phase of olfactory memory (Ueno, 2020).

In ex vivo studies, this study found that DA release requires coincident activation of postsynaptic MB neurons by cholinergic and glutamatergic stimuli. However, other in vivo imaging studies have shown that DA neurons can be activated and release DA on odor stimulation or shock application alone. Notably, projection of DA terminals is compartmentalized on the MB lobes and shows distinct responses and DA release during sensory processing. In these studies, dopaminergic neurons innervating the the a3/a93 compartments at the tips of the MB vertical lobes were examined, whereas other studies focused on compartments located on the MB horizontal lobes. This suggests that plasticity in different MB compartments may be regulated by different mechanisms. Unfortunately, due the location of the microelectrode for AL stimulation which caused interference in fluorescent imaging of the horizontal lobes, it was not possible to obtain reliable imaging data from these lobes in this study. Another difference between ex vivo and in vivo studies is that in vivo imaging studies use living, tethered, dissected flies that are likely in different states of arousal/distress, are exposed to many different stimuli, and can form unintended associations. In contrast, brains in ex vivo preparations are in a more controlled environment and are likely exposed to fewer unintended sensory stimuli. This may also explain apparent discrepancies between ex vivo and previous in vivo results (Ueno, 2020).

In mammals, the role of CO in synaptic plasticity is unclear. Application of CO paired with low-frequency stimulation induces LTP, while inhibiting HO blocks LTP in the CA1 region of the hippocampus. However, HO-2-deficient mice have been reported to have normal hippocampal CA1 LTP. In contrast to CO, a role for NO in synaptic plasticity and learning has been previously reported. Thus, at this point, it is an open question whether CO or NO evokes DA release in mammals. Downstream from CO or NO, RyRs have been shown to be required for hippocampal and cerebellar synaptic plasticity (Ueno, 2020).

The current results suggest that DA neurons release DA via two distinct mechanisms: canonical exocytosis and on-demand release. Canonical exocytosis is evoked by electrical activity of presynaptic DA neurons, requires Ca2+ influx, and may be involved in volume transmission. This mode of release can activate widespread targets over time, and is suited for regulating global brain functions. In contrast, on-demand release is evoked by activity of postsynaptic neurons, requires Ca2+ efflux via RyR, and can regulate function of specific targets at precise times. DA neurons may differentially use these two modes of SV exocytosis in a context-dependent manner. Understanding how DA neurons differentially use these modes of transmission will provide new insights into how a relatively small number of DA neurons can control numerous different brain functions (Ueno, 2020).

Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning

Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. This study showed that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, it was demonstrated that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly this work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, these results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning (Ganguly, 2020).

Dopaminergic mechanism underlying reward-encoding of punishment omission during reversal learning in Drosophila

Animals form and update learned associations between otherwise neutral sensory cues and aversive outcomes (i.e., punishment) to predict and avoid danger in changing environments. When a cue later occurs without punishment, this unexpected omission of aversive outcome is encoded as reward via activation of reward-encoding dopaminergic neurons. How such activation occurs remains unknown. Using real-time in vivo functional imaging, optogenetics, behavioral analysis and synaptic reconstruction from electron microscopy data, this study identified the neural circuit mechanism through which Drosophila reward-encoding dopaminergic neurons are activated when an olfactory cue is unexpectedly no longer paired with electric shock punishment. Reduced activation of punishment-encoding dopaminergic neurons relieves depression of olfactory synaptic inputs to cholinergic neurons. Synaptic excitation by these cholinergic neurons of reward-encoding dopaminergic neurons increases their odor response, thus decreasing aversiveness of the odor. These studies reveal how an excitatory cholinergic relay from punishment- to reward-encoding dopaminergic neurons encodes the absence of punishment as reward, revealing a general circuit motif for updating aversive memories that could be present in mammals (McCurdy, 2021).

Associative learning in larval and adult Drosophila is impaired by the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine

Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly Drosophila melanogaster, dopaminergic reinforcement has largely been studied using genetics, whereas pharmacological approaches have received less attention. This study applied the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine (3IY), which causes acute systemic inhibition of dopamine signaling, and investigated its effects on Pavlovian conditioning. 3IY feeding impairs sugar reward learning in larvae while leaving task-relevant behavioral faculties intact, and additional feeding of a precursor of dopamine (L-3,4-dihydroxyphenylalanine, L-DOPA), rescues this impairment. Concerning a different developmental stage and for the aversive valence domain, it was furthermore demonstrated that punishment learning by activating the dopaminergic neuron PPL1-γ1pedc in adult flies is also impaired by 3IY feeding and can likewise be rescued by L-DOPA. These findings exemplify the advantages of using a pharmacological approach in combination with the genetic techniques available in D. melanogaster to manipulate neuronal and behavioral function (Thoener, 2021).

Dopamine-based mechanism for transient forgetting

Active forgetting is an essential component of the memory management system of the brain. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. This study identified a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval (Sadandal, 2021).

Memory formation, consolidation and retrieval are well-known functions that support memory expression; however, the processes that limit these functions -- including forgetting -- are less understood. Forgetting has been characterized as either passive or active, and is crucial for memory removal, flexibility and updating. Memory may be removed completely, resulting in permanent forgetting; or temporarily irretrievable, resulting in transient forgetting (Sadandal, 2021).

One form of active forgetting-known as intrinsic forgetting-involves one dopamine neuron (DAN) that innervates the γ2α'1 compartment of the axons of mushroom body neurons (MBNs) and the dendrites of the downstream, compartment-specific mushroom-body output neurons (MBONs). This DAN resides in a cluster of 12 DANs in each brain hemisphere that is known as the protocerebral posterior lateral 1 (PPL1) cluster. Current evidence indicates that the ongoing activity of these DANs after aversive olfactory conditioning slowly and chronically erodes labile and nonconsolidated behavioural memory, as well as a corresponding cellular memory trace that forms in the MBONs. This intrinsic forgetting mechanism is shaped by external sensory stimulation and sleep or rest, and is mediated by a signalling cascade in the MBNs that is initiated by the activation of the dopamine receptor DAMB, which leads to the downstream activation of the actin-binding protein Cofilin and the postulated reorganization of the synaptic cytoskeleton (Sadandal, 2021).

By contrast, there is little understanding of the mechanisms that arbitrate transient forgetting. Neuropsychological studies of failures or delays in retrieval in humans have primarily focused on lexical access. Phonological blockers or interfering stimuli produce a tip-of-the-tongue state-the failure to recall the appropriate word or phrase. Tip-of-the-tongue states are resolved when the distracting signals dissipate. Several brain regions have been implicated in tip-of-the-tongue states from functional magnetic resonance imaging studies, but the neurobiological mechanisms that produce a temporary state of impaired retrieval are unknown. This study offers an entry point into this area of brain function (Sadandal, 2021).

Memory retrieval has been proposed to consist of an interplay between internal or external cues and memory engrams, with cue-induced reactivation of engrams across multiple regions of the brain facilitating memory expression. But a central question about this process is how interfering stimuli temporarily block memory retrieval, resulting in transient forgetting. This study offers insights into one such mechanism. Behavioural and functional imaging data reveal that PPL1-α2α'2, working through the DAMB receptor expressed in the α2α'2 MBN axonal compartment, mediates the transient forgetting of PSD-LTM. This effect occurs without altering a cellular memory trace in the postsynaptic MBON-α2sc. This process can be triggered by distracting stimuli, illustrating a neural-genetic-environmental interplay that modifies memory expression (Sadandal, 2021).

This study considered why the cellular memory trace remains unaffected by DAN stimulation despite the occurrence of behavioural forgetting. Because blocking synaptic output from MBON-α2sc reduces PSD-LTM expression, the simplest hypothesis posits that cellular memory traces form with conditioning in the MBON in addition to the cytoplasmic Ca2+-based memory trace that was detected in this study. This is expected: neurons undergo broad changes in physiology as they adopt new states, so it is plausible that such plastic mechanisms-especially ones that gate synaptic release-are inactivated by DAN activity while leaving the Ca2+-based memory trace intact (Sadandal, 2021).

The discovery that loss of function of DAMB leads to enhanced PSD-LTM was surprising, because of a previous study reporting that this insult attenuates PSD-LTM. The experiments argue strongly that DAMB functions normally to suppress expression of PSD-LTM. However, this leads to the question of why a receptor involved in transient forgetting would lead to enhanced PSD-LTM when inactivated. Previous experiments have shown that PPL1-α2α'2-similar to PPL1-γ2α'1-exhibits ongoing activity, leading to a slow release of dopamine onto MBNs. This activity should slowly degrade or suppress existing memory so that when the receptor is inactivated memory expression is enhanced (Sadandal, 2021).

PPL1-α2α'2 has no important role in the forgetting of labile nonconsolidated memory. Instead, previous studies have identified a different DAN (PPL1-γ2α'1) as having a role in this process and the apparent erasure of the downstream cellular memory trace-perhaps an indication of 'permanent forgetting'. This process is modulated by internal and external factors, and is mediated by key molecules expressed in the MBN that receive PPL1-γ2α'1 input. No robust decrement was found in expression of PSD-LTM after PPL1-γ2α'1 stimulation, which points to the existence of two separate dopamine-based circuits for permanent and transient forgetting. This functional separation may indicate a fundamental principle in the organization of circuits that mediate several forms of forgetting (Sadandal, 2021).

However, the DAMB receptor is used for both permanent and transient forgetting. DAMB is widely expressed across the MBN axons but alters synaptic plasticity differently across MBN compartments. It is possible that DAMB signalling may be distinct for the two forms of forgetting. DAMB preferentially couples with Gq, the knockdown of which inhibits the potent erasure of memory, but its potential role in transient forgetting is unknown. The scaffolding protein Scribble orchestrates the activities of Rac, Pak and Cofilin, all of which are important for the permanent forgetting pathway. However, Scribble knockdown or inhibition of Rac1 does not enhance the PSD-LTM as is the case in DAMB-knockdown flies, which suggests that this scaffolding signalosome does not have a large role in transient forgetting. In summary, the two distinct forms of forgetting-transient and permanent-share a dopaminergic mechanism and a common dopamine receptor, but differ in upstream and downstream neural circuits and in downstream signalling pathways within MBNs (Sadandal, 2021).

Models of heterogeneous dopamine signaling in an insect learning and memory center

The Drosophila mushroom body exhibits dopamine dependent synaptic plasticity that underlies the acquisition of associative memories. Recordings of dopamine neurons in this system have identified signals related to external reinforcement such as reward and punishment. However, other factors including locomotion, novelty, reward expectation, and internal state have also recently been shown to modulate dopamine neurons. This heterogeneity is at odds with typical modeling approaches in which these neurons are assumed to encode a global, scalar error signal. How is dopamine dependent plasticity coordinated in the presence of such heterogeneity? This study developed a modeling approach that infers a pattern of dopamine activity sufficient to solve defined behavioral tasks, given architectural constraints informed by knowledge of mushroom body circuitry. Model dopamine neurons exhibit diverse tuning to task parameters while nonetheless producing coherent learned behaviors. Notably, reward prediction error emerges as a mode of population activity distributed across these neurons. These results provide a mechanistic framework that accounts for the heterogeneity of dopamine activity during learning and behavior (Jiang, 2021).

Learning with reinforcement prediction errors in a model of the Drosophila mushroom body

Effective decision making in a changing environment demands that accurate predictions are learned about decision outcomes. In Drosophila, such learning is orchestrated in part by the mushroom body, where dopamine neurons signal reinforcing stimuli to modulate plasticity presynaptic to mushroom body output neurons. Building on previous mushroom body models, in which dopamine neurons signal absolute reinforcement, it is proposed instead that dopamine neurons signal reinforcement prediction errors by utilising feedback reinforcement predictions from output neurons. Plasticity rules were formulated that minimise prediction errors, verify that output neurons learn accurate reinforcement predictions in simulations, and postulate connectivity that explains more physiological observations than an experimentally constrained model. The constrained and augmented models reproduce a broad range of conditioning and blocking experiments, and this study demonstrated that the absence of blocking does not imply the absence of prediction error dependent learning. These results provide five predictions that can be tested using established experimental methods (Bennett, 2021).

Effective decision making benefits from an organism's ability to accurately predict the rewarding and punishing outcomes of each decision, so that it can meaningfully compare the available options and act to bring about the greatest reward. In many scenarios, an organism must learn to associate the valence of each outcome with the sensory cues predicting it. A broadly successful theory of reinforcement learning is the delta rule, whereby reinforcement predictions (RPs) are updated in proportion to reinforcement prediction errors (RPEs): the difference between predicted and received reinforcements. RPEs are more effective as a learning signal than absolute reinforcement signals because RPEs diminish as the prediction becomes more accurate, adding stability to the learning process. In mammals, RPEs related to rewards are signalled by dopamine neurons (DANs) in the ventral tegmental area and substantia nigra, enabling the brain to implement approximations to the delta rule. In Drosophila melanogaster, DANs that project to the mushroom body (MB) (see Valence-specific model of the mushroom body) provide both reward and punishment modulated signals that are required for associative learning5. However, to date, MB DAN activity is typically interpreted as signalling absolute reinforcements (either positive or negative) for two reasons: (1) a lack of direct evidence for RPE signals in DANs, and (2) limited evidence in insects for the blocking phenomenon, in which conditioning of one stimulus can be impaired if it is presented alongside a previously conditioned stimulus, an effect that is indicative of RPE-dependent learning. This study has incorporated anatomical and functional data from recent experiments into a computational model of the MB, in which MB DANs do compute RPEs. The model provides a circuit-level description for delta rule learning in the MB, which is use to demonstrate why the absence of blocking does not necessarily imply the absence of RPEs (Bennett, 2021).

The MB is organised into lateral and medial lobes of neuropil in which sensory encoding Kenyon cells (KCs) innervate the dendrites of MB output neurons (MBONs) that modulate behaviour. Consistent with its role in associative learning, DAN signals modulate MBON activity via synaptic plasticity at KC -> MBON synapses. Current models of MB function posit that the MB lobes encode either positive or negative valences of reinforcement signals and actions. Most DANs in the protocerebral anterior medial (PAM) cluster (called D+ in the model presented in this study) are activated by rewards, or positive reinforcement (R+), and their activation results in depression at synapses between coactive KCs (K) and MBONs that are thought to induce avoidance behaviours (M−). DANs in the protocerebral posterior lateral 1 (PPL1) cluster (D−) are activated by punishments, i.e., negative reinforcement (R−), and their activation results in depression at synapses between coactive KCs and MBONs that induce approach behaviours (M+). A fly can therefore learn to approach rewarding cues or avoid punishing cues as a result of synaptic depression at KC inputs to avoidance or approach MBONs, respectively (Bennett, 2021).

To date, there is only indirect evidence for RPE signals in MB DANs. DAN activity is modulated by feedforward reinforcement signals, but some DANs also receive excitatory feedback from MBONs, and it is likely this extends to all MBONs whose axons are proximal to DAN dendrites. The difference between approach and avoidance MBON firing rates is intrepeted as a RP that motivates behaviour, consistent with the observation that behavioural valence scales with the difference between approach and avoidance MBON firing rates. As such, DANs that integrate feedforward reinforcement signals and feedback RPs from MBONs are primed to signal RPEs for learning. These latter two features have yet to be incorporated in computational models of the MB (Bennett, 2021).

This study incorporate the experimental data described above to formulate a reduced computational model of the MB circuitry, demonstrate how DANs may compute RPEs, derive a plasticity rule for KC -> MBON synapses that minimises RPEs, and verify in simulations that the MB model learns accurate RPs. A limitation to the model was identified that imposes an upper bound on RP magnitudes, and demonstrate how putative connections between DANs, KCs and MBONs help circumvent this limitation. Introducing these additional connections yields testable predictions for future experiments as well as explaining a broader range of existing experimental observations that connect DAN and MBON stimulus responses to learning. Lastly, this study shows that both incarnations of the model—-with and without additional connections—-capture a wide range of observations from classical conditioning and blocking experiments in Drosophila. Different behavioural outcomes in the two models for specific experiments provide further strong experimental predictions (Bennett, 2021).

Successful decision making relies on the ability to accurately predict, and thus reliably compare, the outcomes of choices that are available to an agent. The delta rule, as developed by Rescorla and Wagner (1972), updates beliefs in proportion to a prediction error, providing a method to learn accurate and stable predictions. This work investigated the hypothesis that, in Drosophila melanogaster, the MB implements the delta rule. It is posited that approach and avoidance MBONs together encode RPs, and that feedback from MBONs to DANs, if subtracted from feedforward reinforcement signals, endows DANs with the ability to compute RPEs, which are used to modulate synaptic plasticity. A plasticity rule was formulated that minimises RPEs, and the effectiveness of the rule was verified in simulations of MAFC tasks. This study demonstrated how the established valence-specific circuitry of the MB restricted the learned RPs to within a given range, and postulated cross-compartmental connections, from MBONs to DANs, that could overcome this restriction. Such cross-compartmental connections are found in Drosophila larvae, but their functional relevance is unknown. Two MB models are presented that yield RPEs in DAN activity and that learn accurate RPs: (1) the VSλ model, in which plasticity incorporates a constant source of synaptic potentiation; (2) the MV model, in which mixed-valence connectivity between DANs, MBONs and KC -> MBON synapses is proposed. Both the VSλ and the MV models receive equally good support from behavioural experiments in which different genetic interventions impaired learning, while the MV model provides a mechanistic account for a greater variety of physiological changes that occur in individual neurons after learning. It is plausible, and can be beneficial, for both the VS&lambda and MV models to operate in parallel in the MB, as separately learning positive and negative aspects of decision outcomes, if they arise from independent sources, is important for context-dependent modulation of behaviour. Such learning has been proposed for the mammalian basal ganglia. This study also demonstrated why the absence of strong blocking effects in insect experiments does not necessarily imply that insects do not utilise RPEs for learning (Bennett, 2021).

The models yield predictions that can be tested using established experimental protocols. Below, which model supports each prediction is specified (Bennett, 2021).

Responses in single DANs too the unconditioned stimulus (US), when paired with a CS+, should decay towards a baseline over successive CS ± US pairings, as a result of the learned changes in MBON firing rates. Only one previous study has measured DAN responses throughout several CS-US pairings in Drosophila. Consistent with DAN responses in the current model, Dylla (2017) reported such decaying responses in DANs in the γ- and β'-lobes during paired CS+ and US stimulation. However, they reported similar decaying responses when the CS+ and US were unpaired (separated by 90 s) that were not significantly different from the paired condition. The paper concluded that DANs do not exhibit RPEs, and that the decaying DAN responses were a result of non-associative plasticity. An alternative interpretation is that a 90 s gap between CS+ and US does not induce DAN responses that are significantly different from the paired condition, and that additional processes prevent the behavioural expression of learning. Ultimately, the evidence for either effect is insufficient. Furthermore, Dylla observed increased CS+ responses in DANs after training. Conversely, after training in these models, when the US was set to zero, DAN responses to the CS+ decreased. Interpreting post-training activity in DANs as responses to the CS+ alone, or alternatively as responses to an omitted US, are equally valid in the current model because the CS+ and US always occurred together. Resolving time within trials in the models would allow better addressing of this conflict with experiments. The Dylla results are, however, consistent with the temporal difference (TD) learning rule (as are studies on second order conditioning in Drosophila), of which the Rescorla-Wagner rule used in this work is a simplified case (Bennett, 2021).

DANs of both valence modulate plasticity at MBONs of a single valence. In contrast, anatomical and functional experimental data suggest that, in each MB compartment, the DANs and MBONs have opposite valences. However, the GAL4 lines used to label DANs in the PAM cluster often include as many as 20-30 cells each, and it has not yet been determined whether all labelled DANs exhibit the same valence preference. Similarly, the valence encoded by MBONs is not always obvious. It is not clear whether optogenetically activated MBONs biased flies to approach the light stimulus, or to exhibit no-go behaviour that kept them within the light. In larval Drosophila, there are several examples of cross-compartmental DANs and MBONs, but a full account of the valence encoded by these neurons is yet to be provided. In adult Drosophila, γ1-pedc MBONs deliver cross-compartmental inhibition, such that M4/6 MBONs are effectively modulated by both aversive PPL1-γ1-pedc DANs and appetitive PAM DANs (Bennett, 2021).

This is not the first publication to present a MB model that makes effective decisions after learning about multiple reinforced cues. However, these models utilise absolute reinforcement signals, as well as bounded synapses that cannot strengthen indefinitely with continued reinforcements. Thus, given enough training, these models would not differentiate between two cues that were associated with reinforcements of the same sign, but different magnitudes. Carefully designed mechanisms are therefore required to promote stability as well as differentiability of same sign, different magnitude reinforcements. The model builds upon these studies by incorporating feedback from MBONs to DANs. This allows KC -> MBON synapses to accurately encode the reinforcement magnitude and sign with stable fixed points that are reached when the RPE signalled by DANs decays to zero. Alternative mechanisms that may promote stability and differentiability are forgetting (e.g., by synaptic weight decay), or adaptation in DAN responses. Exploring these possibilities in a MB model for comparison with the RPE hypothesis is well worth while, but goes beyond the scope of this work (Bennett, 2021).

Central to this work is the assumption that the MB has only a single objective: to minimise the RPE. In reality, an organism must satisfy multiple objectives that may be mutually opposed. In Drosophila, anatomically segregated DANs in the γ-lobe encode water rewards, sugar rewards, and motor activity, suggesting that Drosophila do indeed learn to satisfy multiple objectives. Multi-objective optimisation is a challenging problem, and goes beyond the scope of this work. Nevertheless, for many objectives, the principle that accurate predictions aid decision making, which forms the basis of this work, still applies (Bennett, 2021).

For simplicity, the simulations compress all events within a trial to a single point in time, and are therefore unable to address some time-dependent features of learning. For example, activating DANs either before or after cue exposure can induce memories with opposite valences; in locusts, the relative timing of KC and MBON spikes is important, though not necessarily in Drosophila. Nor has this study addressed the credit assignment problem: how to associate a cue with reinforcement when they do not occur simultaneously. A candidate solution is TD learning, whereby reinforcement information is back-propagated in time to all cues that predict it. While DAN responses in the MB hint at TD learning, it is not yet clear how the MB circuity could implement it. An alternative solution is an eligibility trace, which enables synaptic weights to be updated upon reinforcement even after presynaptic activity has ceased (Bennett, 2021).

Lastly, this work addresses memory acquisition, but not memory consolidation, which is supported by distinct circuits within the MB. Incorporating memory stabilising mechanisms may help to better align simulations of genetic interventions with fly behaviour in conditioning experiments (Bennett, 2021).

By incorporating the fact that KC responses to compound stimuli are non-linear combinations of their responses to the components, the model described in this paper was used to demonstrate why the lack of evidence for blocking in insects cannot be taken as evidence against RPE-dependent learning in insects. The model provides a neural circuit instantiation of similar arguments in the literature, whereby variable degrees of blocking can be explained if the brain utilises representations of stimulus configurations, or latent causes, which allow learned associations to be generalised between a compound stimulus and its individual elements by varying amounts. The effects of such configural representations on blocking are more likely when the component stimuli are similar, for example, if they engage the same sensory modality, as was the case in previous studies. By using component stimuli that do engage different sensory modalities, experiments with locusts have indeed uncovered strong blocking effects (Bennett, 2021).

This study has developed a model of the MB that goes beyond previous models by incorporating feedback from MBONs to DANs, and has shown how such a MB circuit can learn accurate RPs through DAN mediated RPE signals. The model provides a basis for understanding a broad range of behavioural experiments, and reveals limitations to learning given the anatomical data currently available from the MB. Those limitations may be overcome with additional connectivity between DANs, MBONs and KCs, which provide five strong predictions from this work (Bennett, 2021).

Differential conditioning produces merged long-term memory in Drosophila

Multiple spaced trials of aversive differential conditioning can produce two independent long-term memories (LTMs) of opposite valence. One is an aversive memory for avoiding the conditioned stimulus (CS+), and the other is a safety memory for approaching the non-conditioned stimulus (CS-). This study shows that a single trial of aversive differential conditioning yields one merged LTM (mLTM) for avoiding both CS+ and CS-. Such mLTM can be detected after sequential exposures to the shock-paired CS+ and unpaired CS-, and be retrieved by either CS+ or CS-. The formation of mLTM relies on triggering aversive-reinforcing dopaminergic neurons and subsequent new protein synthesis. Expressing mLTM involves αβ Kenyon cells and corresponding approach-directing mushroom body output neurons (MBONs), in which similar-amplitude long-term depression of responses to CS+ and CS- seems to signal the mLTM. These results suggest that animals can develop distinct strategies for occasional and repeated threatening experiences (Zhao, 2021).

To survive in a complex environment, animals need to learn from threatening experiences to avoid potential dangers. From invertebrates to humans, aversive differential conditioning is widely used to study memories produced by threatening experiences. After repetitive spaced trials of conditioning, animals form two complementary long-term memories (LTMs) of opposite valence, including the aversive memory to the conditioned stimulus (CS+) and the rewarding memory to the non-conditioned stimulus (CS-). Such complementary LTMs result in enhanced long-lasting discrimination between CS+ and CS- through guiding avoidance to CS+ and approach to CS-. However, it remains unclear whether and how occasional threatening experiences, such as single-trial conditionings, would induce long-lasting changes in future escape behavior (Zhao, 2021).

From invertebrates to humans, experience-dependent long-lasting behavioral modifications mainly rely on the formation of LTMs. In Drosophila, there are at least two categories of aversive olfactory LTMs that last for more than 7 days. One is the spaced training-induced LTM that can be observed after repetitive spaced training with inter-trial rests (multiple trials with a 15 min rest interval between each), but not after either single-trial training or repetitive massed training without interval. Forming such aversive LTM requires new protein synthesis and the paired posterior lateral 1 (PPL1) cluster of dopaminergic neurons (DANs) to depress the connection between odor-activated Kenyon cells (KCs) in the mushroom body (MB) αβ lobe and downstream α2sc (MB-V2) MB output neurons (MBONs). The other is a recently reported context-dependent LTM that forms after single-trial training, which does not require protein synthesis-dependent consolidation. The expression of context-dependent LTM relies on multisensory integration in the lateral horn and is not affected by blocking KCs. However, all these observations derived from the same design principle that evaluates memory performance through testing the discrimination between CS+ and CS-. Thus, direct responses to CS+ and CS- have been largely overlooked (Zhao, 2021).

The current study introduced a third-odor test, in which flies were given a choice between either CS+ and a novel odor, or CS- and a novel odor. It was therefore identified that the single-trial differential conditioning produces a merged LTM (mLTM) guiding avoidances of both CS+ and CS- for several days after training. The encoding and expression of such mLTM involve new protein synthesis, PPL1 DANs, αβ KCs, and α2sc MBONs. These findings suggest that animals utilize distinct escape strategies for facing occasional and repeated dangers (Zhao, 2021).

In the current study, the use of third-odor test leads to a conclusion that single-trial training produces an mLTM for guiding flies to avoid both CS+ and CS- for more than 7 days. Three categories of evidence in support of this conclusion are outlined below (Zhao, 2021)

First, throughout this study, the amplitudes of long-term avoidances of CS+ and CS- are always at a similar level under various conditions, including pharmacological treatment, cold-shock treatment, odor re-exposure, paradigm alteration, and neural circuitry manipulations. Second, re-exposure to either one of CS+ and CS- alone can extinguish both CS+ avoidance and CS- avoidance. Third, the long-term avoidances of CS+ and CS- can be recorded as the depression of odor-evoked responses in the same α2sc MBONs, meanwhile, CS+ avoidance and CS- avoidance both involve the same PPL1 DANs, αβ KCs, and α2sc MBONs. Thus, CS+ avoidance and CS- avoidance derive from the same aversive mLTM, instead of two parallel LTMs of the same valence. The significance of these findings is further discussed below (Zhao, 2021).

Combining with a recent report that uses a similar third-odor test to dissect LTMs induced by multi-trial spaced training, it is concluded that spaced multi-trial aversive differential conditioning produces two independent LTMs of opposite valence for avoiding CS+ and approaching CS-, whereas single-trial aversive differential conditioning yields one mLTM that guides avoidances of both CS+ and CS-. Thus, animals can develop distinct escape strategies for different categories of dangers. When the same dangerous situation has been experienced repeatedly, animals would remember the detailed information to guide behavior in the next similar situation. However, when the dangerous event has only been experienced occasionally, animals would choose to avoid all potentially dangerous cues as a more reserved survival strategy (Zhao, 2021).

Moreover, the differences between single-trial training-induced mLTM and multi-trial training-induced complementary LTMs lead to the question of how these differences are induced by different training sessions. Jacob (2020) reported that multi-trial spaced training induces depressed responses to CS+ in α2sc MBONs and α3 MBONs are required for aversive LTM to CS+, whereas the modulated responses to CS- in β'2mp MBONs and γ3, γ3β'1 MBONs appears to be responsible for the safety memory to CS-. In contrast, this study found that single-trial training is sufficient to induce the depressed responses to both CS+ and CS- in α2sc MBONs. Therefore, the results suggest a lower threshold and specificity of the plasticity between KCs-α2sc MBONs, compared to KCs-α3 MBONs, KCs-β'2mp MBONs, and KCs-γ3, γ3β'1 MBONs connections. Consequently, changing these synaptic connections requires involving more training sessions (Zhao, 2021).

A pair of dopamine neurons mediate chronic stress signals to induce learning deficit in Drosophila melanogaster

Chronic stress could induce severe cognitive impairments. Despite extensive investigations in mammalian models, the underlying mechanisms remain obscure. This study shows that chronic stress could induce dramatic learning and memory deficits in Drosophila melanogaster The chronic stress-induced learning deficit (CSLD) is long lasting and associated with other depression-like behaviors. This study demonstrates that excessive dopaminergic activity provokes susceptibility to CSLD. Remarkably, a pair of PPL1-γ1pedc dopaminergic neurons that project to the mushroom body (MB) γ1pedc compartment play a key role in regulating susceptibility to CSLD so that stress-induced PPL1-γ1pedc hyperactivity facilitates the development of CSLD. Consistently, the mushroom body output neurons (MBON) of the γ1pedc compartment, MBON-γ1pedc>&alpha/β neurons, are important for modulating susceptibility to CSLD. Imaging studies showed that dopaminergic activity is necessary to provoke the development of chronic stress-induced maladaptations in the MB network. The data supports PPL1-γ1pedc mediates chronic stress signals to drive allostatic maladaptations in the MB network that lead to CSLD (Jia, 2021).

Response competition between neurons and antineurons in the mushroom body

The mushroom bodies of Drosophila contain circuitry compatible with race models of perceptual choice. When flies discriminate odor intensity differences, opponent pools of αβ core Kenyon cells (on and off αβ(c) KCs) accumulate evidence for increases or decreases in odor concentration. These sensory neurons and "antineurons" connect to a layer of mushroom body output neurons (MBONs) which bias behavioral intent in opposite ways. All-to-all connectivity between the competing integrators and their MBON partners allows for correct and erroneous decisions; dopaminergic reinforcement sets choice probabilities via reciprocal changes to the efficacies of on and off KC synapses; and pooled inhibition between αβ_c KCs can establish equivalence with the drift-diffusion formalism known to describe behavioral performance. The response competition network gives tangible form to many features envisioned in theoretical models of mammalian decision making, but it differs from these models in one respect: the principal variables-the fill levels of the integrators and the strength of inhibition between them-are represented by graded potentials rather than spikes. In pursuit of similar computational goals, a small brain may thus prioritize the large information capacity of analog signals over the robustness and temporal processing span of pulsatile codes (Vrontou, 2021).

Two-alternative forced-choice tasks, in which a subject must commit to one of two alternatives, sometimes under time pressure and nearly always with uncertain information, are a commonly studied laboratory simplification of real-world decision making. The neural processes that culminate in a binary choice have been compared to the deliberations of a jury before a verdict: neurons, like jurors, gather evidence from witnesses over the course of a trial and then reconcile their divergent views in a majority vote (Vrontou, 2021).

The problem of how neural circuits implement this form of trial by jury has been approached in a range of species, from primates and rodents to fish and flies. A pioneering and influential body of work is built on a two-alternative forced-choice task in which monkeys distinguish directions of motion in a noisy random dot display. Recordings of correlated neuronal activity suggest that motion-sensitive neurons in the middle temporal visual area (MT or V5) provide momentary evidence that is temporally integrated in lateral intraparietal cortex (LIP) before passing an unspecified thresholding mechanism. Although the precise role attributed to LIP is a matter of debate, the principle that ephemeral sensory signals flow into integrators whose fill levels rise to a response threshold appears general; similar arrangements have been inferred to support visual motion discrimination in zebrafish and odor intensity discrimination in the fly (Vrontou, 2021).

In Drosophila, a rate-limiting integration step takes place in a particular group of third-order olfactory neurons. When flies decide on the direction of an odor concentration change, the membrane potentials of Kenyon cells (KCs) in the αβ core (αβ_c) division of the mushroom bodies drift noisily toward action potential threshold, just as accumulating evidence would drift toward a response bound. Consistent with the proposed correspondence of membrane voltage and integrated sensory information, and of action potential and decision thresholds, neurometric functions based on the average timing of the first odor-evoked spikes in the αβ_c KC population can account for the speed and accuracy of the decision-making animal; psychophysical estimates of noise in the decision process match the measured membrane potential noise of αβ_c KCs; and genetically targeted manipulations that alter the latencies of αβ_c KC spikes have the expected impact on reaction times (Vrontou, 2021).

Two functionally separate groups of αβ_c KCs, termed up and down or on and off cells, respond to increases or decreases in odor concentration and can therefore represent the strength of evidence for either of the two alternatives in the choice. This explicit representation of support for each of the competing hypotheses (as opposed to an aggregate representation of the extent to which one hypothesis is favored over the other) suggests that a decision involves a race between two integrators-one built from neurons that accumulate evidence for an increase in odor concentration and another composed of 'antineurons' that do the opposite. Changes in odorant receptor occupancy at the periphery alter the baseline activity of olfactory receptor neurons and the second-order projection neurons (PNs) with which they form receptor-specific glomerular channels. Large odor concentration changes in a channel's preferred direction drive high-frequency transmission from PNs to αβ_c KCs that promotes steep depolarizations to spike threshold and fast, accurate decisions, whereas small concentration changes in the preferred direction, or any change in the null direction, cause only a trickle of synaptic release; shallow, undulating membrane potential rises; and long spike delays that lead to slow, error-prone choices (Vrontou, 2021).

This study examined whether the circuitry downstream of αβ_c KCs is compatible with a model of two competing integrators. Three predictions of such a model were tested. First, to adjudicate the rival hypotheses advocated by on and off αβ_c KCs, mushroom body output neurons (MBONs) sampling the cores of the αβ lobes must listen to both. It is therefore expected that each core-innervating MBON is excited by increases as well as decreases in odor concentration. Second, as an animal learns the rules of the two-alternative forced-choice task-that an increase in odor intensity predicts imminent electric shock, whereas a decrease signals protection-the influence of αβ_c KCs championing the correct choice should be enhanced while that of proponents of the incorrect choice should be diminished. In other words, antagonistic changes are expected in the strengths of connections of on and off αβ_c KCs with the same action selection neurons if evidence for the competing alternatives is accumulated separately. Third, race models become equivalent to a drift-diffusion process-the formalism shown accurately to describe the psychophysics of the decision-making animal-only if they include an element of mutual or pooled inhibition to establish response competition between the integrators. Inhibition is needed to ensure that the integrators are anti-correlated so that evidence for one choice simultaneously counts as evidence against the other. This study therefore predicts the existence of inhibitory interactions between αβ_c KCs (Vrontou, 2021).

The idea that decisions are based on the accumulated spikes of oppositely tuned sensors was born in early attempts to unite psychophysical and neurophysiological measurements under the umbrella of signal detection theory. The recorded spike count distributions of direction-selective units in the monkey's area MT to motion in the preferred or null directions were taken to represent the responses of two neurons-the recorded neuron and its imagined antineuron conjugate-to movement in the neuron's preferred direction. The likely direction of motion can then be inferred as the probability that a draw from the neuron's response distribution yields a larger spike count than a draw from the antineuron's. At minimal motion strengths, when the two distributions are congruent, these odds are even and choices are random, but as the neuron responds ever more vigorously to increasingly coherent motion while the antineuron's response stays flat, the distributions unmix and the probability of a correct choice rises toward one. Comparing the spike counts of two sensors rather than thresholding the output of one removes shared sources of variation and with them the need of adjusting the discrimination threshold to achieve the best separation of the changing response distributions: a neuron-antineuron pair always returns a quantity proportional to likelihood ratio, the optimal hypothesis test. Although opponent sensory channels in one or another guise feature prominently in many decision-making models, their involvement in the brain is unproven: neurons and antineurons owe their status to each other, as inputs to comparator circuits, but these circuits remain uncharacterized (Vrontou, 2021).

This study draws back the curtain on one such circuit in the fly. Changes in odor intensity are registered by pools of on and off αβ_c KCs, which represent the strengths of the accumulated evidence for an increase or decrease in odor concentration. These pools of sensory neurons and antineurons couple to a second layer of neurons and antineurons, the core-innervating MBONs, which bias behavioral intent in opposite ways. Members of both neuronal pools in the sensory layer connect to both types of MBON in the action selection layer via plastic synapses. With two sets of neurons and antineurons and all-to-all feedforward connectivity between them, the comparator circuit allows for approach or avoidance following judgments of upward or downward changes in odor intensity-that is, it comprises neural pathways representing the possible contingencies seen behaviorally. The perceptual decision is won-correctly or incorrectly-by the αβ_c KC pool that reaches spike threshold first, and it is expressed in the behavior instructed by that pool's favored MBON partners (Vrontou, 2021).

Unlike neurons comprising the ON and OFF pathways of motion vision, on and off αβ_c KCs cannot be distinguished and manipulated genetically. This study has therefore exploited the sensitivity of KC-to-MBON synapses to the timing of reinforcement to reveal the convergence of separate on and off channels onto the same MBONs. KC-to-MBON synapses in their ground state exert finely balanced drive on the MBON ensemble, so that votes cast by its members cancel one another as in a hung jury, but experience can shift the synaptic weight distribution and the resulting pattern of MBON activation away from net zero. This study has documented such shifts for the approach-advocating MBON-γ1pedc>αβ: pairing odor on- or offset with electric shock weakens transmission from the on αβ_c KC pool and strengthens transmission from the off αβ_c KC pool (or vice versa), synergistically changing odor preference. The underlying mechanism is a switch from synaptic depression to synaptic potentiation when the order of odor-evoked KC activation and dopaminergic reinforcement is reversed. This mechanism operates at KC connections with all core-innervating MBONs but is likely engaged at different timescales that may reflect sequential memory phases; to demonstrate the mechanism's ubiquity, this temporal sequence was artificially collapsed by photostimulating DANs directly. Within the short time frame of these behavioral experiments, only PPL1-γ1pedc, but not PPL1-α'2α2, shows significant pain responses that modulate its sensitivity to a predictive odor, consistent with the view that PPL1-γ1pedc and its cognate MBON-γ1pedc>αβ represent the core circuit for the storage and expression of short-term aversive memories (Vrontou, 2021).

A crucial element of many neural network models of decision making is inhibitory feedback from a common interneuron pool driven by the competing integrators, which helps to amplify small differences in conflicting sensory evidence until, eventually, one integrator prevails. The response competition circuit this study has delineated contains such an inhibitory element but with the intriguing twist that the key variables are represented by membrane voltages rather than spikes. Analog processing may be a consequence of numerical constraints: if the mushroom bodies lack the neuron numbers needed to approximate continuous quantities with discrete-time action potentials, there may be little choice but to swap the advantages regenerative spikes could provide (such as long time windows for adding and retaining sensory evidence) for the greater information capacity of graded potentials. Perhaps more is different (Vrontou, 2021).

Presynaptic inhibition of dopamine neurons controls optimistic bias

Regulation of reward signaling in the brain is critical for appropriate judgement of the environment and self. In Drosophila, the protocerebral anterior medial (PAM) cluster dopamine neurons mediate reward signals. This study shows that localized inhibitory input to the presynaptic terminals of the PAM neurons titrates olfactory reward memory and controls memory specificity. The inhibitory regulation was mediated by metabotropic gamma-aminobutyric acid (GABA) receptors clustered in presynaptic microdomain of the PAM boutons. Cell type-specific silencing the GABA receptors enhanced memory by augmenting internal reward signals. Strikingly, the disruption of GABA signaling reduced memory specificity to the rewarded odor by changing local odor representations in the presynaptic terminals of the PAM neurons. The inhibitory microcircuit of the dopamine neurons is thus crucial for both reward values and memory specificity. Maladaptive presynaptic regulation causes optimistic cognitive bias (Yamagata, 2021).

Regulation of reward signaling in the brain is critical for maximizing positive outcomes and for avoiding futile costs of the behaviors at the same time. Across animal phyla, dopamine neurons are primarily involved in reward processing. In the fruit fly Drosophila melanogaster, a subset of dopamine neurons in the protocerebral anterior medial (PAM) cluster mediates the reinforcement property of sugar reward. In olfactory learning, dopamine input to the mushroom body (MB) causes changes in preference of a simultaneously presented odor by modulating the output of odor-representing MB intrinsic neurons, Kenyon cells (KCs). Such associative presentations of odor and electric shocks were reported to change the activity of MB-projecting dopamine neurons. Recent studies suggest that axon terminals of the dopamine neurons locally integrate olfactory inputs to function as multiple independent units, though such subcellular reward processing has yet to be examined (Yamagata, 2021).

The results of this study indicate that presynaptic modulation of the PAM neurons is a critical component for determining the magnitude of dopaminergic reward signals. Notably, abolition of the local GABAergic input to the PAM terminals not only enhanced the internal reward intensity but compromised memory specificity. These behavioral alterations can be explained by a dual physiological role of GABA-B-R3, that is, the gain control and the spatial segmentation of dopaminergic reward signals in the PAM terminals. As the behavioral traits caused by the downregulation of GABA-B-R3 are characteristic in optimism, presynaptic control of reward signals may underlie such a cognitive bias. It would be fruitful to examine if a similar subcellular modulation of punishment-mediating neurons conversely leads to the pessimistic bias (Yamagata, 2021).

Dopamine, sleep, and neuronal excitability modulate amyloid-beta-mediated forgetting in Drosophila

Alzheimer disease (AD) is one of the main causes of age-related dementia and neurodegeneration. However, the onset of the disease and the mechanisms causing cognitive defects are not well understood. Aggregation of amyloidogenic peptides is a pathological hallmark of AD and is assumed to be a central component of the molecular disease pathways. Pan-neuronal expression of Aβ42 Arctic peptides in Drosophila melanogaster results in learning and memory defects. Surprisingly, targeted expression to the mushroom bodies, a center for olfactory memories in the fly brain, does not interfere with learning but accelerates forgetting. This study shows that reducing neuronal excitability either by feeding Levetiracetam or silencing of neurons in the involved circuitry ameliorates the phenotype. Furthermore, inhibition of the Rac-regulated forgetting pathway could rescue the Aβ42Arctic-mediated accelerated forgetting phenotype. Similar effects are achieved by increasing sleep, a critical regulator of neuronal homeostasis. Results provide a functional framework connecting forgetting signaling and sleep, which are critical for regulating neuronal excitability and homeostasis and are therefore a promising mechanism to modulate forgetting caused by toxic Aβ peptides (Kaldun, 2021).

An incentive circuit for memory dynamics in the mushroom body of Drosophila melanogaster

Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting, and assimilation of memories. It is proposed that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short term to long term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control (Gkanias, 2022).

Dopaminergic neurons mediate male Drosophila courtship motivational state

Motivational states are important determinants of behavior. In Drosophila melanogaster, courtship behavior is robust and crucial for species continuation. However, the motivation of courtship behavior remains unexplored. This study first found the phenomenon that courtship behavior is modulated by motivational state. A male fly courts another male fly when it first courts a decapitated female fly, however, male-male courtship behavior rarely occurs under normal conditions. Therefore, in this phenomenon, the male fly's courtship motivational state is induced by its exposure to female flies. Blocking dopaminergic neurons synaptic transmission by expressing Tetanus toxin light chain (TNTe) decreases motivational state induced male-male courtship behavior without affecting male-female courtship behavior. Vision cues are another key component in sexually driven Drosophila male-male courtship behavior. This study has identified a base theory that the inner motivational state could eventually decide Drosophila behavior (Wang, 2022).

Differential mechanisms underlie trace and delay conditioning in Drosophila

Two forms of associative learning-delay conditioning and trace conditioning-have been widely investigated in humans and higher-order mammals. In delay conditioning, an unconditioned stimulus (for example, an electric shock) is introduced in the final moments of a conditioned stimulus (for example, a tone), with both ending at the same time. In trace conditioning, a 'trace' interval separates the conditioned stimulus and the unconditioned stimulus. Trace conditioning therefore relies on maintaining a neural representation of the conditioned stimulus after its termination (hence making distraction possible), to learn the conditioned stimulus-unconditioned stimulus contingency; this makes it more cognitively demanding than delay conditioning. By combining virtual-reality behaviour with neurogenetic manipulations and in vivo two-photon brain imaging, this study shows that visual trace conditioning and delay conditioning in Drosophila mobilize R2 and R4m ring neurons in the ellipsoid body. In trace conditioning, calcium transients during the trace interval show increased oscillations and slower declines over repeated training, and both of these effects are sensitive to distractions. Dopaminergic activity accompanies signal persistence in ring neurons, and this is decreased by distractions solely during trace conditioning. Finally, dopamine D1-like and D2-like receptor signalling in ring neurons have different roles in delay and trace conditioning; dopamine D1-like receptor 1 mediates both forms of conditioning, whereas the dopamine D2-like receptor is involved exclusively in sustaining ring neuron activity during the trace interval of trace conditioning (Grover, 2022).

Nutrient responding peptide hormone CCHamide-2 consolidates appetitive memory

CCHamide-2 (CCHa2) is a protostome excitatory peptide ortholog known for various arthropod species. In fruit flies, CCHa2 plays a crucial role in the endocrine system, allowing peripheral tissue to communicate with the central nervous system to ensure proper development and the maintenance of energy homeostasis. Since the formation of odor-sugar associative long-term memory (LTM) depends on the nutrient status in an animal, CCHa2 may play an essential role in linking memory and metabolic systems. This study shows that CCHa2 signals are important for consolidating appetitive memory by acting on the rewarding dopamine neurons. Genetic disruption of CCHa2 using mutant strains abolished appetitive LTM but not short-term memory (STM). A post-learning thermal suppression of CCHa2 expressing cells impaired LTM. In contrast, a post-learning thermal activation of CCHa2 cells stabilized STM induced by non-nutritious sugar into LTM. The receptor of CCHa2, CCHa2-R, was expressed in a subset of dopamine neurons that mediate reward for LTM. In accordance, the receptor expression in these dopamine neurons was required for LTM specifically. It is thus concluded that CCHa2 conveys a sugar nutrient signal to the dopamine neurons for memory consolidation. This finding establishes a direct interplay between brain reward and the putative endocrine system for long-term energy homeostasis (Yamagata, 2022).

Lateral axonal modulation is required for stimulus-specific olfactory conditioning in Drosophila

Effective and stimulus-specific learning is essential for animals' survival. Two major mechanisms are known to aid stimulus specificity of associative learning. One is accurate stimulus-specific representations in neurons. The second is a limited effective temporal window for the reinforcing signals to induce neuromodulation after sensory stimuli. However, these mechanisms are often imperfect in preventing unspecific associations; different sensory stimuli can be represented by overlapping populations of neurons, and more importantly, the reinforcing signals alone can induce neuromodulation even without coincident sensory-evoked neuronal activity. This paper reports a crucial neuromodulatory mechanism that counteracts both limitations and is thereby essential for stimulus specificity of learning. In Drosophila, olfactory signals are sparsely represented by cholinergic Kenyon cells (KCs), which receive dopaminergic reinforcing input. KCs were found to have numerous axo-axonic connections mediated by the muscarinic type-B receptor (mAChR-B). By using functional imaging and optogenetic approaches, it was shown that these axo-axonic connections suppress both odor-evoked calcium responses and dopamine-evoked cAMP signals in neighboring KCs. Strikingly, behavior experiments demonstrate that mAChR-B knockdown in KCs impairs olfactory learning by inducing undesired changes to the valence of an odor that was not associated with the reinforcer. Thus, this local neuromodulation acts in concert with sparse sensory representations and global dopaminergic modulation to achieve effective and accurate memory formation (Manoim, 2022).

Hierarchical architecture of dopaminergic circuits enables second-order conditioning in Drosophila

Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. This study identified a feedforward circuit formed between dopamine subsystems and showed that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. A slow and stable memory compartment can serve as an effective 'teacher' by instructing other faster and transient memory compartments via a single key interneuron, which was identified by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the 'student' compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists (Yamada, 2023).

Optogenetic induction of appetitive and aversive taste memories in Drosophila

Tastes typically evoke innate behavioral responses that can be broadly categorized as acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses also exhibit plasticity through experience-dependent changes in mushroom body circuits. This study developed a novel taste learning paradigm using closed-loop optogenetics. Appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory neurons or dopaminergic neurons encoding reward or punishment. As with olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. This paradigm affords new opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses depend on experience (Jelen, 2023).

Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention

Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, this study reports that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Burgundy (Bur), which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. These data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior (Takakura, 2023).

Animal behaviors are shaped by past experiences, which are stored as memories in the brain. Since the surrounding environment changes occasionally, animals should be equipped with neuronal and molecular mechanisms that actively decay memories for the subsequent behavioral adaptation, while retaining robust and reliable memories by a process known as consolidation via gene expression. The active process of memory decay, defined as "forgetting," is well documented at the molecular level in Drosophila. In the olfactory aversive training paradigm, where an odor is associated with electric shocks, associative memory is formed in the olfactory memory center, mushroom body (MB) neurons, mediated by a coincident activation of dopamine neurons. The olfactory aversive memory rapidly undergoes forgetting within 3 h, which is mediated by a member of the Rho GTPase family of small G proteins, Rac1, and an upstream kinase of mitogen-activated protein kinase, Raf. Although inhibition of the Rac1-forgetting pathway prolongs memory, the memory eventually decays, raising the question of whether memory passively decays over time or whether an additional active process of memory decay exists. Elucidating the neuronal and molecular mechanisms underlying the conflicting functions of memory, consolidation, and decay will help to understand the dynamic and adaptive nature of animal behavior (Takakura, 2023).

The mechanisms of memory formation must converge with those of memory decay. Dopamine and its G-protein-coupled receptor activate adenylate cyclase (AC), leading to an increase in cAMP, which is one of the general rules in learning and memory. At the synaptic level in Drosophila, synapses between MB and the postsynaptic neurons, MB output neurons, undergo synaptic depression. How synaptic plasticity in MBs is linked to the memory decay mechanisms: particularly how synaptic depression could be recovered by forgetting mechanisms or other mechanisms of memory decay, if any, remains unclear (Takakura, 2023).

While gene expression has been primarily associated with memory consolidation, this study demonstrated a causal link between gene expression and synaptic plasticity for memory decay, in which activation is independent of Rac1 or Raf. Since this decay follows the time of Rac1-dependent forgetting and is also mechanistically distinct from it, this memory decay has been termed 'gene expression-based forgetting'. Gene expression-based forgetting is mediated by cGMP (cyclic guanine monophosphate) signaling in MB neurons, which is stimulated by dopamine neurons, resulting in gene expression in MB neurons and presynaptic changes. These results demonstrate another role of gene expression via cGMP signaling from dopamine neurons, apart from cAMP signaling, that could fill in the gap in the understanding between memory formation and decay (Takakura, 2023).

Memory undergoes retention and decay, and their imbalance negatively affects behavioral adaptation. Understanding how intracellular mechanisms orchestrate these seemingly opposite functions of memory and how those mechanisms are linked to synaptic plasticity presents challenges in explaining animal behavior. This study focused on gene expression mediated by an epigenetic regulator, Kdm4B. It was demonstrated that gene expression-based forgetting is controlled by cGMP signaling from dopamine neurons through activation of Kdm4B/Bur. Given that cAMP signaling from dopamine neurons is a major pathway for memory formation, these findings suggest a simple model with two signaling pathways for conflicting memory processes: cAMP signaling for memory formation and cGMP signaling for gene expression-based forgetting, both of which are mediated by dopamine neurons (see Activation of Kdm4B/Bur results in presynaptic changes.). Furthermore, the presynaptic changes through activation of Kdm4B/Bur illustrated the pathway from gene expression to synapses (Takakura, 2023).

Memory formation is mediated by the specific sets of MB synapses which are activated by the odor presentation, when dopamine neurons are simultaneously activated, supporting the synapse specificity. In contrast, gene expression-based forgetting may not be equipped with synaptic specificities. Activation of dopamine neurons without activation of MB neurons was sufficient to induce the interaction of Kdm4B/Bur and kek2 mRNA expression. In addition, the expression of Bur-S139E, which binds to Kdm4B, increased the number and size of presynapses in naive flies. In this case, anterograde memory interference could occur: when two learning events happened closely enough, the latter learning could be impaired by the previous activation of gene expression-based forgetting. However, this may not be the case as the latter learning itself activates gene expression-based forgetting, and preactivation of this pathway may not have any additive effects. In addition, retrograde memory interference may also occur, and preexisting memory may be nonspecifically decayed by new learning. This would not be the case if the preexisting memory is consolidated as consolidated memory via spaced training was resistant to the Bur-S139E expression. Thus, although gene expression-based forgetting may occur synapse-nonspecifically, animals would not have significant unanticipated memory interference (Takakura, 2023).

In Drosophila, rapid decay of memory is an active process mediated by dopamine, Rac1, or Raf, which is defined as memory forgetting. While Rac1 targets ASM within 3 h after learning, Kdm4B/Bur-mediated forgetting effectively targets ASM at a later time point. The Rac1- and Raf-mediated memory forgetting pathways did not affect the Kdm4B/Bur interaction, indicating that activation of Kdm4B/Bur is independent of Rac1. However, memory retention was not synergistically enhanced by the expression of Rac1-DN and the knockdown of Kdm4B or bur. This could be a ceiling effect in memory enhancement. Alternatively, Kdm4B/Bur-mediated forgetting could only target synapses destabilized by Rac1. Rac1 activates cofilin and changes the dynamics of the actin cytoskeleton, which may be required for forgetting via Kdm4B/Bur (Takakura, 2023).

The current study highlighted the differential functions of second messenger signaling pathways, cAMP for memory consolidation and cGMP for gene expression-based forgetting through dopamine neurons. A recent report demonstrated that increased soluble guanylyl cyclase (sGC) expression during aging is related to age-related memory impairment, suggesting that cGMP pathway for gene expression-based forgetting can change according to the physiological state. Although regulation of gene expression by Bur acting as GMP synthetase was unexpected, GMP synthetase has been described in gene regulation and control of stability of nuclear protein. It could be important to investigate the biological meaning of the convergence of GMP synthetase to gene expression-mediated forgetting in the future study (Takakura, 2023).

Transient Administration of Dopaminergic Precursor Causes Inheritable Overfeeding Behavior in Young Drosophila melanogaster Adults

Imbalances in dopaminergic signaling during development have been indicated as part of the underlying neurobiology of several psychiatric illnesses. Yet, how transient manipulation of dopaminergic signaling influences long-lasting behavioral consequences, or if these modifications can induce inheritable traits, it is still not understood. This study used the Drosophila model to test if transient pharmacological activation of the dopaminergic system leads to modulations of feeding and locomotion in adult flies. Transient administration of a dopaminergic precursor, levodopa, at 6 h, 3 days or 5 days post-eclosion, induced overfeeding behavior, while no significant effects on locomotion were found. Moreover, this phenotype was inherited by the offspring of flies treated 6 h or 3 days post-eclosion, but not the offspring of those treated 5 days post-eclosion. These results indicate that transient alterations in dopaminergic signaling can produce behavioral alterations in adults, which can then be carried to descendants. These findings provide novel insights into the conditions in which environmental factors can produce transgenerational eating disorders (Moulin, 2020.

A decision underlies phototaxis in an insect

Like a moth into the flame-phototaxis is an iconic example for innate preferences. Such preferences probably reflect evolutionary adaptations to predictable situations and have traditionally been conceptualized as hard-wired stimulus-response links. Perhaps for that reason, the century-old discovery of flexibility in Drosophila phototaxis has received little attention. This study reports that across several different behavioural tests, light/dark preference tested in walking is dependent on various aspects of flight. If flying ability is temporarily compromised, walking photopreference reverses concomitantly. Neuronal activity in circuits expressing dopamine and octopamine, respectively, plays a differential role in photopreference, suggesting a potential involvement of these biogenic amines in this case of behavioural flexibility. It is concluded that flies monitor their ability to fly, and that flying ability exerts a fundamental effect on action selection in Drosophila. This work suggests that even behaviours which appear simple and hard-wired comprise a value-driven decision-making stage, negotiating the external situation with the animal's internal state, before an action is selected (Gorostiza, 2016).

Interestingly, experiments described by McEwen in 1918 and Benzer in 1967 demonstrated that wing defects affect phototaxis also in walking flies. These early works showed that flies with clipped wings did not display the phototactic response to light, whereas cutting the wings from mutants with deformed wings did not decrease their already low response to light any further. The fact that manipulating an unrelated organ, such as wings, affects phototaxis contradicts the assumed hard-wired organization of this behaviour, suggesting that it may not be a simple matter of stimulus and rigid, innate response, but that it contains at least a certain element of flexibility. This work systematically addressed the factors involved in this behavioural flexibility and begin to explore the neurobiological mechanisms behind it (Gorostiza, 2016).

McEwen's discovery is of interest because of its implications for the supposed rigidity of simple behaviours. The findings of McEwen and Benzer that wing manipulation leads to a decrease in Drosophila phototaxis were reproduced in this study. Slightly altering the conditions of Benzer's countercurrent paradigm (BCP) and comparing performance between two additional experiments, this study found that the decrease in phototaxis is not due to hypoactivity of wing-manipulated flies, but to a more general change in the flies' assessment of their environment. Evidence was discovered that the BCP is just one of several experiments that can measure a fly's general photopreference. Manipulating the wings modulated this preference in all of the selected experiments such that compromised wing utility yielded a decreased preference for brightness (bright stimuli) and an increased preference for darkness (dark stimuli) across the experiments chosen. However, of these experiments, only the BCP can be argued to test phototaxis proper. In Buridan's paradigm, the flies walk between two unreachable black stripes; and in the T-maze, the flies choose between a dark tube and a bright one where the light is coming from an angle perpendicular to their trajectory. Neither of the two paradigms is testing taxis to nor away from a light source. Interestingly, in pilot experiments, this study tested phototaxis in different variations of the T-maze with various LEDs placed at the end of one of two opaque tubes, and only found a reduction of phototaxis and never negative phototaxis. In fact, in these pilot experiments, every possible difference was observed between flying and manipulated flies. In the end, the experimental design was chosen that yielded positive and negative scores, respectively, in wild-type Berlin (WTB) flies purely for practical reasons. Other wild-type strains, such as some Canton S substrains, do not show a negative photopreference in the T-maze after wing clipping. Taken together, these lines of evidence strongly suggest that photopreference in Drosophila is a strain-specific continuum where experimental design assigns more or less arbitrary values along the spectrum. In some special cases, this photopreference manifests itself as phototaxis. If that were the case, phototaxis would constitute an example of a class of experiments not entailing a class of behaviours (Gorostiza, 2016).

This insight entails that manipulations of different aspects of flight ought to affect this continuum in different ways. Complete loss of flight ought to have more severe effects than manipulations affecting merely individual aspects of flight behaviour, such as wing beat amplitude/frequency (i.e., lift/thrust), torque, flight initiation, flight maintenance, proprioception or motion/wind-speed sensation. This study found some evidence to support this expectation. For instance, clipping only the tips of the wings does not eliminate flight, but affects torque as well as lift/thrust. Flies with the tips of their wings cut behave indifferently in the T-maze and do not avoid the bright tube. Flies without antennae are reluctant to fly and have lost their main sense of air speed detection, but they are still able to fly. Also these flies do not become light averse in the T-maze after the manipulation, but indifferent. Only clipping the wings in these flies abolishes their flight capabilities completely and yields negative scores. Flies with removed gyroscopic halteres, on the other hand, are severely affected in their detection of rotations and usually do not fly, despite being able to still beat their wings and control flight direction using vision alone in stationary flight. These flies avoid the bright arm of the T-maze. Finally, injuries to flight-unrelated parts of the fly's body did not affect photopreference ruling out the preference of darkness being a direct escape response due to bodily harm. Further research is required to establish a quantitative link between the many different aspects of flight behaviour and their relation to photopreference (Gorostiza, 2016).

Taken together, these experiments so far demonstrate that: (1) the physical state of the wings with regard to their shape, form or degree of intactness influences photopreference; (2) the capability to not just move the wings, but specifically to move them in a way that would support flight also influences the flies' photopreference; and (3) the state of sensory organs related to flight such as antennae or halteres also exerts such an influence, while non-flight-related sensory deprivation shows no such consequences. This multitude of flight-related aspects extends the concept of flying ability beyond mere wing utility: manipulating seemingly any aspect of the entire sensorimotor complex of flight will affect photopreference, and do so reversibly). As it appears that any aspect of flight, sensory or motor, is acutely linked to photopreference, it is straightforward to subsume all of these aspects under the term 'flying ability', emphasizing that flying ability encompasses several more factors in addition to wing utility. The observation that each fly, when it is freshly eclosed from the pupal case and the wings are not yet expanded, goes through a phase of reduced phototaxis that extends beyond wing expansion until the stage when its wings render it capable of flying lends immediate ethological value to a neuronal mechanism linking flying ability with photopreference (Gorostiza, 2016).

One possible explanation of how the link between flying ability and photopreference may be established mechanistically is via a process reminiscent of learning: at one time point, the flies register a sensory or motor deficit in their flight system and at a later time point they use this experience when making a decision that does not involve flying. Once flying ability is restored, the same choice situation is solved with a different decision again in the absence of flight behaviour. How the flies accomplish this learning task, if indeed learning is involved, is yet unknown, but it is tentatively concluded that it is unlikely that any of the known learning pathways or areas involved in different forms of learning play more than a contributing role. While the molecular learning mechanism remains unidentified, the process appears to be (near) instantaneous. Even though it is not possible to rule out that an unknown learning mechanism exists which is unaccounted for in the screen, it is concluded that at least none of the known learning mechanisms suffices to explain the complete effect size of the shift in photopreference. These results corroborate the findings above, that the switch is instantaneous and does not require thorough training or learning from repeated attempts to fly, let alone flight bouts. They do not rule out smaller contributions due to these known learning processes or an unknown, fast, episodic learning process. It is also possible that the flies constantly monitor their flying ability and hence do not have to remember their flight status. Despite these ambiguities, this study has been able to elucidate some of the underlying neurobiological mechanisms. Much as in other forms of insect learning and valuation, neurons expressing the biogenic amine neuromodulators OA and DA appear to have opposite functions in the modulation of photopreference (Gorostiza, 2016).

Although both DA and OA play some role in different aspects of flight behaviour, these cannot explain the results. In general, the biogenic amine neuron manipulated flies escape their vial via flight if granted the opportunity. Thus, flight is not abolished in any of the transgenic lines affecting OA, TA or DA neurons. However, there may be more subtle deficits in less readily perceived aspects of flight. Experiments performed with mutant flies lacking OA demonstrated that OA is necessary for initiation and maintenance of flight. However, in the paradigm used in this study, silencing OA/TA neurons promoted approaching light, the opposite effect of what would be expected for a flightless fly. Activating these OA/TA neurons, however, rendered the flies indifferent in the T-maze. OA/TA appear to be involved in flight initiation and maintenance via opponent processes. Transient activation of OA/TA neurons may lead to a subtle alteration of flight performance and reduce photopreference in these flies. Similarly, it has been shown that altering the development of specific DA neurons results in flight deficits (reduction of flight time or loss of flight, depending on the treatment. the manipulations lasted for approximately 30 min during adulthood, ruling out such developmental defects. Work in the laboratory of Gaiti Hasan has also found that silencing of three identified TH-positive interneurons for several days in the adult animal compromises flight to some extent (wing coordination defects during flight initiation and cessation) (Sadaf, 2015). The much shorter manipulation of the current study does not lead to any readily observable flight defect. However, one need not discuss whether or not the aminergic manipulations may have had subtle effects on some aspects of flight behaviour, as these flies can be compared to the wing-clipped siblings with which they were tested simultaneously (i.e., the flies with the maximum shift in photopreference due to completely abolished flight). Comparing the intact DA-inactivated flies and OA/TA-activated flies with their respective wingless siblings (reveals that the choice indices of the pairs of groups become essentially indistinguishable at the restrictive temperature. In other words, intact flies where DA neurons have been inactivated or OA/TA neurons have been activated behave as if their wings had been clipped and their flight capabilities abolished completely, despite them being capable of at least some aspects of flight. Hence, even if there were some contribution of some aspect of flight behaviour being subtly affected by manipulating these aminergic neurons, there is a contribution of activity in these neurons that goes beyond these hypothetical flight deficits. Therefore, it is concluded that neither the OA/TA nor the DA effects can be explained only by subtle defects in one or the other aspect of flight behaviour in the manipulated flies (Gorostiza, 2016).

The precise neurobiological consequences of manipulating OA/TA and DA neurons, respectively, are less certain, however. The two driver lines (th-GAL4 and tdc2-GAL4) only imperfectly mimicking the expression patterns of the genes from which they were derived. The effectors, moreover, only manipulated the activity of the labelled neurons. One manipulation (shiTS) prevents vesicle recycling and probably affects different vesicle pools differentially, depending on their respective release probabilities and recycling rates. The other effector (TrpA1) depolarizes neurons. It is commonly not known if the labelled neurons may not be co-releasing several different transmitters and/or modulators in the case of supra-threshold depolarization. Hence, without further research, the involvement can be stated only of the labelled neurons, which as populations are likely to be distinct mainly by containing either DA or OA/TA, respectively. Whether it is indeed the release of these biogenic amines or rather the (co-)release of yet unknown factors in these neuronal populations remains to be discovered. Further research will also elucidate the exact relationship between the activities of these two neuronal populations and whether/how it shifts after manipulations of flying ability (Gorostiza, 2016).

In conclusion, the current findings provide further evidence that even innate preferences, such as those expressed in classic phototaxis experiments, are not completely hard-wired, but depend on the animal's state and presumably other factors, much like in the more complex behaviours previously studied. This endows the animal with the possibility to decide, for example, when it is better to move towards the light or hide in the shadows. Moreover, the fact that flies adapt their photopreference in accordance with their flying ability raises the tantalizing possibility that flies may have the cognitive tools required to evaluate the capability to perform an action and to let that evaluation impact other actions - an observation reminiscent of meta-cognition (Gorostiza, 2016).

Discrete serotonin systems mediate memory enhancement and escape latencies after unpredicted aversive experience in Drosophila place memory

Feedback mechanisms in operant learning are critical for animals to increase reward or reduce punishment. However, not all conditions have a behavior that can readily resolve an event. Animals must then try out different behaviors to better their situation through outcome learning. Learned helplessness, as a type of outcome learning, manifests in part as increases in escape latency in the face of repeated unpredicted shocks. Little is known about the mechanisms of outcome learning. When fruit flies are exposed to unpredicted high temperatures in a place learning paradigm, flies both increase escape latencies and have a higher memory when given control of a place/temperature contingency. This study describes discrete serotonin neuronal circuits that mediate aversive reinforcement, escape latencies, and memory levels after place learning in the presence and absence of unexpected aversive events. The results show that two features of learned helplessness depend on the same modulatory system as aversive reinforcement. Moreover, changes in aversive reinforcement and escape latency depend on local neural circuit modulation, while memory enhancement requires larger modulation of multiple behavioral control circuits (Sitaraman, 2017).

Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. This study surveyed the spectrum of G protein coupling of dDA1 and Damb, and it was confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca(2+) signaling with greater efficiency and dopamine sensitivity. The knockdown of Galphaq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. These findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting (Himmelreich, 2017).

This study provides biochemical and behavioral evidence that the Drosophila DA receptor Damb couples preferentially to Gαq to mediate signaling by Damb for active forgetting. This conclusion offers an interesting contrast to the role of the dDA1 receptor in MBns for acquisition, and it resolves the issue of how MBns distinguish DA-mediated instructions to acquire memory versus those to forget. Prior studies had classified both dDA1 and Damb as cAMP-stimulating receptors, similar to mammalian D1/D5 DA receptors that work through Gαs/olf. The results extend prior studies of dDA1 by examining coupling of this receptor with multiple heterotrimeric G proteins to show that the receptor strongly and preferentially couples to Gs proteins. This affirms the receptor's role in the acquisition of memory, consistent with the tight link between acquisition and cAMP signaling. This study found that the Damb receptor can weakly couple to Gs proteins but preferentially engages Gq to trigger the Ca2+-signaling pathway, a feature not displayed by dDA1. Comparing the two Gαq paralogs of Drosophila (G and D) with a human ortholog shows that Drosophila GαqG and human Gαq share a conserved C terminus, essential for selective coupling to GPCRs, but quite distinct in sequence compared to the GαqD C terminus. Since GαqD is a photoreceptor-selective G protein that couples with rhodopsin, it is proposed that GαqG is the isoform that relays Damb's signals to spur forgetting (Himmelreich, 2017).

It is envisioned that memory acquisition triggered by strong DA release from electric shock pulses used for aversive conditioning drives both cAMP and Ca2+ signaling through dDA1 and Damb receptors in the MBns. Forgetting occurs from weaker DA release after the acquisition through restricted Damb/Gαq/Ca2+ signaling in the MBns. The coupling of Damb to Gs at high DA concentrations also explains why Damb mutants have a slight acquisition defect after training with a large number of shocks. Although the model allows the assignment of acquisition and forgetting to two distinct intracellular signaling pathways, it does not preclude the possibility that other differences in signaling distinguish acquisition from forgetting. These include the possible use of different presynaptic signals, such as a co-neurotransmitter released only during acquisition or forgetting (Himmelreich, 2017).

Cyclic AMP-dependent plasticity underlies rapid changes in odor coding associated with reward learning

Learning and memory rely on dopamine and downstream cAMP-dependent plasticity across diverse organisms. Despite the central role of cAMP signaling, it is not known how cAMP-dependent plasticity drives coherent changes in neuronal physiology that encode the memory trace, or engram. In Drosophila, the mushroom body (MB) is critically involved in olfactory classical conditioning, and cAMP signaling molecules are necessary and sufficient for normal memory in intrinsic MB neurons. To evaluate the role of cAMP-dependent plasticity in learning, this study examined how cAMP manipulations and olfactory classical conditioning modulate olfactory responses in the MB with in vivo imaging. Elevating cAMP pharmacologically or optogenetically produced plasticity in MB neurons, altering their responses to odorants. Odor-evoked Ca(2+) responses showed net facilitation across anatomical regions. At the single-cell level, neurons exhibited heterogeneous responses to cAMP elevation, suggesting that cAMP drives plasticity to discrete subsets of MB neurons. Olfactory appetitive conditioning enhanced MB odor responses, mimicking the cAMP-dependent plasticity in directionality and magnitude. Elevating cAMP to equivalent levels as appetitive conditioning also produced plasticity, suggesting that the cAMP generated during conditioning affects odor-evoked responses in the MB. Finally, this plasticity was found to be dependent on the Rutabaga type I adenylyl cyclase, linking cAMP-dependent plasticity to behavioral modification. Overall, these data demonstrate that learning produces robust cAMP-dependent plasticity in intrinsic MB neurons, which is biased toward naturalistic reward learning. This suggests that cAMP signaling may serve to modulate intrinsic MB responses toward salient stimuli (Louis, 2018).

Learning generates plasticity in neuronal responses to input stimuli, which is distributed across multiple cells and synapses in the brain. Molecularly, dopamine and downstream cAMP signaling are involved in multiple forms of memory, including olfactory learning. For instance, dopamine is required in the amygdala for olfactory classical conditioning in mammals. Similarly, dopamine and downstream cAMP signaling molecules play a central role in olfactory classical conditioning in Drosophila. This pathway is particularly critical in the mushroom body (MB), a brain region that receives olfactory information and is required for olfactory learning. Dopaminergic neurons are postulated to convey a reinforcement signal to the MB-stimulating certain subsets of MB-innervating dopaminergic neurons drives aversive or appetitive reinforcement in lieu of a physical reinforcer. The dopamine released from these neurons acts directly on intrinsic MB neurons, and possibly other neurons in the area as well. The D1-like receptor DopR, type I adenylyl cyclase Rutabaga (Rut), catalytic domain of protein kinase A, and Dunce phosphodiesterase (Dnc) are all required for olfactory classical conditioning. Importantly, rescuing the expression of DopR or Rut -- specifically in intrinsic MB neurons of otherwise mutant animals -- restores normal olfactory learning and memory. Further downstream, both Epac and PKA, as well as phosphorylation targets such as synapsin, have been shown to regulate learning and memory via effects in MB neurons. Thus, dopamine and cAMP are critical in intrinsic MB neurons for normal memory. Furthermore, broadly elevating cAMP generates plasticity in MB neurons, demonstrating that this pathway influences the responsivity of MB neurons. However, the role of this pathway in driving coherent patterns of plasticity that encode memory is unknown (Louis, 2018).

Recent advances have opened up the possibility of understanding how olfactory memory is encoded in exquisite detail. Recent studies of memory encoding in the Drosophila MB have suggested that mushroom body neurons are highly plastic, exhibiting learning-related changes in odor responses. This is supported by observations of memory traces using in vivo Ca2+ imaging of neurons innervating the MB. However, the neuronal changes associated with cAMP-dependent, short-term memory are unclear. Conditioning generates plasticity in α'/β'-neurons within a few minutes of training, a time point at which the animals exhibit robust short-term memory. However, the Rut cyclase is not required in α'/β'-neurons for learning, leaving the functional role of cAMP-dependent plasticity in the MB unclear. MB γ-neurons exhibit depression in response to an aversive conditioned odor that is sensitive to manipulations of G αo-signaling, though it is not clear how this relates to dopaminergic modulation via G αs. Finally, blocking the synaptic output of MB neurons during conditioning does not impair aversive learning, suggesting that a significant proportion of the engram resides in the MB neurons and/or upstream connections (Louis, 2018).

In contrast, other studies have described a major role for plasticity in downstream MB output neurons (MBONs), which may arise via pre- and/or postsynaptic plasticity. Robust, dopamine-dependent plasticity has been observed in MBONs, but not at the cellular level in MB neurons. This emphasizes the role of the MB in encoding sparse, relatively invariant olfactory representations. Learning-induced plasticity is then layered in at the MB-MBON synapses, possibly via synaptic depression. This leaves the requirement of cAMP signaling molecules in the MB, and the dispensability of MB output during memory acquisition, unresolved. Thus, there is a paradoxical dissociation of anatomical loci between where cAMP signaling is required and where robust, short-term, learning-induced plasticity has been reported. This study has examined the role of cAMP-dependent plasticity in the MB using in vivo imaging, combined with pharmacological and optogenetic manipulation of cAMP levels. Results suggest that cAMP-dependent plasticity localizes to intrinsic MB neurons and mirrors the plasticity induced during olfactory classical conditioning, with a bias toward appetitive conditioning (Louis, 2018).

The present data support several major conclusions about the role of cAMP-dependent plasticity in the memory-encoding MB: (1) Intrinsic MB neurons exhibit robust cAMP-dependent plasticity; (2) cAMP-dependent plasticity is heterogeneous, both across and within anatomical classes of MB neurons; (3) the directionality and magnitude of plasticity parallel Rut-dependent associative changes in MB responsivity following appetitive classical conditioning; and (4) appetitive conditioning produces changes in cAMP of a magnitude that generates plasticity in odor-evoked responses. Thus, cAMP-dependent plasticity plays a major role in modulating intrinsic MB neurons, directly linking the physiology of MB neurons to the behavioral roles for cAMP signaling molecules in learning and memory. One caveat to the interpretation of imaging studies is that the preparations require tethering the animal under a microscope. Future developments enabling recording of brain activity in freely behaving animals will be necessary to test how responses in the MB neurons facilitate behavioral output in real time (Louis, 2018).

In the context of olfactory learning, the MB encodes a sparse representation of olfactory space, which is computationally advantageous for learning and potentially modulated by learned valence. If neurons responded homogeneously to input stimuli, coincidence detection would result in uniform plasticity across the sparse set of neurons that encode the odor and receive a reinforcement signal. However, the heterogeneity observed in cAMP-dependent plasticity in this study suggests that olfactory memory traces may be driven to specific subsets of 'eligible' neurons in the MB. This could play an analogous role to memory allocation, which drives memory traces to subsets of eligible neurons in the mammalian amygdala during fear conditioning. Molecularly, heterogeneity may be driven by differential expression of genes that function downstream of cAMP/Epac/PKA to regulate neuronal excitability or presynaptic function. Such differences in expression could be set up via developmental or epigenetic mechanisms (Louis, 2018).

It is proposed that the cAMP-dependent plasticity in the MB plays two roles during olfactory learning: filtering MB responses based on salience and encoding valence. A role for MB plasticity in salience filtering is suggested by the observation that appetitive conditioning produced enhancement of MB responses across spatial compartments. These compartments have been suggested to route olfactory signals to valence-encoding output neurons, driving learned approach or avoidance via heterosynaptic plasticity. Therefore, the broad pattern of plasticity observed in this study would affect multiple downstream output pathways of opposing valence, suggesting that it does not encode valence per se. Rather, it may function to heighten relative MB sensitivity to salient stimuli. Across multiple sensory systems, ascending information is filtered according to salience, typically enhancing responses to stimuli that are biologically important. Alternatively, appetitive conditioning may modulate MB neurons in a fundamentally different way from aversive conditioning. While these opposing forms of memory require many overlapping MB-associated neurons, there are some differences in the circuits recruited during these forms of learning, and plasticity across MB neurons may be one difference. Regardless of the interpretation, the data reveal cAMP-dependent plasticity at the cellular level in intrinsic MB neurons. This may be layered on top of synaptic plasticity at MB output synapses, which have been proposed to encode valence by altering how olfactory signals flow through the neuronal networks that mediate behavioral approach or avoidance (Louis, 2018).

Several additional lines of evidence support the idea that cAMP-dependent plasticity serves as an overall gain control, regulating MB responses based on stimulus salience. First, the MB and MB-innervating dopaminergic neurons modulate salience-based decision making in a visual flight simulator paradigm. Second, dopaminergic neurons innervating the MB respond broadly to sensory stimuli that do not have an acquired valence, and exhibit activity that is correlated with locomotion. Activation of these neurons elevates cAMP in the downstream MB neurons in a compartmentalized manner, which in turn modulates their sensitivity and neurotransmission at the MB-MBON synapses. Thus, the MB neurons receive dynamically regulated dopaminergic inputs that alter the function of both the MB and downstream network components as a function of behavioral state. This may facilitate learning in situations in which the animal is likely to experience biologically important events (e.g., during foraging). Similar modulatory mechanisms modulate plasticity and memory in other animals as well. For instance, in honeybees, appetitive conditioning prolongs odor responses in MB neurons. Likewise, in the mammalian amygdala, coactivation of neuromodulatory and Hebbian plasticity is necessary for plasticity and memory (Louis, 2018).

Aversive conditioning produced no significant plasticity in the current study, consistent with results from some optogenetic reinforcement substitution imaging experiments. However, since Rut is required in MB neurons for normal aversive memory, cAMP-dependent plasticity is likely present in some form. Indeed, a previous study detected plasticity in the γ-lobe following aversive conditioning, which could be tightly localized to specific output synapses or neuronal subsets. Pairing odor with stimulation of tyrosine hydroxylase Gal4-labeled dopaminergic neurons produces aversive memory and detectable plasticity in MB γ-neurons. In this study, robust plasticity differentially following appetitive conditioning. This may be due to a bias toward learning about stimuli that guide motivationally relevant behaviors, such as approaching food-associated odors. Consistent with such an interpretation, appetitive conditioning produces memory that is more stable over time than aversive memory. A single trial of appetitive conditioning leads to the formation of long-term memory, while aversive conditioning requires multiple-spaced trials. The cAMP-dependent plasticity during appetitive conditioning could trigger downstream molecular pathways necessary to engage long-term memory formation. This presumably interacts with Ca2+ levels in neurons to regulate short- and long-term memory. In honeybees, elevating intracellular Ca2+ during a single-trial conditioning, which normally only triggers short-term memory, can induce long-term memory, whereas decreasing intracellular Ca2+ during multiple-spaced training impaired long-term memory formation. In addition, appetitive memory retrieval is motivationally gated by hunger state, suggesting a tie-in with motivational state. Integrating these observations, this suggests that motivationally relevant stimuli may enhance the sensitivity of MB neurons via cAMP-dependent plasticity, modulating the overall gain of the system in a salience-dependent manner (Louis, 2018).

The MB is involved in multiple distinct yet potentially interrelated behaviors, including several forms of learning and memory, regulating sleep and activity, context generalization, habituation, temperature preference, context dependence of olfactory behaviors, and salience-based decision making. The common thread among these behaviors is that they revolve around selection of an appropriate action based on context. Thus, a primary function of the MB and its modulatory input may to be alter the probability of action based on integrating environmental cues and internal state. In such a scenario, modulating the overall gain of the circuit could function in concert with fine-scale synapse-specific plasticity to alter the flow of information to downstream motor areas. Thus, these data support a model in which dopaminergic neurons and downstream cAMP-dependent plasticity modulate MB responses to stimuli based on their salience, priming the animal to engage in appropriate goal-oriented behaviors (Louis, 2018).

The neural circuit linking mushroom body parallel circuits induces memory consolidation in Drosophila

Memory consolidation is augmented by repeated learning following rest intervals, which is known as the spacing effect. Although the spacing effect has been associated with cumulative cellular responses in the neurons engaged in memory, this study reports the neural circuit-based mechanism for generating the spacing effect in the memory-related mushroom body (MB) parallel circuits in Drosophila. To investigate the neurons activated during the training, expression was monitored of phosphorylation of mitogen-activated protein kinase (MAPK), ERK [phosphorylation of extracellular signal-related kinase (pERK)]. In an olfactory spaced training paradigm, pERK expression in one of the parallel circuits, consisting of gammam neurons, was progressively inhibited via dopamine. This inhibition resulted in reduced pERK expression in a postsynaptic GABAergic neuron that, in turn, led to an increase in pERK expression in a dopaminergic neuron specifically in the later session during spaced training, suggesting that disinhibition of the dopaminergic neuron occurs during spaced training. The dopaminergic neuron was significant for gene expression in the different MB parallel circuits consisting of alpha/betas neurons for memory consolidation. These results suggest that the spacing effect-generating neurons and the neurons engaged in memory reside in the distinct MB parallel circuits and that the spacing effect can be a consequence of evolved neural circuit architecture (Awata, 2019).

Spaced learning, which consists of repeated learning with appropriate rest intervals, facilitates memory consolidation to a greater extent than repeated learning without rest. This augmentation of memory, known as the spacing effect, has been demonstrated in the animal kingdom. The central issue of this type of memory consolidation is how the neural circuit recognizes the temporally distributed same learning experience as spaced learning without recognizing each learning session as a novel experience and induce memory consolidation. Numerous studies have aimed to elucidate the mechanism by which the neurons recognize spaced learning through the cumulative cellular responses, such as the oscillatory activation of PKA and mitogen-activated protein kinase (MAPK). However, animals encounter various sensory stimuli in the natural environment, and it remains unclear how repeated experiences among intermingled stimuli are specifically subjected to memory consolidation. A recent study has identified the neural correlates of novelty and familiarity in the olfactory system of Drosophila, raising another possibility that the spacing effect may be produced by distinguishing the initial novel training experience from subsequent training experiences at the neural circuit level (Awata, 2019).

The spacing effect in Drosophila has been demonstrated using an aversive training paradigm in which an odor [the conditioned stimulus (CS)] is associated with electric shocks (the unconditioned stimulus). When flies are repeatedly subjected to aversive training with rest intervals, LTM formation occurs, depending on de novo gene expression. In contrast, single aversive training or repeated aversive training without rest intervals (massed training) does not induce LTM formation. Olfactory memory in flies is mediated by parallel circuits in the MB, each of which circuit consists of different types of neurons, including ~ 500 α/β surface (α/βs) neurons, 600 γmain (γm) neurons, and others (see The making of the Drosophila mushroom body and The neuronal architecture of the mushroom body provides a logic for associative learning). Given that retrieval of aversive LTM requires α/βs neurons, the spacing effect may target α/βs neurons for LTM formation. Importantly, MB axons are compartmentalized, and each compartment projects to a different single MB output neuron (MBON). Each MBON exhibits projections to different brain areas, some of which are known to innervate dopamine neurons (DANs) and form feedback loops with MB neurons. This layered structure linking the MB parallel circuits may be important for producing the spacing effect (Awata, 2019).

The present study explored the neural mechanisms underlying the spacing effect by focusing on the MB parallel circuits. The findings suggested that the reduced activity of the MB parallel circuit consisting of γm neurons is important for LTM formation, which affects the activity of the downstream MBON-DAN network. The results suggest that the spacing effect does not only solely depend on the cumulative cellular responses, but also relies on the neural circuit-based computation via the MB parallel circuits (Awata, 2019).

This study adopted an olfactory spaced training paradigm in Drosophila to investigate the neural circuit underlying the spacing effect. Advantage of immunohistochemistry by monitoring phosphorylation of MAPK (ERK), which allowed mapping the neurons activated in the normal training paradigm. Although an increase or decrease in pERK expression may result from either the change in the neural activation or of the ERK-signaling pathway, the optogenetic manipulation in this study suggested that the neural activity change in the MB-MBON-DAN network is significant in LTM formation. While previous studies have demonstrated that γm neurons are actively involved in memory formation, the present study suggests that a decrease in γm activation is also required for LTM formation. As a result, a single GABAergic neuron (MBON-γ1pedc) postsynaptic to γm neurons became inactivated, which, in turn, led to activation of a dopamine neuron (PPL1-α'2α2). The findings further revealed that the PPL1-α'2α2 neuron innervates another MB parallel circuit consisting of α/βs neurons to induce gene expression required for LTM. This study suggests the model in which the multistep linear circuit in the MB would be significant to index spaced learning of the environment. This neural circuit may act in concert with the cumulative cellular responses, such as the previously proposed oscillatory kinase activity during spaced learning. Dopamine-dependent synaptic suppression between MB neurons and MBON as previously demonstrated may also affect the MBON-DAN network (Awata, 2019).

PPL1-α'2α2 activation in the latter sessions of spaced training was required for gene expression in LTM formation. PPL1-α'2α2 activation was observed via calcium imaging during single training. However, increases in PPL1-α'2α2 activation during spaced training via MBON-γ1pedc inactivation may be necessary to provide sufficient signaling for inducing gene expression. Backward spaced training significantly increased pERK expression in the PPL1-α'2α2 neuron, although Arc2 mRNA was not induced, suggesting that association of an odor and electric shocks is also required for Arc2 expression. Consistently, although dTRPA1-dependent activation induced pERK in all α/βs neurons, artificial activation of the PPL1-α'2α2 neuron, and α/βs neurons induced Arc2 protein expression in only a few α/βs neurons, which would be the result of bypassing the requirement of the association due to the artificial activation. Thus, the multiple mechanisms for gene expression should be converged during spaced training, which include activation of the PPL1-α'2α2 neuron (spacing effect information), α/βs neurons (odor information), and other dopamine neurons (electric shock information). A previous study demonstrated that the cfos-expressing neurons show pERK expression upon memory retrieval. In contrast, this study never found pERK expression in the Arc2-expressing neurons upon retraining, memory retrieval, or reverse training. Accordingly, it was found that the pERK-expressing α/βs neurons were slightly reduced following spaced training, compared to single training. There are 2 possibilities. First, the neural activity of the Arc2-expressing neurons could be suppressed by spaced training. Given that synaptic depression between MBs and MBONs has been proposed as the neural correlates of memory, the decreased activity of the Arc2-expressing neurons may play an important role in LTM. Second, the Arc2-expressing neurons could undergo down-regulation in the ERK signaling, although the neurons are activated during memory retrieval. These should be examined in the future study to understand the physiological role of gene expression involved in LTM (Awata, 2019).

Previous studies have suggested that olfactory information relies on sparse coding in the parallel circuits of the MB, although the plasticity of these sparse codings has yet to be explored. In the present study, it was demonstrated that spaced learning preferentially targets sparse coding in the MB parallel circuit consisting of γm neurons via dopamine signaling, leading to memory consolidation in another MB parallel circuit consisting of α/βs neurons. Thus, the neurons responsible for generating the spacing effect and the neurons engaged in memory reside in the different MB parallel circuits. This neural circuit-based computation is accomplished by the MBON-DAN network linking these parallel circuits. This may be generalized to other types of sensory input in Drosophila and may provide insight into the neural representations within parallel neural circuits in other animals (Awata, 2019).

A connectome of a learning and memory center in the adult Drosophila brain

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. This study reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's alpha lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. It was found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). Two unanticipated classes of synapses, KC>DAN and DAN>MBON, were identified. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall (Takemura, 2017).

Associative memory helps animals adapt their behaviors to a dynamically changing world. The molecular mechanisms of memory formation are thought to involve persistent changes in the efficiency of synaptic transmission between neurons. In associative learning, persistent changes in synaptic efficacy correlated with memory formation have been found at points of convergence between two neuronal representations: one providing information from sensory inputs about the outside world and a second indicating whether the current environment is punitive or rewarding. Such sites of convergence have been identified for multiple forms of associative learning. However, a comprehensive synaptic level description of connectivity at such a site of convergence is not available for an animal as complex as the fruit fly, Drosophila (Takemura, 2017).

The mushroom body (MB) is the center of associative learning in insects. Sensory information enters the MB via the calyx, where the dendritic claws of Kenyon cells (KCs) receive synaptic inputs from projection neurons of olfactory and other modalities including visual, gustatory and thermal. The parallel axonal fibers of the KCs form the MB-lobes, the output region of the MB. A pattern of sparse activity in the KC population represents the identity of the stimulus. This sparseness is maintained through two mechanisms. First, individual KCs generally only spike when they receive simultaneous inputs from multiple projection neurons. Second, overall KC excitability is regulated by feedback inhibition from a GABAergic neuron, MB-APL, that arborizes throughout the MB. Thus, only a small subset of KCs respond to a given sensory stimulus. Upon this representation of the sensory world, dopaminergic or octopaminergic neurons convey information of punishment or reward and induce memories that associate the sensory stimulus with its valence (Takemura, 2017).

The functional architecture of the MB circuit is best understood in adult Drosophila (see Diagram of the α lobe of the mushroom body). In each MB, the parallel axonal fibers of ~2000 KCs can be divided into 16 compartmental units by the dendrites of 21 types of MB output neurons (MBONs) and the axon terminals of 20 types of dopaminergic neurons (DANs). A large body of behavioral and physiological studies suggests that these anatomical compartments are also parallel units of associative learning (see e.g., Hige, 2015; Lin, 2014). In each compartment, the dendrites of a few MBONs overlap with axon bundles of hundreds of KCs. Punishment and reward activate distinct sets of DANs. DAN input to a compartment has been shown to induce enduring changes in efficacy of KC>MBONs synapses in those specific KCs that were active in that compartment at the time of dopamine release. The valence of the memory appears to be determined by which compartment receives dopamine during training, while the sensory specificity of the memory is determined by which KCs were active during training (Takemura, 2017).

Compartments can have distinct rates of memory acquisition and decay, and the 16 compartments together appear to form a set of parallel memory units whose activities are coordinated through both direct and indirect inter-compartmental connections. The DANs which project to the α1 compartment, the ventral-most compartment of the vertical lobe, play a key role in the formation of appetitive long-term memory of nutritional foods. DANs that project to the other α lobe compartments, α2 and α3, play roles in aversive long-term memory. All three of these compartments receive feedforward inputs from GABAergic and glutamatergic MBONs whose dendrites lie in other MB compartments known to be involved in aversive or appetitive memory. In addition, two types of MB-intrinsic neurons send arbors throughout the MB-lobes: a large GABAergic neuron, MB-APL, which provides negative feedback important for sparse coding, and the MB-DPM neuron, which is involved in memory consolidation and sleep regulation (Takemura, 2017).

Previous EM studies in the MB lobes of cockroaches, locusts, crickets, ants, honey bees and Drosophila identified KCs by their abundance, fasciculating axons and small size. Additionally, large GABA immunoreactive neurons that contact KC axons were identified in the locust pedunculus. While these data provided early insights to guide modeling of the MB circuit, the volumes analyzed were limited and most neuronal processes could not be definitively assigned to specific cell types. This paper reports a dense reconstruction of the three compartments that make up the α lobe of an adult Drosophila male. Because a dense reconstruction was performed, with the goal of determining the morphology and connectivity of all cells in the volume, it can be confidently stated that all cell types with processes in the α lobe have been identified (Takemura, 2017).

Comprehensive knowledge of the connectivity in the α lobe has allowed addressing of several outstanding issues. The first concerns the nature of KC>MBON connectivity. Although each KC passes through all three compartments, it is not known if individual KCs have en passant synapses in each compartment. Thus, it remains an open question whether the sensory representation provided to each compartment and each MBON within a compartment is the same or whether different MBONs within a compartment might sample from non-overlapping sets of KCs, and thus use independent sensory representations for learning. It was also not known which, if any, other cell types are direct postsynaptic targets of KCs (Takemura, 2017).

The second concerns dopamine modulation. What are the locations of dopaminergic synapses and what does this distribution imply about the targets of dopaminergic modulation as well as volume versus local transmission? Cell-type-specific rescue of dopamine receptor mutants suggests that dopamine acts presynaptically in the KCs of KC>MBON synapses. However, postsynaptic mechanisms have also been proposed and a recent study detected expression of dopamine receptors in MBONs, raising the possibility that MBONs might also be direct targets of DAN modulation. Behavioral, imaging and electrophysiological data indicate that dopamine modulation respects the borders between compartments, but it is not known whether these borders have a distinct structure, such as a glial sheet (Takemura, 2017).

The third concerns the two MBON types that send feedforward projections into the α lobe. These MBONs have important roles in associative learning as revealed by behavioral assays and have been postulated to integrate memories of opposing valence and different time scales. However, it is not known which cell types these feedforward MBON projections targets within the MB (Takemura, 2017).

The fourth concerns the two neurons, MB-APL and MB-DPM, which arborize throughout the MB and are thought to regulate MB function globally. What is their local synaptic connectivity within the α lobe and what can this inform about how they perform their roles (Takemura, 2017)?

Finally, the three compartments of the α lobe differ in important aspects, including valence of the memory formed, the time course of memory formation and retrieval, and the numerical complexity of their DAN inputs and MBON outputs. Are there obvious differences in the microcircuits of different compartments (Takemura, 2017)?

In this paper reports the answers to these questions. In addition, the utility of detailed anatomy at the electron microscopic level to provide novel insights is demonstrated: It is shown that nearly all cell types in the α lobe contain more than one morphological class of synaptic vesicle, raising the possibility that these cells utilize multiple neurotransmitters. In addition, two prevalent sets of synaptic motifs - from DANs to MBONs and from KCs to DAN s- are described that were unanticipated despite the extensive anatomical, physiological, behavioral and theoretical studies that have been performed on the insect MB. These novel DAN to MBON connections are characterized using behavioral and physiological assays and find that DAN activation produces a slow depolarization of postsynaptic MBONs and can weaken memory recall (Takemura, 2017).

The connections between the neurons observed in this study are are summarized in a Summary diagram of the connectome reconstruction of the α lobe. In each of the lobe's three compartments, parallel axonal fibers of ~1000 KCs project through the dendrites of a few MBONs and the terminal arbors of a few DANs. The results provide support for several aspects of the generally accepted model for MB circuit function. First, it was found that each KC forms en passant synapses with multiple MBONs down the length of its axon, making it possible for parallel processing across the different compartments of the MB lobes. Secondly, with the assumption that released dopamine diffuses locally, KC>MBON synapses would receive dopaminergic input close to the sites of vesicle release, consistent with the prevailing hypothesis that plasticity occurs at the presynaptic terminals of KCs. However, several circuit motifs were found that were not anticipated by previous work. For example, synaptic connections were found from KCs to DANs, indicating that DANs get axo-axonal inputs within the MB lobes themselves. A recent report provides evidence that these KC>DAN synapses are functional (Cervantes-Sandoval, 2017). An even more unexpected motif was the direct synaptic contacts from DAN to MBON found in every compartment. Functional connectivity experiments confirmed that these connections are monosynaptic, and showed that they give rise to a slow depolarization in the MBON. Moreover, stimulating DANs in freely behaving flies yields effects consistent with a net excitatory DAN>MBON connection. Finally, the synaptic connections are described of two feedforward MBONs, which have been proposed to mediate the interaction of the various parallel memories within the MB lobes, as well as two intrinsic MB neurons, APL and DPM (Takemura, 2017).

This work not only provides definitive evidence for, and quantitative detail about, many previously observed circuit motifs, but also reveals several motifs not anticipated by prior anatomical, behavioral or theoretical studies. These additional circuit motifs provide new insights and raise new questions about the computations carried out by the MB. It is noted that these same novel connections were also found in a parallel study of the larval MB (Eichler, 2017). Not only were the same circuit motifs found in the larval MB and adult α lobe, but also the relative prevalence of these connections was strikingly similar: DAN>MBON synapses were 4.5% the number of KC>MBON synapses in the adult α lobe and 3.4% in the larval MB. KC>DAN synapses were 1.5 times as prevalent as DAN>KC synapses in the adult α lobe, as compared with 1.1 in the larval MB. KCs make 48% of their synapses onto other KCs in the adult α lobe and 45% in the larval upper vertical lobe compartments. It is tempting to speculate that the conservation of the relative abundances of these connections across developmental stages reflects important functional constraints on the circuit (Takemura, 2017).

A large body of work supports the idea that individual KC>MBON synapses are the elemental substrates of associative memory storage in the MB. The dominant hypothesis in the field is that coincidence detection occurs within the presynaptic terminals of the KCs. The Conditioned Stimulus (CS, for example an odor) evokes a spiking response in a sparse subset of KCs, which in turn leads to Ca2+ influx. The Unconditioned Stimulus (US, for example electric shock) activates dopaminergic inputs to the MB lobes, where they likely activate G-protein-coupled dopamine receptors on the KC cell membrane. The coincidence of these two events is thought to be detected by the Ca2+ sensitive, calmodulin-dependent adenylate cyclase rutabaga, which initiates a cAMP signaling cascade that leads to the biochemical changes underlying synaptic plasticity (Takemura, 2017).

The tiling of MBON and DAN projections down the length of the KC axons suggests that each of these compartments serves as an independent module, with the association of reinforcement with sensory input taking place in parallel across several different modules. One important assumption in this model is that each KC sends parallel input to each compartment by making synapses all the way down the length of its axon. Light microscopic imaging established that the axons of individual α/β KCs do indeed run through all three compartments of the α lobe. However, they also revealed that the axonal branching patterns differ between KC classes. For example, the axons of α/βp KCs branch in α2, whereas those of α/βc and α/βs KCs do not, raising the question of how extensive KC outputs are across the different compartments. The dense EM reconstruction established that in fact all α/β KCs form en passant synapses on MBONs in each of the three α lobe compartments (Takemura, 2017).

In many cases, these synapses were found at enlarged boutons that contained the presynaptic machinery. However, output sites were also found on the smooth axons of the α/βc KCs, which lack obvious bouton-like swellings. Only occasional, short (generally <5 μm) segments of KC axons where the axon became thinner than 300 nm in diameter lacked presynaptic sites. Of course, it is not known whether all these synapses are functional. EM analysis showed that within each compartment, every KC passing through a layer of the compartment that was extensively innervated by an MBON made at least one synapse with that MBON. Previous electrophysiological measurements of connectivity in the α2 compartment indicated that only about 30% of KCs connect to MBON-α2sc, suggesting the possibility that the majority of KC>MBON synapses are functionally silent, as they are in cerebellar cortex, where 98% of the parallel fiber-to-Purkinje cell synapses are believed to be silent. However, a more trivial explanation cannot be ruled out: These measurements were made in the presence of cholinergic antagonists that could have partially blocked synaptic events and lead to an underestimate of total connectivity levels (Takemura, 2017).

The EM data revealed that the the number of synapses made by individual KCs was well-described by a Poisson distribution, where each synapse connects with a uniform, independent, and random probability to one of the KCs. Although the predicted distributions strongly depend on the number of connections between two cell types, almost all KC connections to other cells obeyed Poisson statistics. This was true of every KC in the α1 and α3 compartments, where each MBON has compartment-filling dendrites. The α2 compartment is somewhat unusual in that its MBONs innervate only subzones of the compartment. While light microscopy showed that MBON-α2sc primarily innervates the surface and core of the compartment, MBON-α2sp was found to project more to the surface and posterior. The connectome results bore out these observations from the light and electron microscopy, although EM reconstructions also showed that these borders were not sharp, and these MBONs receive less extensive and weaker connections outside these subzones. Nevertheless, within the primary area of innervation, it was again the case that every KC made synapses with all MBONs along its axon. Thus each of the 949 α/β KCs can deliver information to the MBONs in each of the three α lobe compartments (Takemura, 2017).

A strictly feed-forward view of the circuit may miss important processing, however, as earlier studies suggested, and the current results re-emphasize. Firstly, gap junctions between KCs have been reported. This opens up the possibility for lateral propagation of signals across KCs, either biochemical or electrical. For example, in mammalian systems, axo-axonal gap junction coupling can synchronize firing between neurons. Secondly, chemical synapses between KCs have been reported in the MB pedunculus in the locust. The reconstructions show that such KC>KC connections are also present in the lobes, where they are surprisingly prevalent. In fact, the most frequent outputs of the α/βs KCs are other α/βs KCs, assuming the morphologically defined KC>KC connections are functional synapses (Takemura, 2017).

A high percentage (55%) of these putative KC>KC synapses occur in rosette-like structures where multiple KCs also converge on a single dendritic process of an MBON . These are relatively unusual structures, not observed in EM reconstructions of the Drosophila visual system and, indeed, there is no direct evidence that they are functional synapses. At present it is only possible to speculate on their role. As points of heavy convergence, they might allow the effects of synapses from different KCs onto the same dendrite to act synergistically. Activity of a single KC may spread to its neighbors within the rosette, potentially generating a large compound synaptic release event onto the MBON in the middle. Such a signal amplification mechanism may be important to ensure that individual KCs can have a significant impact on MBON membrane potential by recruiting their rosette partners. How the specificity of learning could be maintained in this scenario is, however, unclear. Several basic questions will need to be answered before it is possible to begin to understand the functional significance of these rosettes. For example, can a single KC in the rosette indeed activate its neighbors? And how similar are the response properties of the different KCs that contribute to one rosette (Takemura, 2017)?

In conclusion, the connectivity of the KCs that carry olfactory and other sensory representations supports a model where parallel distributed memory processing occurs in each compartment. However, several circuit motifs that seem designed to spread and possibly amplify signals at the sites of KC output indicate that this circuit is likely more complicated than a simple feed-forward view of the system suggests (Takemura, 2017).

Dopamine-induced plasticity of the KC>MBON synapse is thought to be central to associative learning in this system. The reconstructions showed that dopaminergic neurons make well-defined synaptic contacts within the α lobe, with closely apposed post-synaptic membranes. This contrasts somewhat with dopaminergic innervation in the mammalian system, where there is typically not such close contact with a single clear post-synaptic partner, and volume transmission is the predominant model for dopamine release. It is not known whether the direct and indirect dopaminergic release sites have different functional consequences. Nevertheless, it seems likely that some type of volume transmission happens in the mushroom body. First, ~10 times more KC>MBON synapses than presynaptic sites of dopamine release were found in the α lobe, but previous work showed that learning-induced plasticity depresses MBON responses so strongly that most inputs are likely affected. Second, dopamine would need to diffuse only ~2 μm to reach every KC>MBON synapse within a compartment, but would also be sufficiently short range to prevent significant spill-over of dopamine to neighboring compartments, ensuring that the modularity of plasticity is maintained (Takemura, 2017).

Functional connectivity measurements showed that stimulating the DANs elicits large amplitude calcium signals from MBONs, similar to previous results. Intracellular recordings revealed that this was a surprisingly strong connection, sufficient to elicit spikes in the MBON. The response persisted when both spiking and nicotinic transmission was blocked, to limit the possibility that the DANs act through the KCs, which are cholinergic. Conversely, the response was strongly reduced by adding a dopamine receptor antagonist. Taken together, these results indicate that the response is likely a direct action of dopamine released by the DANs on the MBON, although a more complex mechanism or a role for the transmitter contained in the dense core vesicles observed in the DANs cannot be formally rule out. The depolarization exhibited markedly slow dynamics, peaking >2 s after stimulation offset, and then decaying over tens of seconds. Dopaminergic responses of similar amplitude and time course have been reported in both mammalian systems and in Aplysia, where it is mediated by cAMP-driven changes in a non-selective cation conductance (Takemura, 2017).

It is possible to induce memory formation in this circuit by pairing odor delivery with artificial activation of DANs. Targeting this optogenetic training procedure to DANs that innervate different compartments within the α lobe gives rise to memories with different valence, induction threshold and persistence. In the α1 compartment, a single pairing for 1 min induces an appetitive memory that lasts for 1 day. In contrast, optogenetic training focused on the α3 compartment requires multiple 1 min pairings, repeated at spaced intervals, and induces an aversive memory that lasts for 4 days. Although it seems likely that the different valences reflect the different projection sites of the MBONs for each of these compartments, where the differences in induction threshold and memory persistence might arise is less clear. There is no simple explanation for these differences from the EM-level circuit structure, as the basic wiring motifs were very similar in each compartment. Moreover, any explanation that invokes biochemical differences in KC>MBON synapses would require crisp spatial localization of the signaling pathway machinery that triggers plasticity, as exactly the same KCs participate in memory formation in different compartments. However, the observation that there are DAN>MBON synapses raises the possibility that biochemical differences in the MBONs might contribute to these differences in plasticity induction and maintenance. Indeed, RNAseq data from a set of four different MBONs showed expression of dopamine receptors. An alternative possibility, suggested by the findings in this study, is that the cotransmitter found in the dense core vesicles in the DANs is responsible for these differences. The size of these vesicles differs between DANs innervating the different compartments. Thus, these cells might release distinct co-transmitters, as has been observed in mammalian brain, which could trigger different signaling cascades in either the KCs or the MBONs to differentially modulate the induction and expression of plasticity across compartments (Takemura, 2017).

Models of MB function have generally considered the role for DANs to be confined to relaying signals about punishment or reward to the MB. However, in the mammalian brain, DANs can dynamically change their responses to both US and CS. In this study, it was found that the axonal terminals of the DANs receive many inputs from KCs within the lobes. In other words, both MBONs, DANs and even KCs receive extensive synaptic input from KCs in each compartment. If the current model that plasticity is pre-synaptic proves to be correct, this suggests that the responses of the DANs themselves would be subject to plasticity. If the synaptic depression observed at KC>MBON synapses also acts at KC>DAN connections, odor-evoked DAN responses would be diminished as a result of learning. This would serve as a negative feedback loop, reducing the strength of plasticity on successive training cycles with the same odor. Indeed, a gradually plateauing of the learning curve is a common feature of memory formation in different systems, including olfactory conditioning in Drosophila (Takemura, 2017).

One of the more surprising findings of this study was the observation that there are many direct DAN>MBON synaptic connections. Moreover, the functional connectivity measures indicate that these were relatively strong excitatory inputs. The excitatory sign of the DAN>MBON connection is also consistent with the behavioral effects of DAN activation that was observed. What role these DAN>MBON connections play in overall circuit function is an important question for future work. There are two general possibilities that are felt to be interesting to consider. Dopaminergic modulation has been proposed to play a general role in routing of information through the MB to different downstream neurons. Although changes in KC>MBON strength contribute to this process, the current results suggest that such state changes could also potentially be conveyed to the MBONs directly from the DANs. State-dependent changes in DAN activity have indeed been observed with calcium imaging. The slow synaptic dynamics observed in the DAN>MBON connection in MBON-α1 suggest the possibility that small changes in DAN firing might be capable of producing sustained changes in MBON membrane potential reflecting the current internal state of the animal (Takemura, 2017).

A second possibility, suggested from the framework of reinforcement learning established in vertebrates, is related to motivation and the comparison of expected versus actual reward. In Drosophila, prior work on odor-sugar conditioning in larvae provided evidence that flies form a comparison between the current state of reward and the reward expected from the conditioned cue. This work showed that animals behaviorally express memories only when the expected reward intensity is higher than the currently available reward. This is similar to the results presented in this study; just as the presence of reward diminished memory expression in the larvae, stimulating the DANs suppressed performance of animals trained by the optogenetic conditioning. The need to compare current and expected reward could potentially explain why there is an opponent relationship between the depression of KC>MBON synapses that drives associative learning and the excitatory effects of the DAN>MBON connection. If depression dominates, the association drives behavior, but this can be overridden by sufficient levels of DAN activity. In this respect, it is noteworthy that DANs appear to be able to act directly on the MBON, without participation of the KCs. Overall, this comparison could ensure that learned behavior is motivated not strictly by the expectation of reward, but rather the expected increase in reward, assessed at the moment of testing (Takemura, 2017).

The organization of the MB into a set of compartments arranged in series along the KC axons is well suited for simultaneously storing multiple independent memories of a given sensory stimulus. However, there must be some means by which these modules interact with one another to ensure coordinated, coherent expression of memory. Feedforward connections that link different compartments, first discovered by light microscopic anatomy, have recently been shown to be important for mediating such interactions. In particular, MBON-γ1pedc>α/β is an inhibitory neuron that connects aversive and appetitive learning compartments; it ensures that the circuit can readily toggle between different behavioral outputs (Takemura, 2017).

The EM reconstructions included both MBON-γ1pedc>α/β and MBON-β1>α, two feedforward neurons which project from their respective compartments to widely innervate other parts of the MB. Memories stored in the α lobe compartments are long-term and relatively inflexible, whereas the short-term memories formed in β1 and γ1pedc are readily updated by recent experiences. The feedforward connections are thought to enable the short-term memories in β1 and γ1pedc to temporarily mask expression of the stable memories stored in the α lobe. Indeed training an animal with either a multi-component aversive/appetitive food stimulus, or by simultaneous optogenetic activation of a composite set of DANs covering both appetitive and aversive compartments results in a compound memory that is initially aversive and later transitions to appetitive. The connectome results show that the primary synaptic targets of these feedforward neurons are the MBONs in the downstream compartment. By contrast, relatively few connections onto KCs were observed. Overall, this suggests that the feedforward connections can strongly influence the output from a compartment, but likely have little impact on the sensory information delivered to each compartment from the KCs. This is consistent with observations that MBON-γ1pedc>α/β strongly modulates activity of glutamatergic neurons at the tip of the horizontal lobe, but not their dendritic responses. Targeting these feedforward connections to the MBON may ensure that conflicting memories can form simultaneously in response to a complex sensory input, but with the behavioral manifestation of those memories capable of undergoing a crisp switch (Takemura, 2017).

This study has provided synapse level anatomical information on neuronal circuits involved in learning and memory in Drosophila. The comprehensive nature of this dataset should enable modeling studies not previously possible and suggests many experiments to explore the physiological and behavioral significance of the circuit motifs that were observed. That many of these motifs were not anticipated by over 30 years of extensive anatomical, experimental and theoretical studies on the role of the insect MB argues strongly for the value of electron microscopic connectomic studies (Takemura, 2017).

A dense (complete) reconstruction of neurons and synapses is resource intensive, so it is reasonable to ask if tracing a subset of cells or synapses could have yielded similar results with less effort. This is hard to answer in general, since there are many sparse tracing strategies, and each can be pursued to differing degrees of completeness. It is likely that most sparse tracing strategies would have discovered the new pathways reported in this, as the connections are numerous and connect well known cell types. Conversely, the conclusions that all cell types in this circuit had been identified would have been more difficult to make with confidence and a rare cell type, such as the SIFamide neuron, might have been missed. Perhaps, most importantly, statistical arguments, particularly those that require an accurate assessment of which cells are not connected, such as the absence of network structures such as rings or chains, would have been hard to make from sparse tracing. More generally, the model independent nature of dense tracing helps to discover any 'unknown unknowns', provides the strongest constraints on how neural circuits are constructed, and allows retrospective analysis of network properties not targeted during reconstruction (Takemura, 2017).

Developmental analysis of the dopamine-containing neurons of the Drosophila brain

The Drosophila dopaminergic (DAergic) system consists of a relatively small number of neurons clustered throughout the brain and ventral nerve cord. Previous work shows that clusters of DA neurons innervate different brain compartments, which in part accounts for functional diversity of the DA system. This study analyzed the association between DA neuron clusters and specific brain lineages, developmental and structural units of the Drosophila brain that provide a framework of connections that can be followed throughout development. The hatching larval brain contains six groups of primary DA neurons (born in the embryo), which are assigned to six distinct lineages. All larval DA clusters persist into the adult brain. Some clusters increase in cell number during late larval stages, whereas others do not become DA positive until early pupa. Ablating neuroblasts with hydroxyurea (HU) prior to onset of larval proliferation (generates secondary neurons) confirms that these added DA clusters are primary neurons born in the embryo, rather than secondary neurons. A single cluster that becomes DA positive in the late pupa, PAM1/lineage DALcm1/2, forms part of a secondary lineage that can be ablated by larval HU application. By supplying lineage information for each DA cluster, this analysis promotes further developmental and functional analyses of this important system of neurons (Hartenstein, 2017).

DA neurons of the early and late larval brain were labeled by anti-TH and TH-Gal4 driving UAS-mCD8::GFP. Both markers, used in the same brain specimen, labeled identical clusters of neurons, confirming previous studies thet concluded that the expression of TH and DA 'probably label the same neurons.' Neurons labeled by anti-DA or anti-TH/TH-Gal4-positive neurons can be expected to contain DA, even though that in itself is admittedly no proof that they are dopaminergic, in the sense that they utilize dopamine as a transmitter. That being said, this study will in the following refer to these neurons as 'DA neurons', following the convention established in the recent literature. The pattern was compared of DA clusters in the early larva (L1) and late larva (L3). The DA population comprises two dorsomedial clusters (DM1/2) and two lateral clusters (DL1/2). DM1 and DL2 are split into two smaller units each (DM1a/b and DL2a/b, respectively) (see Lineage identity of larval DA neurons). Since most larval DA neurons appear as differentiated cells already in L1, they constitute embryonically born primary neurons, a supposition confirmed by the analysis of Blanco (2011) who could assign the seven clusters to seven separate neuroblast clones induced in the embryo. One cluster, DM2, is not yet represented at L1; cells of this cluster gradually express TH-Gal4 during the L3 stage. In mid-L3 larvae, DM2 is typically represented by a single TH-Gal4-positive neuron; by late L3 the number has increased to about 6. It is concluded that, even though all DA neurons are born in the embryo, they initiated TH expression at different stages in the larva. As shown below, several groups of primary neurons remain TH-Gal4-negative throughout the larva and initiate expression during metamorphosis (Hartenstein, 2017).

To determine the specific lineage identity of the DA clusters, the trajectory of their primary axon tracts was mapped relative to the secondary tracts in the late larval brain, for which a complete lineage atlas has been established. The underlying assumption is that the axon tracts of secondary lineages fasciculate with, or are at least are very close to, the axon tracts formed by primary neurons. Such a spatial relationship has been shown for many lineages for which both primary and secondary components were labeled together. To visualize the secondary axon tracts (SATs), brains were labeled with anti-Neurotactin (Nrt). It is apparent that most of the GFP-positive axons of DA neurons fasciculate with specific SATs, which was then taken as the criterion to assign a given DA neuron to the corresponding lineage (Hartenstein, 2017). The first objective of this paper was to assign the brain DA neurons to neural stem cell lineages and follow them from their time of appearance to the adult stage. At least six different lineages contain primary neurons that express TH during the larval stage. Primary neurons of four additional lineages initiate TH expression past the onset of metamorphosis. The second goal was to establish whether primary DA neurons visible in the larva are maintained in the adult, or are replaced by secondary neurons. The former is the case, as shown by the continued presence of labeled (antibody or TH-Gal4> UAS-mCD8::GFP) clusters of neurons at corresponding positions at closely spaced pupal stages, and the fact that application of HU during larval stages does not eliminate DA neurons in the adult. Only one group of DA neurons, PAM1, consists of secondary neurons of lineage DALcm1/2, born in the larva, and differentiating during late pupal stages. Interestingly, a recent study (Rohwedder, 2015) in which a different antibody directed against TH was used, discovered four additional primary neurons (pPAM) that express DA, and that, based on their branching pattern (around the medial lobe of the mushroom body) belong to the lineage DALcm1/2 (Hartenstein, 2017).

Projections of the different groups/lineages of DA neurons can be assigned to different compartments of the brain. Notably, PPL1 (CP2/3 lineage) innervates the lobes of the mushroom body and the adjacent IPa compartment in the anterior protocerebrum; PPM3 (CM4 lineage) branches widely in the different compartments of the central complex and adjacent LAL, and PPL2 (BLVa1/2) projects to the posterior SLP. Within a given group, DA neurons differ further in regard to their exact branching pattern. PPL1 neurons fall into five different types innervating different domains within the vertical lobe. PPM3 neurons project to different compartments of the central complex; for example, one subset innervates the upper layers of the fan shaped body (FB) and the ellipsoid body (EB); another one is restricted to the lower FB and noduli (NO). These findings suggest that primary neurons of the CP2/3 lineage have a unique identity, which coincides with numerous previous studies of primary neurons belonging to other lineages (Hartenstein, 2017).

At present, not much information is available about the primary lineages that include the clusters of DA neurons of the larval brain. Primary neurons are produced in the embryo by the proliferatory activity of approximately 100 pairs of neuroblasts. These cells undergo 5-8 rounds of division, producing lineages of 10-16 cells each. Approximately 30% of primary neurons undergo programmed cell death at the embryo-larva transition, reducing the average size per larval primary lineage to about 10. Still, this number exceeds the number of DA neurons in any of the clusters in the larval brain, implying that not all cells of a given lineage express the DA phenotype. Future studies, employing lineage-specific markers, are required to establish these additional details about the DA neuronal genealogy (Hartenstein, 2017).

Location and projection pattern of DA neurons have been mapped for numerous insect species, among them locusts and bees. In all species, DA neurons are distributed in discrete clusters, scattered over much of the brain surface, suggesting that as in Drosophila, these neurons form part of multiple lineages. In some cases, it is evident that lineages producing DA neurons are homologous. For example, the C1/C2 clusters of DA neurons in the bee brain (Schäfer and Rehder, 1989) quite clearly correspond in cell body location and projection (to the lobes of the mushroom body) to PAM1/DALcm1/2. Likewise, C3 of the bee may correspond to a combination of PPL1/CP and PPM3/CM4. C3 emits two separate fiber bundles, one of which projects and ramifies towards the central complex; this could represent the PPM3/CM4 group in fly which projects via the MEF fascicle to the central complex. The second contingent of bee C3 DA neurons projects towards the lobes of the mushroom body in the anterior neuropil; this group may correspond to PPL1/CP in Drosophila. Aside from the above mentioned similarities, one very obvious difference exists in the antennal lobe with regards to in the distribution of DA neurons and their terminal arbors. In Drosophila, only two neurons which start expressing TH during metamorphosis are associated with this compartment, whereas in other insects (like bee), the antennal lobe receives dense projections from DA neurons located adjacent to it. It will be an interesting and rewarding task to establish, lineage by lineage, the similarities and differences in the anatomy of the DA system (as well as other groups of neurons sharing similar neurotransmitter expression patterns) among different insect taxa, and to correlate these patterns with physiological and behavioral parameters (Hartenstein, 2017).

DA neurons play a role in many aspects of insect brain function, which is not surprising since most parts of the brain are innervated by these neurons. Predictably, DA neurons projecting towards the mushroom body will be involved in learning and memory, whereas DA neurons associated with other compartments will have other functions. It is of course possible that intricate, not yet resolved interactions take place between the different DA neurons. One fundamental role of DA neurons appears to be to modulate overall motor activity (endogenous arousal; sleep vs wakefulness), as well as the responsiveness of brain neurons to specific sensory stimuli (Hartenstein, 2017).

Three clusters of DA neurons of the adult brain, PPL1, PPL2, and PAM1, innervate the vertical lobe/spur (also called 'heel'), the calyx, and the medial lobe of the mushroom body (MB), respectively. Experimental studies link each of these clusters to memory formation. PPL1 neurons were identified as part of the circuit required for aversive odor memory, but (in part) also play a role in reward learning. Thus, several PPL1 neurons carry information reflecting energy build up during the formation of long term memory (Musso, 2015). In the larva, the DL1 (=PPL1 in adult), but also other DA groups (DM1, DM2) sense amino acid imbalances and control food intake (Bjordal, 2014). PAM1 neurons, innervating different domains of the medial lobes of the MB, also form part of the pathway that carries both aversive (electric shock) and reward stimuli (e.g., sweet taste; water) towards the MB. Different subsets of PAM1 neurons appear to be involved in the formation of short term and long term memories (Yamagata, 2015). Aggressive behavior is modulated by DA neurons of the PPM3 group whose neurons widely innervate the central complex. Members of the PPM3 cluster of DA neurons also control arousal and ethanol-induced locomotion (Hartenstein, 2017).

An important step forward in understanding of DA neuronal function would be to decipher how DA synapses, as well as different DA receptors, are integrated into the microcircuitry of the brain. Progress towards this has been made in the mammalian prefrontal cortex, where DA axons innervate both projection neurons (glutamatergic, excitatory pyramidal cells) as well as local interneurons (inhibitory GABAergic cells). Pyramidal neurons form direct, reciprocal excitatory connections; in addition, local different types of interneurons form recurrent inhibitory and disinhibitory loops. The electrical activity of this structured network depends on the balance between excitatory and inhibitory elements; for example, elevated excitatory synapse activity, or depressed inhibitory synapse activity, would result in an abnormal spread of activation throughout the prefrontal cortex. Dopamine plays a key role in modulating this balance. The exact pattern of dopamine receptor expression in pyramidal neurons and inhibitory interneurons, and of the distribution of DA neurons within the microcircuitry of the prefrontal cortex, specifies the role of the DA system (Hartenstein, 2017).

The relationship between DA terminals, DA receptors, projection neurons, and local interneurons has not been worked out in the Drosophila brain. Thus, it is not clear for the mushroom body, central complex, or any other compartment innervated by DA neurons what type of neuron is the direct target of the DA input. Attempts to reconstruct neuronal connectivity, in the larval brain, from serial section electron microscopy will hopefully provide answers to these questions. Thus, given their characteristic projection, branching pattern, and possibly ultrastructural characteristics (such as size and shape of transmitter vesicles), it should be possible to identify DA neurons in the serial EM stacks, and reconstruct their exact position within the neuronal circuits of different brain compartments. The association of DA neurons with specific lineages plays an important role within this undertaking. Thus, trajectories of primary lineages, including those containing the DA neurons, have been mapped for the early larval brain. As a result, DA lineages can be identified and their connectivity was abolished with the help of a serial EM stack (Hartenstein, 2017).

Dopamine and serotonin are both required for mate-copying in Drosophila melanogaster

Mate-copying is a form of social learning in which the mate-choice decision of an individual (often a female) is influenced by the mate-choice of conspecifics. Drosophila melanogaster females are known to perform such social learning, and in particular, to mate-copy after a single observation of one conspecific female mating with a male of one phenotype, while the other male phenotype is rejected. This study shows that this form of social learning is dependent on serotonin and dopamine. Using a pharmacological approach, dopamine or serotonin synthesis in adult virgin females with 3-iodotyrosine (3-IY) and DL-para-chlorophenylalanine (PCPA), respectively, and then their mate-copying performance was tested. While control females without drug treatment copied the choice of the demonstrator, drug-treated females with reduced dopamine or serotonin chose randomly. To ensure the specificity of the drugs, the direct precursors of the neurotransmitters, either the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) or the serotonin precursor 5-L-hydroxytryptophan (5-HTP) were given together with the drug, (respectively 3-IY and PCPA) resulting in a full rescue of the mate-copying defects. This indicates that dopamine and serotonin are both required for mate-copying. These results give a first insight into the mechanistic pathway underlying this form of social learning in D. melanogaster (Monier, 2018).

Aminergic signaling controls ovarian dormancy in Drosophila

In response to adverse environmental conditions many organisms from nematodes to mammals deploy a dormancy strategy, causing states of developmental or reproductive arrest that enhance somatic maintenance and survival ability at the expense of growth or reproduction. Dormancy regulation has been studied in C. elegans and in several insects, but how neurosensory mechanisms act to relay environmental cues to the endocrine system in order to induce dormancy remains unclear. This fundamental question was examined in Drosophila by genetically manipulating aminergic neurotransmitter signaling in Drosophila melanogaster. Both serotonin and dopamine were found enhance adult ovarian dormancy, while the downregulation of their respective signaling pathways in endocrine cells or tissues (insulin producing cells, fat body, corpus allatum) reduces dormancy. In contrast, octopamine signaling antagonizes dormancy. These findings enhance understanding of the ability of organisms to cope with unfavorable environments and illuminate some of the relevant signaling pathways (Andreatta, 2018).

A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe

Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species where it functions in cognition, motor control, and sensory processing. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LN)s. However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. This study identified a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABAB receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. These results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL (Suzuki, 2020).

Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species where it functions in cognition, motor control, and sensory processing. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LNs). However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. This study identified a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABAB receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. The results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL (Suzuki, 2020).

This study identified a subset of GABAergic LNs that are responsible for signaling low levels of 5-HT in the AL. The 5-HT7R is well suited for detecting changes in basal concentrations of 5-HT, as it is the highest affinity serotonin receptor in Drosophila. Interestingly, 5-HT7R-expressing LNs in the R70A09 promoter line do not appear to express any other 5-HTRs, thus suggesting they may be dedicated cellular sensors of low 5-HT levels. LNs are an ideal target for modulation as they influence several aspects of olfactory coding in both flies and mice. Serotonin is also a potent modulator of GABAergic transmission in vertebrates , and 5-HT targets local interneurons in the olfactory bulb (OB) as well. LNs expressing 5-HT7R are non-specific in their glomerular innervation patterns and odor responses, suggesting a general role for their modulation. Indeed, it was observed that manipulations of basal levels of serotonin signaling had similar effects across glomeruli encoding pheromones, private odors, and broadly activating public odors. However, it is critical to note 5-HT7R expression likely differs across glomeruli. This study demonstrated that DA1 PNs do not express 5-HT7R, but other PNs likely do. Multiple cell classes in the AL express 5-HT7R and may do so in a glomerulus-specific manner. Thus, it is likely the exact effect of 5-HT differs slightly across glomeruli and is dependent on the unique expression pattern of 5-HT7R across cell types within each glomerulus. Glomerular-specific effects of 5-HT have been reported previously. Importantly, data regarding expression patterns of 5-HT7R come from approaches that label neurons according to promoters for these receptors. Although it was possible to discern which cell classes express specific 5-HTRs, there are no data to verify whether individual LNs can traffic receptors to dendrites innervating specific glomeruli. Future studies employing physiology or novel molecular tools will be vital for determining how 5-HTRs are dispersed across the AL to modulate PNs innervating different glomeruli (Suzuki, 2020).

The best characterized form of inhibition in the AL and OB is presynaptic inhibition that targets ORN terminals and mediates divisive gain control. Divisive gain control is important, as it prevents the saturation of downstream PN and mitral cell responses, allowing olfactory circuits to encode odors over a wider range of concentrations. This form of gain control also allows the postsynaptic cell to utilize its entire dynamic range as the strength of presynaptic input increases. Finally, inhibition targeting ORNs leaves the window of temporal integration in PNs unaltered by inhibitory conductances. Interestingly, the results suggest that low-concentration 5-HT may specifically shift the balance between pre- and post-synaptic inhibition and mediate subtractive gain control. The postsynaptic inhibition arises in part from a monosynaptic connection between 5-HT7R-expressing LNs and PNs. Such direct connections from LNs onto PNs are supported by electron microscopy (EM) reconstructions of the AL in both the adult fly and larvae. The properties of postsynaptic inhibition are distinct from those described above. Postsynaptic inhibition will suppress PN firing from a broader distribution of inputs, rather than just its cognate ORNs. Subtractive gain control reduces the maximum firing rate of a neuron, thus diminishing its influence on perception, regardless of stimulus strength. This may be advantageous for suppressing behavior when sensory stimuli are presented in the wrong context. The utilization of divisive and subtractive gain control within the same circuit is not unique to olfaction, as it has also been described in the attentional regulation of vision (Suzuki, 2020).

An alternative function for the suppression by low-concentration 5-HT may be to provide stability to olfactory processing under modulation. Data from the lobster stomatogastric ganglion (STG) indicate that dopamine has opposing actions on the same neurons, depending on its concentration. This mechanism has a homeostatic effect and stabilizes the motor output. Similarly, this study found that low and high concentrations of serotonin also have opposing effects, which are likely mediated by different receptors on different cells. Bidirectional modulation by the same transmitter has been reported in numerous other systems, suggesting it may be a common feature of the central nervous system across diverse phylogenetic groups. Indeed, as in flies, odor responses in vertebrate mitral cells are also boosted by methysergide and exogenous 5-HT. This suggests that bidirectional modulation by 5-HT may be a universal theme in olfaction (Suzuki, 2020). a

Neurochemical organization of the Drosophila brain visualized by endogenously tagged neurotransmitter receptors

Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. This study describes a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, the expression of 75 neurotransmitter receptors was profiled in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, T2A-GAL4 was converted into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. These resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain (Kondo, 2020).

The comprehensive collection of T2A-GAL4 lines generated in this study enabled performance of a brain-wide expression profiling of endogenous neurotransmitter receptors at high resolution. The T2A-GAL4 knockin system can faithfully recapitulate endogenous gene expression, without disrupting the function of the tagged protein in most cases (Kondo, 2020).

The configuration of T2A-GAL4 knockin system has several key features that together make it advantageous to existing methods and resources. The first is the choice of the insertion site. C-terminal insertion used in this system is less likely to disturb the endogenous expression of the target than N-terminal or internal insertion, as it does not block communication between the promoter and intronic enhancers. Most neurotransmitter receptor genes contain multiple large introns, which are known to contain important cis-regulatory elements. Second, the inserted GAL4 transgene is followed by the endogenous 3' UTR of the target gene in this system. 3' UTR sequences are known to affect gene expression by regulating mRNA stability and subcellular localization. Importantly, recent studies in Drosophila have revealed that many neuronally expressed genes have long 3' UTR sequences that play critical roles in proper expression. Thus, the T2A-GAL4 knockin system recapitulates gene expression not only at the transcriptional level but also at the post-transcriptional level. Last, recombinase sites flanking the T2A-GAL4 cassette allow replacement of T2A-GAL4 with any other reporter genes by recombinase-mediated cassette exchange (RMCE). Unidirectional RMCE in this system offers a more rapid and straightforward way of transgene replacement than other systems. A series of exchange vectors was developed for converting T2A-GAL4 lines into other transcriptional activators, fluorescent fusions, and activity reporters. This versatility of this resource allowed the characterization not only expression patterns but also the subcellular localization of dopamine receptor proteins, as well as to visualize the dynamic regulation of receptor levels and activity (Kondo, 2020).

Recently, a large-scale collection of gene-trap lines based on the MiMIC system and a T2A-GAL4 collection similar to the one described in this study have been developed. Both the resources and these collections represent versatile platforms for endogenous gene tagging. MiMIC insertions are located in coding introns and produce an internal GFP fusion. A previous study showed that ~30% of MiMIC-GFP insertions disrupt protein function and they could also disrupt subcellular localization. Although C-terminal tags are less likely to affect protein folding, they could disturb the function of certain proteins, in which the C terminus is modified or is important for interaction with other proteins. Thus, these collections complement each other in many respects, allowing users to choose reagents optimal for their studies (Kondo, 2020).

Systematic clustering analysis of neuropils identified several brain regions with characteristic expression profiles of neurotransmitter receptors. Among others, neuropils constituting the MB and the central complex were conspicuously different from the rest of the brain. This observation is in line with their unique inter-neuropil connectivity previously revealed by connectome analysis: both the MB and the central complex lack overt connectivity with the peripheral nervous system and are considered higher order integrative centers of the brain. It is speculated that the unique combinations of receptors in these centers provide the basis of complex information processing in multi-modal integration (Kondo, 2020).

At the level of individual cells, combinatorial expression of receptors could define the diverse responses of neurons to inputs from outside. Enormous complexity was observed in the expression profiles of transmitter receptors across the brain. The result of clustering analysis shows that receptors for the same ligand molecule or the same ligand type have similar expression patterns. Certain receptors expressed in the same neuron might function cooperatively by forming heterooligomers to recruit different G proteins or to form ion channels with different property. Specific functions of such receptor complexes will further contribute to the diversity of cellular responses, thereby augmenting the computational capacity of the neuronal circuit (Kondo, 2020).

All dopamine receptors were expressed in the MB despite the fact that they have clearly distinct functions in fly behavior. This study confirmed co-expression of multiple dopamine receptors in individual Kenyon cells by simultaneous visualization of two receptors. This observation corroborates the results of a recent singe-cell transcriptome analysis in the fly brain. It is, however, in stark contrast to the situation in the mammalian striatum, where D1 and D2 receptors are expressed in segregated cell populations and mediate the bidirectional dopamine inputs. In the fly, segmentation was found of the neuronal cell membrane by differential subcellular localization of receptor proteins. Furthermore, various tagging approaches of endogenous receptors revealed unexpected subcellular localization of DopEcR as well as experience-dependent regulation of protein levels and activity. It is thus proposed that differential receptor localization along the cell membrane underlies distinct subcellular responses to dopamine input in different contexts (Kondo, 2020).

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission

Many neurons influence their targets through co-release of neuropeptides and small molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release, as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both STED super-resolution and transmission electron microscopy. Ptp4E also controls presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content (Tao, 2019).

Synaptic complexity is enhanced by co-transmission with small molecules and bioactive peptides. The two transmitter classes differ in their postrelease distances traveled and durations of action, thus providing mechanisms for rapid point-to-point control and slow neuromodulation of circuits, development and behavior. Furthermore, from a cell biology perspective, transmission by small molecules and neuropeptides is distinguished by different vesicular loading mechanisms. The genetic results presented here are remarkable because (a) they reveal increased transmitter packaging, when past genetic screens have only yielded mutants that reduce vesicular packaging; (b) control of vesicular packaging varied between neuron subtypes based on differential Rptp expression, which represents a new mechanism for generating variation in co-transmission in the nervous system. Furthermore, this result is intriguing in the context of monoaminergic neurons because Ptp4E interacts genetically with α-synuclein toxicity in Drosophila. Given that synuclein is implicated in Parkinson's disease, DCV fusion pore dynamics and the early secretory pathway, the results here suggest that the mechanistic relationship between Rptps and synuclein may be broader than previously recognized; and (c) presynaptic abundance of a small-molecule vesicular membrane transporter and luminal neuropeptides are regulated in parallel. This shows that regulation of co-transmission is not limited to control of activity-induced vesicle exocytosis. Instead, an Rptp regulates vesicular packaging of both small-molecule and peptide neurotransmitters that underlies co-release (Tao, 2019).

How can a single Rptp simultaneously modify vesicular loading of both monoamines and neuropeptides? Peptidergic neurotransmission relies on packaging of neuropeptides in the soma, where they condense in the TGN and are sorted into DCVs. There is little DCV circulation in octopamine terminals because of their extensive axonal arbors and numerous boutons. Therefore, Rptp regulation of neuropeptide content of individual DCVs likely originates prior to axonal transport. VMAT is also processed in the TGN to be sorted into DCVs and small synaptic vesicles, rather than proceeding through the constitutive secretory pathway. A recent study found that knockdown of the TGN protein HID-1 reduces DCV luminal cargo and VMAT in DCVs by affecting sorting and DCV production. The coordinated effects on neuropeptides and VMAT are reminiscent of the results presented in this study, but the Ptp4E effect on packaging was not associated with a change in DCV number or transport. Therefore, the uncoupling of DCV number from packaging is indicative of a novel cell biological mechanism. With this in mind, a possible explanation for the effect of inhibiting Ptp4E is that the tyrosine phosphorylation stimulates TGN sorting of luminal and vesicle membrane content without changing DCV number or size. By this mechanism, Rptp regulation of vesicular packaging in the soma could scale co-release at the distal synaptic ending (Tao, 2019).

These results pose the question of the site of Ptp4E function. Rptps often mediate signaling triggered by cell-cell contacts. For example, the presynaptic Rptp Lar is activated by muscle Syndecan during development of the NMJ. By analogy, it is possible that presynaptic Ptp4E governs retrograde signaling (e.g., by interrupting tyrosine kinase-dependent mechanisms). In favor of this hypothesis, the closely related Rptp Ptp10D is found on axons in the embryo, where it is positioned to regulate axonal guidance during development. However, the potential involvement of vesicle biogenesis and the unknown localization of Ptp4E raise the possibility that somatic Ptp4E is responsible for synaptic effects. Novel tools to differentially control of Rptp activity by compartment (i.e. soma versus terminal) will be needed to distinguish between these possibilities. Regardless of the cellular location of Ptp4E signaling, the mechanism discovered here (i.e. coincident control of packaging of neuropeptides and small-molecule transmitters) represents a previously unknown cell biological strategy for regulating synaptic co-transmission (Tao, 2019).

Previous experiments have shown that increased VMAT leads to enlargement of vesicles and greater vesicular monoamine storage, but this effect was not seen in this study. Notably, the mechanistic basis of the VMAT expression effect on vesicle size is not understood because thermodynamics with a simple system suggests that maximal vesicular monoamine concentration should be reached even with one VMAT per vesicle. Therefore, to explain the previously observed effect on vesicle size, some other factor, such as monoamine leakage or membrane flexibility, must come into play. It is suggested that these parameters might differ in the synaptic terminals examined in this study. Alternatively, in contrast to the spherical vesicles studied previously, the DCVs in octopamine neurons are ovoid. Therefore, the current analysis of largest dimension cannot exclude that the narrow axis of these DCVs increased. According to the latter scenario, increases in vesicular volume and membrane surface area could have been undetected with the methodology used in the current study (Tao, 2019).

What would the expected consequences be of upregulating VMAT and neuropeptides in vesicles? Upregulating VMAT will increase the speed of vesicle loading when there is exocytosis-endocytosis cycling or kiss-and-run release. Hence, increased VMAT will affect release more when vesicle emptying by release is most marked. In Drosophila, the effect of increased VMAT on behavior has not been examined. However, the dopamine precursor L-DOPA increases vesicular monoamine content and ameliorates Parkinsonian symptoms in humans. Thus, by analogy, it is suggested that octopaminergic signaling would be boosted by increased vesicular octopamine packaging induced by synaptic VMAT upregulation. Of course, increased co-transmission by neuropeptides could further alter octopamine action. Therefore, it would be interesting to explore how Rptps in the brain affect octopamine-dependent fly behaviors such as feeding and egg laying (Tao, 2019).

Bidirectional regulation of glial potassium buffering: glioprotection versus neuroprotection

Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K(+) and water in Drosophila, however upstream regulatory mechanisms are unknown. This study identified an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctβR to upregulate glial buffering capacity, while under pathological K(+) stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability (Li, 2021).

K+ homeostasis in the nervous system is required to maintain a healthy level of neuronal activity. Neurons release K+ ions as they repolarize during action potentials. If this K+ builds up in the extracellular space, it can disrupt neuronal firing and set the stage for neuronal hyperexcitability. Conventional treatments for conditions of hyperexcitability mostly target neuronal channels. However, more than one-third of epilepsy patients suffer from medically intractable seizures, while others experience debilitating side effects as neuron-targeting treatments often interfere with healthy neuronal functions. Hence, there is an urgent need for innovative therapeutic approaches that improve seizure control. This study explored glial mechanisms that regulate K+ balance to modulate neuronal excitability and seizure sensitivity (Li, 2021).

Glia buffer K+ stress by taking in excess K+ ions and osmotically obliged water molecules from the extracellular space. This function allows glia to control the extracellular level of K+ and thereby modulate neuronal excitability, raising the hope that glia may be targeted to restore K+ homeostasis and suppress hyperexcitability in epilepsy and stroke. Glial-centric therapeutic strategies for these conditions have not been possible, largely due to a lack of understanding of mechanisms controlling the glial K+ buffering capacity. Previous work discovered a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). SIK3 (salt inducible kinase 3) is an AMPK family kinase that sequesters histone deacetylase 4 (HDAC4) in the cytoplasm, thereby promoting myocyte enhancer-factor 2 (Mef2)-dependent expression of K+ and water transport molecules, including the Drosophila orthologs of aquaporin-4 (Drip) and SPAK (Frayed) a kinase necessary for activating the Na+/K+ transporter NKCC1 Ncc69. This glial program suppresses extracellular nerve edema and prevents neuronal hyperexcitability and seizure in Drosophila, suggesting that it plays an important role in glial maintenance of K+ and water homeostasis and is required for a healthy level of neuronal activity. This glial program is best characterized in wrapping glia, which ensheathe axons in the Drosophila peripheral nervous system (PNS) and are akin to vertebrate non-myelinating Schwann cells, and so are well positioned to regulate the ionic composition around PNS axons (Li, 2021).

While this study has elucidated downstream effectors by which SIK3 regulates K+ and water buffering, the upstream signals that work through the SIK3 pathway to control glial buffering capacity are unknown. In other contexts, SIK3 integrates signals from different signal transduction pathways, coupling extracellular signals to changes in cellular responses (Choi, 2015; Wang, 2011; Wein, 2018). One of these pathways is G-protein-coupled receptor (GPCR)-cyclic AMP (cAMP)-protein kinase A (PKA) signaling, in which PKA directly inhibits SIK3 activity (Wang, 2011). A better understanding of upstream signaling mechanisms that regulate the SIK3 pathway and extracellular cues that modulate glial K+ buffering capacity will inform approaches to leverage this glial function for therapeutic benefits in diseases of hyperexcitability (Li, 2021).

This study identified an octopaminergic circuit linking neuronal activity to glial K+ and water buffering. Octopamine regulates the glial buffering program via GPCR-dependent regulation of PKA, which inhibits SIK3. At baseline, octopamine acts through the inhibitory octopaminergic GPCR OctβR to activate the glial SIK3 pathway to promote K+ and water buffering and healthy levels of excitability. Under pathological K+ stress, in contrast, octopamine functions through the stimulatory octopaminergic GPCR OAMB1 to inhibit SIK3 and downregulate the glial capacity to buffer K+ and water. While loss of the SIK3 pathway leads to extracellular edema, constitutive activation of the pathway results in intracellular glial swelling and activation of stress signaling, suggesting that turning down this pathway protects glia. In eag shaker, a classic Drosophila hyperexcitable mutant with defective K+ channels, the SIK3-mediated glial buffering program is turned off, likely as a self-protective mechanism for glia. However, reactivation of this glial K+ and water buffering program dramatically suppresses neuronal hyperexcitability, seizures, and shortened life span in eag shaker. Therefore, the maintenance of a robust glial K+ and water buffering program in response to extreme neuronal excitability is beneficial to the organism despite the risks posed to glial health. Taken together, this study identifies a neuromodulatory circuit that links neuronal activity to glial K+ buffering and highlights the promise of a glial-centric therapeutic strategy that restores K+ and water homeostasis for control of hyperexcitability in diseases such as epilepsy and stroke (LiH, 2021).

K+ dysregulation drives edema and neuronal hyperexcitability and can lead to brain damage in patients with epilepsy and stroke. Glia buffer K+ stress by taking up excess K+ ions and water to maintain a healthy extracellular environment for neuronal excitability. Previous work identified SIK3 as the central node of a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). This study uncovered upstream regulatory mechanisms controlling the SIK3 pathway and demonstrated that activation of this glial program can dramatically ameliorate the pathological consequences of neuronal hyperexcitability. An octopaminergic circuit couples neuronal activity to glia buffering, exerting a dual effect on SIK3-mediated K+ buffering: low levels of octopamine enhance the glial K+ buffering capacity, while high levels of octopamine inhibit this buffering program, likely to protect glia against extreme K+ stress. Glial-specific inhibition of HDAC4, a central repressor of SIK3 signaling, results in a constitutively active glial buffering program that dramatically suppresses seizure and extends life span in a classic epilepsy model. Hence, augmenting glial K+ and water buffering holds promise as a therapeutic approach for the treatment of seizures (Li, 2021).

The glial SIK3/HDAC4/Mef2 pathway regulates expression of both fray/SPAK, a kinase controlling ion transporter activity, and aquaporin 4, a water channel essential for volume regulation (Leiserson, 2011; Li, 2019; Papadopoulos, 2004). The original analysis of SIK3 LOF mutants demonstrated that glial SIK3 inhibits extracellular edema, presumably by promoting the uptake of ions and water into glial cells. This is an essential activity of glia, since accumulation of K+ in the extracellular space around axons leads to hyperexcitability and heightened seizure susceptibility. Since peripheral nerves must continuously fire, this raised the question of why expression of fray and aquaporin 4 would be regulated by SIK3, which is best understood to integrate opposing signals such as feeding and fasting to control the levels of downstream transcriptional targets. If ion and water buffering were so essential, then why is expression of essential buffering proteins not constitutive? The findings of this study suggest that the downregulating glial K+ buffering capacity is a protective response that prevents pathological glial swelling. Activity-dependent glial swelling is a commonly observed physiological phenomenon, but when taking in an excess load of ions and water, glia may swell to the point of cytolysis. This study has shown that glia with enhanced K+ buffering capacity undergo cell swelling that is exacerbated by extreme K+ stress. These glia also exhibit enhanced JNK pathway activity, which responds to a variety of stressors, including mechanical stress caused by cell stretching. JNK plays a critical role in the regulation of apoptosis, suggesting that pathological swelling that results from enhanced K+ buffering could promote cell death. Taken together, these findings demonstrate that overactivity of glial SIK3 activity leads to intracellular edema while underactivity of glial SIK3 signaling leads to extracellular edema. Hence, SIK3 balances the need for robust extracellular buffering with the health of the glial cell. It is suggested that the ability of the SIK3 pathway to integrate disparate upstream signals is important for maintaining this balance between an extracellular milieu conducive to neuronal firing with intracellular volume regulation that maintains glial health (Li, 2021).

Norepinephrine is a stress hormone that can exert anticonvulsant effects in the mammalian nervous system. Moreover, norepinephrine regulates astrocyte transporter activity. In Drosophila, octopamine is structurally and functionally analogous to norepinephrine and regulates a variety of stress responses, including aggression, alertness, and starvation. Octopamine is also similar to norepinephrine in that it exerts dual effects on cellular responses by signaling through receptors with antagonistic functions. In this study, it is proposed that octopamine bidirectionally controls SIK3-mediated glial K+ buffering in response to changes in neuronal excitability (Li, 2021).

Like its mammalian counterpart, octopamine is normally maintained at low levels and is released upon physiological stimuli to help the system mount a stress response. Octopamine release can be enhanced by a surge in the level of extracellular K+, suggesting that its concentration correlates with the level of K+ stress in the nervous system. This study proposes a model in which octopamine release links activity-dependent K+ stress to glial K+ buffering. This model is supported by a number of key findings. First, octopamine is required for SIK3 signaling and this regulation occurs through the inhibitory octopaminergic GPCR Octβ1R. Second, elevated levels of either octopamine or K+ each inhibit the glial SIK3 pathway, and both of these effects require the stimulatory octopaminergic GPCR OAMB. Hence, elevated K+ works through octopaminergic signaling to regulate the SIK3 pathway and glial buffering. Finally, the glial SIK3 pathway is inhibited in the hyperexcitable eag,Sh mutant. It is proposed that basal levels of octopamine promote SIK3-dependent glial K+ buffering to cope with physiological K+ stress and maintain a healthy level of neuronal activity. When neurons become hyperexcitable, as would occur under pathological conditions, levels of extracellular K+ rise and more octopamine is released. High levels of octopamine will inhibit SIK3 activity and turn down glial K+ buffering, likely to protect glia from pathological cell swelling (Li, 2021).

This model posits that different levels of octopamine differentially activate Oct&beta1R and OAMB to exert these dual effects on glial K+ buffering. Octβ1R couples to the inhibitory Gαi protein, whereas OAMB can function through the excitatory Gαs protein . How might octopamine differentially signal through both Octβ1R and OAMB when they bind to the same ligand, reside in the same cell, and have antagonistic functions? Potentially, Octβ1R and OAMB could have different binding affinities for octopamine, with Octβ1R having a higher affinity such that Octβ1R is preferentially activated at low levels of octopamine. Inhibitory Gαi-coupled Octβ1R would decrease cAMP levels and inhibit PKA activity, thereby relieving the inhibition on SIK3 to upregulate glial K+ buffering capacity. Conversely, octopamine would bind to the lower affinity OAMB when octopamine levels are high, as would occur during hyperexcitability. High levels of octopamine signaling through the excitatory Gαs-coupled OAMB would enhance PKA activity and downregulate the glial capacity to buffer K+ stress. Hence, different binding affinities would allow Octβ1R and OAMB to be activated by different octopamine concentrations. A similar differential regulation via opposing octopaminergic receptors residing in the same cell is described at the larval NMJ, where octopamine signals through inhibitory Octβ1R and excitatory Octβ2R to bidirectionally control synaptic function (Li, 2021).

This study has defined an octopaminergic circuit that links neuronal activity to glial K+ buffering to maintain K+ homeostasis and neuronal excitability. Neuromodulators such as octopamine are useful for integrating synaptic activity over time and space rather than signaling rapidly and locally like classical neurotransmitters. Moreover, altering the glial transcriptional response will be reasonably slow to change physiological function since such changes require transcription and translation. Hence, this system is likely tuned to respond to average neuronal activity rather than to transient changes in neuronal firing. This pathway may work in concert with additional mechanisms for rapid tuning of glial ion transporter function as described in mammalian astrocytes (Li, 2021).

This model for octopaminergic regulation of the glial SIK3-dependent ionic buffering program leaves open a number of important questions. First, is the octopamine released locally or does release from the central octopaminergic neurons diffuse widely to set the glial tone for ionic buffering? Second, while this study showed that a glial octopamine receptor is necessary for pathway modulation by octopamine, it would also be interesting to test whether direct activation of octopaminergic neurons is sufficient to regulate the glial SIK3 pathway. Similarly, is direct inhibition of octopaminergic neurons capable of rescuing the whole animal phenotypes of the eag, Sh mutants? Third, while this study has focused on octopaminergic regulation, in other systems the SIK3 pathway is regulated by additional upstream signals including the kinase Lkb1 and the insulin receptor. Input from these additional pathways would allow for regulation of glial ion and water buffering in locations that may not be accessible for octopaminergic neuromodulation. Finally, this analysis focused on peripheral nerves and peripheral glia. In future studies it will be important to assess the role of this SIK3 buffering program in the cortex glia that buffer ions around neuronal cell bodies (Li, 2021).

With hyperexcitability, waves of action potentials can lead to increases in extracellular K+ that, if not properly buffered, will lead to neuronal depolarization and further hyperexcitability. This study has explored the role of glial buffering in a paradigmatic Drosophila seizure mutant, eag,Sh, that exhibits dramatic neuronal hyperexcitability, seizure behaviors, and shortened life span. Although it would be expected that eag,Sh larvae have an increased need for glial buffering, this study found that the glial SIK3 signaling pathway is downregulated, likely as part of the glial protective response described above. Since downregulating the glial SIK3 pathway is sufficient to cause hyperexcitability and seizures, whether this inhibition of SIK3 could be exacerbating the eag,Sh phenotypes was tested. HDAC4 is the key repressor of SIK3-regulated glial K+ buffering, and so SIK3 signaling was activated by glial knockdown of HDAC4. Reactivation of the glial buffering program dramatically suppressed neuronal hyperexcitability, seizures, and shortened life span in eag,Sh. This result is quite remarkable, since the neurons are still deficient for two extremely important K+ channels. Nonetheless, the pathological consequences for the organism are almost wholly mitigated by enhanced glial buffering. This highlights the potential of glial-centric therapeutic strategies for diseases of hyperexcitability (Li, 2021).

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade

Drosophila has been used as a model system to study mechanisms of sleep regulation. The first studies on sleep in Drosophila revealed that they periodically enter a quiescence state that meets a set of criteria for sleep. Drosophila sleep is monitored normally by a Drosophila activity monitoring system (DAMS) and is defined as immobility for 5 min or longer which is a sleep bout. Drosophila sleep mainly happens at night, while a period of siesta is in the mid-day. For example, total sleep time is around 380 min (male) and 250 min (female) during the day time, and 480 min (male) and 490 min (female) during the night time in w1118 (Zhao, 2021).

In Drosophila, central complex structures, especially the ellipsoid body (EB) and fan-shaped body (FSB), are important for sleep homeostasis regulation. Activation of dorsal FSB neurons is sufficient to induce sleep. The dorsal FSB also integrates some sleep inhibiting signals. Both dorsal FSB and EB ring 2 are important in sleep homeostasis. Recently, the helicon cells were found to connect the dorsal FSB and EB Ring 2, indicating that these EB and FSB are connected (Zhao, 2021).

Multiple studies indicate that the epigenetic mechanisms are involved in circadian regulation. However, a direct link between epigenetic regulation and sleep homeostasis is not yet established (Zhao, 2021).

Octopamine (OA) in Drosophila is a counterpart of vertebrate noradrenaline. Previous studies in Drosophila showed that OA is a wake-promoting neurotransmitter and plays an important role in regulating both sleep amount and sleep homeostasis. The mutants of the OA synthesis pathway show an increased total sleep. Activation of OA signaling inhibits sleep homeostasis, while in OA synthesis pathway mutants, an enhanced sleep homeostasis is observed. Study of the neural circuit responsible for the sleep/wake effect of OA showed that octopaminergic ASM neuronsproject to the pars intercerebralis (PI), where OAMB (one of the OA receptors)-expressing insulin-like peptide (ILP)-secreting neurons act as downstream mediators of OA signaling. However, the effects of manipulating ASM neurons or ILP-secreting neurons are much weaker than those observed by manipulating all OA secreting neurons. Moreover, the effect of octopamine is not completely suppressed in the OAMB286 mutant, arguing that another receptor or circuit may participate in this process (Zhao, 2021).

Eight OA receptors are identified to date: OAMB, Octβ1R, Octβ2R, Octβ3R, TAR1, TAR2, TAR3, and Octα2R. Although the expression pattern of OA is identified, the endogenous expression profile of these receptors is lacking. A previous study demonstrated that the mushroom body-expressed OAMB mediates the sleep:wake effect of OA. Recently, Octβ2R was shown to be important for the OA effect on endurance exercise adaptation. How the versatility of OA function is mediated by the diverse array of its receptors needs further study. Moreover, the upstream regulatory mechanisms of OA receptors are still unknown (Zhao, 2021).

A previous study showed that Stuxnet (Stx) is important in mediating Polycomb (Pc) protein degradation in the proteasome (Du, 2016). Stx, which is an ubiquitin like protein, mediates Polycomb (Pc) protein degradation through binding to the proteasome with a UBL domain at its N terminus and to Polycomb through a Pc-binding domain. stx level changes result in a series of homeotic transformation phenotypes. Pc is an epigenetic regulator functioning in Polycomb Group (PcG) Complexes. Although it is reported that PcG component E(Z) is involved in circadian regulation, the role of stx in adult physiological process is unknown (Zhao, 2021).

This study identified the role of the epigenetic regulator stx in sleep regulation. stx positively regulates Octβ2R through regulation of Polycomb in the EB of the adult fly brain. Further study demonstrated that the Stuxnet-Polycomb-Octβ2R cascade plays an important role in sleep regulation. In order to elucidate the role of this Stuxnet-Polycomb-Octβ2R cascade in sleep regulation, the role of various Octβ receptors was systematically identified in sleep regulation. Octβ2R was found to be one of the receptors that mediates OA function in sleep homeostasis. More interestingly, it was found that stx was OA-responsive depending on the Octβ1R. Based on these data, it is proposed that the Stuxnet-Polycomb-Octβ2R cascade provides a feedback mechanism for OA signals to the EB to regulate sleep homeostasis and sleep amount (Zhao, 2021).

This study highlights the importance of epigenetic regulation on sleep. Although epigenetic regulation was intensively studied in adult pathological processes such as cancer, epigenetic factors have been far less studied in other physiological processes such as sleep. This study provides an example of the maintenance role of PcG complex in sleep regulation. Although the core PcG complex component Pc is ubiquitously expressed, its regulator stx is tissue specifically distributed, and this distribution may keep appropriate activity of Pc as well as the PcG complex in a tissue-specific manner. The factors regulating the tissue specificity of stx expression need to be further investigated (Zhao, 2021).

A previous study found that mutation of Octβ2R does not have an obvious sleep phenotype. The current data were compared with the published Octβ2R^f05679 mutant data. Although Octβ2R^f05679 mutant was shown not significantly affected total sleep, this study found that the Octβ2R^f05679 has mild effect on sleep. Other Octβ2R mutants were tested, and it was found that the male flies from these mutations indeed have sleep phenotype (Zhao, 2021).

Published studies have shown that the sleep phenotype of octopamine pathway mutants is different between video-based method and DAM-based method. For example, based on DAM data, the TβH mutant resulted in increased sleep per day, while the same mutant showed decreased sleep based on video data. This study used the video-based method to repeat the TβH mutant phenotype. The results showed that compared with the control flies, the TβH mutant got significantly less sleep. This result is consistent with the previously published data. Through close observation of TβH mutant and control flies, this study found that this mutant has much more frequent grooming behavior than the controls. The TβH mutant and control flies were video recorded for 10 min between ZT3.5 and ZT4.5. The results showed a statistically significant increase of the total number of grooming case. The difference between video-based method and DAM-based method is that these grooming behaviors can be detected in video-based methods, but not in DAM-based methods. Multiple studies have established a positive correlation between octopamine treatment and grooming behavior. Theoretically, TβH allele should result in a decrease in octopamine synthesis. The opposite phenotype may be caused by increased tyramine in TβH mutant or by other feedback regulation. The alleles for Octβ2R receptor used in this study show a similar grooming behavior as the control flies. The previously published octβ2R knockout allele should be a stronger one. The difference of sleep phenotypes between video-based and DAM-based methods may be due to the grooming behavior induced by the massive decrease of octopamine detection. Or other unrelated effects caused by the compensation effect previously reported. One hypothesis is that the significant change of grooming behavior probably masks the sleep behavior. The relationship between grooming and sleep needs to be further clarified. The detection of the sleep phenotype without significant changes in grooming phenotype may be a better strategy to get reliable sleep phenotype. If the increase of grooming in TβH mutant is a side effect caused by the increased tyramine, the identification of the phenotypes of octopamine treatment or collective phenotype of octopamine receptors may be more reliable ways to draw conclusions on the function of octopamine. Furthermore, whether grooming is epistatic to sleep is a problem worthy of further study (Zhao, 2021).

Two aspects of sleep homeostasis need to be further studied. First, this study found that Octβ2R and stx colocalize in a subset of EB neurons. In a previously study, EB R2 neurons were found to be responsible for sleep homeostasis regulation. The relationship of these two groups of EB neurons needs further study. Second, the OA-treated Octβ2R mutant has more sleep recovery than the control. This indicates that OA induces more sleep recovery in the condition of Octβ2R downregulation. It seems that in this condition OA induces certain pathways to counteract its role in sleep homeostasis. One possibility is that Octβ2R negatively regulates Octβ3R which results in increased sleep pressure in the absence of Octβ2R. Further studies are needed to clarify the mechanism (Zhao, 2021).

The results suggest the stx-Pc-Octβ2R regulatory cascade serves as a buffering step for OA function in sleep homeostasis. Two-way regulation of OA on stx leads to reverse changes of stx-the more OA, the less stx and vice versa. Through the function of stx-Pc-Octβ2R regulatory cascade, the Octβ2R transcription is changed accordingly. Variation of Octβ2R transcription could buffer the OA response. As a result, the unfavorable effect of OA causing dramatic decrease of sleep amount and homeostasis could be compensated by its receptor (Zhao, 2021).

Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster

Dopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe(2+)) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. This study analyzed the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels. Catsup was found to be a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and the increased dopamine production was shown to be due to a reduction in zinc levels in the cytosol. Zinc ion (Zn(2+)) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe(2+) binding to TH, thus rendering the enzyme ineffective or non-functional. These findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe(2+) and Zn(2+) ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases (Xiao, 2021).

Serotonin Signals Modulate Mushroom Body Output Neurons for Sustaining Water-Reward Long-Term Memory in Drosophila

Memory consolidation is a time-dependent process through which an unstable learned experience is transformed into a stable long-term memory; however, the circuit and molecular mechanisms underlying this process are poorly understood. The Drosophila mushroom body (MB) is a huge brain neuropil that plays a crucial role in olfactory memory. The MB neurons can be generally classified into three subsets: γ, αβ, and α'β'. This study reports that water-reward long-term memory (wLTM) consolidation requires activity from α'β'-related mushroom body output neurons (MBONs) in a specific time window. wLTM consolidation requires neurotransmission in MBON-γ3β'1 during the 0-2 h period after training, and neurotransmission in MBON-α'2 is required during the 2-4 h period after training. Moreover, neurotransmission in MBON-α'1α'3 is required during the 0-4 h period after training. Intriguingly, blocking neurotransmission during consolidation or inhibiting serotonin biosynthesis in serotoninergic dorsal paired medial (DPM) neurons also disrupted the wLTM, suggesting that wLTM consolidation requires serotonin signals from DPM neurons. The GFP Reconstitution Across Synaptic Partners (GRASP) data showed the connectivity between DPM neurons and MBON-γ3β'1, MBON-α'2, and MBON-α'1α'3, and RNAi-mediated silencing of serotonin receptors in MBON-γ3β'1, MBON-α'2, or MBON-α'1α'3 disrupted wLTM. Taken together, these results suggest that serotonin released from DPM neurons modulates neuronal activity in MBON-γ3β'1, MBON-α'2, and MBON-&alpha'1&alpha'3 at specific time windows, which is critical for the consolidation of wLTM in Drosophila (Lee, 2021).

Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, this study demonstrated that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. It was further shown that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. These results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD (Miozzo, 2022). dopamine (DA) neurotransmission. This increase is promoted by nonvesicular DA release mediated by reversal of DA transporter (DAT) function. Syntaxin 1 (Stx1) is a SNARE protein that is phosphorylated at Ser(14) by casein kinase II. This study shows that Stx1 phosphorylation is critical for AMPH-induced nonvesicular DA release and, in Drosophila melanogaster, regulates the expression of AMPH-induced preference and sexual motivation. Molecular dynamics simulations of the DAT/Stx1 complex demonstrate that phosphorylation of these proteins is pivotal for DAT to dwell in a DA releasing state. This state is characterized by the breakdown of two key salt bridges within the DAT intracellular gate, causing the opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that is hypothesized to underlie nonvesicular DA release (Shekar, 2023).

Comparative morphology of serotonin-immunoreactive neurons innervating the central complex in the brain of dicondylian insects

Serotonin (5-hydroxytryptamine) acts as a widespread neuromodulator in the nervous system of vertebrates and invertebrates. In insects, it promotes feeding, enhances olfactory sensitivity, modulates aggressive behavior, and, in the central complex of Drosophila, serves a role in sleep homeostasis. In addition to a role in sleep-wake regulation, the central complex has a prominent role in spatial orientation, goal-directed locomotion, and navigation vector memory. To further understand the role of serotonergic signaling in this brain area, this study analyzed the distribution and identity of serotonin-immunoreactive neurons across a wide range of insect species. While one bilateral pair of tangential neurons innervating the central body was present in all species studied, a second type was labeled in all neopterans but not in dragonflies and firebrats. Both cell types show conserved major fiber trajectories but taxon-specific differences in dendritic targets outside the central body and axonal terminals in the central body, noduli, and lateral accessory lobes. In addition, numerous tangential neurons of the protocerebral bridge were labeled in all studied polyneopteran species except for Phasmatodea, but not in Holometabola. Lepidoptera and Diptera showed additional labeling of two bilateral pairs of neurons of a third type. The presence of serotonin in systems of columnar neurons apparently evolved independently in dragonflies and desert locusts. The data suggest distinct evolutionary changes in the composition of serotonin-immunolabeled neurons of the central complex and provides a promising basis for a phylogenetic study in a wider range of arthropod species (Homberg, 2023).

5-HT1A regulates axon outgrowth in a subpopulation of Drosophila serotonergic neurons

Serotonergic neurons produce extensively branched axons that fill most of the central nervous system, where they modulate a wide variety of behaviors. Many behavioral disorders have been correlated with defective serotonergic axon morphologies. Proper behavioral output therefore depends on the precise outgrowth and targeting of serotonergic axons during development. To direct outgrowth, serotonergic neurons utilize serotonin as a signaling molecule prior to it assuming its neurotransmitter role. This process, termed serotonin autoregulation, regulates axon outgrowth, branching, and varicosity development of serotonergic neurons. However, the receptor that mediates serotonin autoregulation is unknown. This study asked if serotonin receptor 5-HT1A plays a role in serotonergic axon outgrowth and branching. Using cultured Drosophila serotonergic neurons, this study found that exogenous serotonin reduced axon length and branching only in those expressing 5-HT1A. Pharmacological activation of 5-HT1A led to reduced axon length and branching, whereas the disruption of 5-HT1A rescued outgrowth in the presence of exogenous serotonin. Altogether this suggests that 5-HT1A is a serotonin autoreceptor in a subpopulation of serotonergic neurons and initiates signaling pathways that regulate axon outgrowth and branching during Drosophila development (Long, 2023).

Local 5-HT signaling bi-directionally regulates the coincidence time window for associative learning

The coincidence between conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning; however, the mechanism regulating the duration of this temporal window remains unclear. This study found that serotonin (5-HT) bi-directionally regulates the coincidence time window of olfactory learning in Drosophila and affects synaptic plasticity of Kenyon cells (KCs) in the mushroom body (MB). Utilizing GPCR-activation-based (GRAB) neurotransmitter sensors, this study found that Kenyon cell (KC)-released acetylcholine (ACh) activates a serotonergic dorsal paired medial (DPM) neuron, which in turn provides inhibitory feedback to KCs. Physiological stimuli induce spatially heterogeneous 5-HT signals, which proportionally gate the intrinsic coincidence time windows of different MB compartments. Artificially reducing or increasing the DPM neuron-released 5-HT shortens or prolongs the coincidence window, respectively. In a sequential trace conditioning paradigm, this serotonergic neuromodulation helps to bridge the CS-US temporal gap. Altogether, this study reports a model circuitry for perceiving the temporal coincidence and determining the causal relationship between environmental events (Zeng, 2023).

A century ago, Ivan Pavlov proposed the associative conditioning theory, stating as follows: “A … most essential requisite for … a new conditioned reflex lies in a coincidence in time of … the neutral stimulus with … the unconditioned stimulus." However, the molecular and circuitry underpinnings that guarantee the maintenance of the coincidence time window have been unknown since then. This study reports that the coincidence time window of olfactory learning in Drosophila is bi-directionally regulated by the 5-HT signal from the single DPM neuron, which forms a feedback inhibitory circuit with the KCs in the MB (Zeng, 2023).

In a natural environment, flies do not experience the precisely controlled conditioned and unconditioned stimuli that can be delivered in a laboratory setting; as a consequence, their learning must be capable of adapting to changing CS/US regimens. Thus, the modulation due to 5-HT signaling improves their ability to successfully extract meaningful cause and effect. Additionally, studies have shown that the DPM neuron is involved in stress, sociality, and aging. Therefore, it is speculated that flies in different brain states shall accordingly exhibit different coincidence time windows due to the changes of serotonergic tone within the MB (Zeng, 2023).

Previously, the DPM neuron was reported to be required specifically during memory consolidation of 3-h middle-term memory after delay conditioning. This study found that the DPM neuron plays a different role in trace conditioning, regulating the coincidence time window during memory formation. Interestingly, people also found that DA has different functions in delay conditioning and trace conditioning of visual learning via distinct receptors (Zeng, 2023).

Another recent finding suggests that the DPM neuron also functions as a bridge between two groups of KCs—encoding visual and olfactory signals, respectively—to improve cross-modal learning. Besides the DPM neuron, there is a serotonergic projection neuron (SPN) innervating DANs in the peduncle of the MB, which gates the formation of long-term memory. Taken together, the 5-HT signals play versatile functions in different computational processes of olfactory learning (Zeng, 2023).

The adenylyl cyclase, rutabaga, and its product, cAMP, have been widely recognized as the key nodes in KCs for olfactory learning, but the regulation of the cAMP signal has not been fully explored. By directly imaging cAMP dynamics with G-Flamp1, it was found that activating the DPM neuron selectively suppressed the tonic level, while the phasic signal remained unchanged, indicating that the cAMP is tightly controlled by the endogenous 5-HT signal (Zeng, 2023).

It also remains unclear how the cAMP-related signaling cascades affect the neurotransmission of KCs. This study found that artificial activation of the Gαi signaling via hM4Di could eliminate physiological stimuli-evoked ACh release and subsequent 5-HT release from the DPM neuron. By contrast, endogenous activation of the Gαi signaling via 5-HT1A—in response to the DPM neuron-released 5-HT—just turned down the phasic and tonic ACh dynamics. These results emphasize the nuance of upstream regulations and downstream functions of the cAMP signal. These results drove the authors to ask how the 5-HT affects intracellular cAMP signaling and regulates the coincidence time window. From the perspective of KCs' ensemble, a computational model suggests that the difference in cAMP levels between odor-responsive KCs and non-responsive KCs determines learning efficiency (Zeng, 2023).

During odor-shock pairing, 5-HT released from the DPM neuron broadly suppresses cAMP in both odor-responsive and non-responsive KCs; thus, 5-HT indeed increases the signal-to-noise ratio and improves learning efficiency. It is hypothesize that this improvement might become more prominent at relatively long ISIs, and in such a way 5-HT extends the coincidence time window. 5-HT serves as an additional timing-regulating factor in the neo-Hebbian learning rule Apart from Drosophila, 5-HT is involved in learning and memory in a wide range of species, including Aplysia, C. elegans, and mammals (Zeng, 2023).

A growing body of evidence supports the notion that 5-HT affects timing during reinforcement learning. Human studies in a trace conditioning paradigm showed that decreasing 5-HT level by tryptophan deprivation specifically impaired learning with a long ISI. By contrast, studies of the nictitating membrane response in rabbits found that the hallucinogenic lysergic acid diethylamide (LSD, a non-selective 5-HT receptor agonist) facilitates learning with a long ISI. These findings are reminiscent of observations in Drosophila in which 5-HT bi-directionally regulates the coincidence time window. Thus, a similar serotonergic mechanism may be recruited by both vertebrates and invertebrates. The classic model of Hebbian plasticity suggests that co-activation of presynaptic and postsynaptic neurons within a short time window enables changes in synaptic plasticity, a phenomenon known as spike timing-dependent plasticity (STDP). Due to the inability of STDP to adequately explain reinforcement learning with a temporal gap, this theoretical framework was updated in the past decade by introducing a third factor encoded by the phasic activity of neuromodulators, mediating reinforcement, surprise, or novelty (Zeng, 2023).

In this updated three-factor neo-Hebbian learning rule, 'co-activation' plants a flag at the synapse called an eligibility trace, which waits for the third factor to implement the change in synaptic strength and determine the direction of that change (i.e., synaptic depression vs. potentiation). The neo-Hebbian learning rule is also applied in the MB of arthropods, where STDP exits between KCs and MBONs, with the dopaminergic reinforcement corresponding to the third factor. However, a putative fourth factor that specifically regulates the length of the eligibility trace remains unknown. Several theories have been proposed suggesting that 5-HT may serve as a timing regulator in a variety of processes, including reinforcement learning (Zeng, 2023).

Consistent with these predictions, this study experimentally showed that 5-HT signaling from the DPM neuron proportionally gates the coincidence time window, therefore serving as a specific timing-regulating factor that provides the missing piece of the puzzle (Zeng, 2023).

Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation

Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. This study reports that stress-induced elevation of ecdysone is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as described in this study for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability. These findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity (Meiselman, 2018).

Evidence presented in this study establishes a new paradigm for Drosophila reproduction, wherein stressful conditions arrest egg production via a hormonal cascade involving reciprocal ecdysone and ETH signaling. As steroid levels fluctuate in response to stress, so too does ETH, a consequence of steroid-regulated changes in Inka cell secretory competence. ETH activates two downstream targets: the JH-producing corpus allatum and modulatory OA neurons innervating the ovary and oviducts. This study characterized the nature of ETH dependence, and assigned function and context to a newly recognized hormonal axis governing reproductive responses to stress (Meiselman, 2018).

Previous report showed that ETH is an obligatory allatotropin, promoting oogenesis and fecundity through JH production; consequently, ETH deficiency results in low JH levels and arrested oogenesis (Meiselman, 2017). The present work demonstrates that ovulation of stage 14 oocytes depends upon ETH activation of OA neurons innervating the ovary and oviduct. A comprehensive explanation is offered for the change in distribution of vitellogenic oocytes reported in EcR mutants or under conditions of high or low ecdysone, depending on stress levels. ETH deficiency or ETHR knockdown results in accumulation of stage 14 oocytes in the ovary due to ovulation block, and a mechanistic link between altered endocrine state and ovulation is provided (Meiselman, 2018).

ETH promotes ovulation through activation OA neurons to induce contractions in the ovary and relaxation of the oviducts. It is interesting that ETH triggers calcium dynamics in vitro on distal axonal projections, suggesting ETH-stimulated OA release results from direct action of ETH on axons and/or nerve terminals. While ovary contractions in response to ETH exposure occur in both virgin and mated females, this study chose virgin females for analysis due to higher spontaneous contractile activity in mated females. This is likely due to actions of ovulin after insemination, which stimulate outgrowth of octopaminergic neurons innervating the oviduct. In virgin females, concentration-dependent ETH actions on the ovary are in the range predicted for activation of ETHR-A receptors (Meiselman, 2018).

Acting through OA neurons, ETH mobilizes calcium in the epithelium enveloping the ovary, initiating bursts of contractions in the peritoneal sheath at the base of the ovary associated with ovulation. Although bath-applied ETH and OA are both sufficient to induce calcium mobilization in the oviduct epithelium, they induce distinctive response patterns. OA causes a rapid, sustained calcium wave with a slowly waning plateau following the peak response. ETH actions occur with longer latency and induce oscillatory calcium dynamics, which could be a consequence of periodic synaptic reuptake of OA by nerve terminals. No changes in intensity were observed between treatments or at different doses, suggesting a possible threshold effect. It is also interesting to note that calcium waves spread through the epithelial layer, suggesting that the epithelium is a functional syncytium, which undoubtedly aids in coordination of relaxation (Meiselman, 2018).

Injection of mated females with either ETH or OA induces ovulation in vivo, whereas injected virgin females respond much more weakly. In order for ovulation to occur, OA causes follicle rupture inside the ovaries, a process requiring one to several hours ex vivo. It is hypothesized that mated females are in the proper endocrine state for ovulation, and thus follicle rupture may already be in progress before application of ETH or OA. As follicle rupture is the critical first step for egg-laying, this limiting factor would explain the length of time (up to 60 min) elapsed after physiological levels of ETH/OA are reached for in vivo ovulation to occur, given that ovary contraction and oviduct relaxation occur within seconds (Meiselman, 2018).

Agents previously implicated in oviduct contractions were also examined, including tyramine, glutamate, and proctolin. While the ineffectiveness of tyramine and glutamate is not surprising, the negative result with proctolin is at variance with prior literature. Examination of proctolin-induced contractions revealed that they are localized to the distal tip (germaria) of the ovaries. Moreover, proctolin does not stimulate ovulation in vitro. It appears that the role of proctolin in Drosophila ovaries is more limited than in the well-studied locust oviduct (Meiselman, 2018).

This study has shown that elevated ecdysone levels in response to heat and nutritional stress are associated with a drop in circulating ETH levels. It was previously hypothesized that the Inka cell secretory competence model governing ecdysis signaling during developmental stages may persist into adulthood (Meiselman, 2017). The results presented in this study support this hypothesis (Meiselman, 2018).

Both stress and ETH deficiency have similar consequences for reproduction, namely arrested oogenesis and reduced ovulation, resulting in increased stage 14 egg retention and lower egg production. Progression of mid-oogenetic oocytes is directly correlated with JH levels, while OA release from reproductive tract neurons is necessary for ovulation. This study shows that arrested oogenesis and ovulation contributing to the ovariole profile observed in heat-stressed flies can be explained by ETH deficiency, which has a dual role in regulating JH levels and activity of OA neurons innervating ovaries and oviducts. Indeed, arrest of both oogenesis and ovulation deficiencies can be rescued by ETH, either through TRPA1 activation of Inka cells or direct injection of ETH1 (Meiselman, 2018).

The mechanism through which elevated ecdysone leads to ETH deficiency was examined by performing rescue experiments designed to (1) suppress steroid signaling in Inka cells and (2) express the transcription factor βFTZ-F1, which confers secretory competence of Inka cells and is suppressed by high ecdysone levels. Although somewhat variable in their effectiveness, these manipulations resulted in clear rescue of oogenesis and ovulation in heat-stressed females, confirming that the thermal stress response operates through the influence of ecdysone on Inka cell secretion (Meiselman, 2018).

Methoprene treatment increases progression of oogenesis but does not increase oviposition in stressed animals. In fact, this study observed a significant increase in eggs retained after methoprene treatment, suggesting that synthesis of mature eggs resumes with JH treatment, but ovulation remains impaired under conditions of elevated ecdysone and ETH deficiency. This suggests that ovulation provides a gating mechanism under stressful conditions, limiting egg production while conditions are suboptimal. A recent report suggested that normal ecdysone levels stimulate follicle rupture and ovulation, but that elevated levels inhibit follicle rupture (Knapp, 2017). The present work provides an additional mechanism for suppression of ovulation associated with elevated ecdysone levels: repression of ETH release leading to reduced OA neuron activity (Meiselman, 2018).

It is interesting to note that wet starvation reduces ecdysone levels and increases ETH levels, whereas sugar starvation increases ecdysone levels and, as is shown in this study, increases ETH levels. Wet-starved females were precisely synchronized in mating on day 4, and began starvation (no nutrient source, wet KimWipe) 24 h later for an additional 24 h. mino acid-deprived females were group-raised until day 3, and groups were placed on agar + 10% sucrose for 24 h. Mating was not controlled in sugar-starved females, though it is known to influence ecdysone levels dramatically in the short term. Arguably the most interesting result is that ecdysone decrease led to elevated circulating ETH. This adds credence to the hypothesis that ETH and ecdysone levels are generally inversely correlated (Meiselman, 2018).

Unique stresses may garner different endocrine responses because different types of cues require differential behavioral adaptation. The ability of a hormone to coordinate a wide variety of target tissues to change in state makes it a perfect tool for stress adaptation. As an organism encounters a new type of stress, they may adapt a new endocrine state to coordinate a tissue-wide response. Many hormones in closely related insects play markedly different roles, which evolve as rapidly as behavioral niches, but an endocrine core in E-ETH-JH is highly conserved, similar to the hypothalamic-pituitary-gonadal (HPG) axis among vertebrates. A hormonal network with competence to adjust reproductive output in response to environmental changes is undoubtedly a common phenomenon among multicellular organisms. The discovery of a stress response hormonal axis and, more aptly, a peptide hormone with the potential to alleviate stress-induced deficits in reproduction could be of particular relevance to the honey bee Apis mellifera. In recent years, Apis reproductives have been producing fewer progeny due to a variety of stressors, including temperature extrema. While proctolin has already been found to be a short-term reproductive stimulant in Apis queens, ETH is attractive as it can alter JH levels, which in turn may rescue poor pheromone production, the proximal cause of supersedure (Meiselman, 2018).

Enabling cell-type-specific behavioral epigenetics in Drosophila: a modified high-yield INTACT method reveals the impact of social environment on the epigenetic landscape in dopaminergic neurons

Epigenetic mechanisms play fundamental roles in brain function and behavior and stressors such as social isolation can alter animal behavior via epigenetic mechanisms. However, due to cellular heterogeneity, identifying cell-type-specific epigenetic changes in the brain is challenging. This study reports the first use of a modified isolation of nuclei tagged in specific cell type (INTACT) method in behavioral epigenetics of Drosophila melanogaster, a method called mini-INTACT. Using ChIP-seq on mini-INTACT purified dopaminergic nuclei, epigenetic signatures were identified in socially isolated and socially enriched Drosophila males. Social experience altered the epigenetic landscape in clusters of genes involved in transcription and neural function. Some of these alterations could be predicted by expression changes of four transcription factors (Hr38, sr, CrebA, and Cbt) and the prevalence of their binding sites in several clusters. These transcription factors were previously identified as activity-regulated genes, and their knockdown in dopaminergic neurons reduced the effects of social experience on sleep. This work work enables the use of Drosophila as a model for cell-type-specific behavioral epigenetics and establishes that social environment shifts the epigenetic landscape in dopaminergic neurons. Four activity-related transcription factors are required in dopaminergic neurons for the effects of social environment on sleep (Agrawal, 2019).

gamma-Oryzanol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in Drosophila melanogaster

Chronic unpredictable mild stress (CUMS) is a valid model for inducing depression-like symptoms in animal models, causing predictive behavioral, neurochemical, and physiological responses to this condition. This work aims to evaluate the possible antidepressant effect of γ-oryzanol (ORY) in the CUMS-induced depressive model in male Drosophila melanogaster. The CUMS protocol was used to continue a previous study, mimicking a depressive state in these insects. Male flies were subjected to various stressors according to a 10-day randomized schedule and concomitantly treated with ORY or fluoxetine (FLX). After the experimental period, in vivo behavioral tests were performed (open field, forced swimming, aggressiveness test, mating test, male virility, sucrose preference index and light/dark test) and ex vivo analyses measuring serotonin (5HT). dopamine (DA). octopamine (OCT) levels and body weight. ORY-treated flies and concomitant exposure to CUMS did not exhibit obvious behaviors such as prolonged immobility or increased aggressive behavior, reduced male mating and virility behavior, and anxiolytic behavior, in contrast to ORY, not altering sucrose preference and body weight flies exposed to CUMS. ORY effectively prevented 5HT and OCT reduction and partially protected against DA reduction. The data are consistent and provide evidence for the use of ORY as a potential antidepressant compound (Araujo, 2020).

Iron overload during the embryonic period develops hyperactive like behavior and dysregulation of biogenic amines in Drosophila melanogaster

Iron (Fe) is used in various cellular functions, and a constant balance between its uptake, transport, storage, and use is necessary to maintain its homeostasis in the body. Changes in Fe metabolism with a consequent overload of this metal are related to neurological changes and cover a broad spectrum of diseases, mainly when these changes occur during the embryonic period. This work aimed to evaluate the effect of exposure to Fe overload during the embryonic period of Drosophila melanogaster. Progenitor flies (male and female) were exposed to ferrous sulfate (FeSO(4)) for ten days in concentrations of 0.5, 1, and 5 ​mM. After mating and oviposition, the progenitors were removed and the treatment bottles preserved, and the number of daily hatches and cumulative hatching of the first filial generation (F1) were counted. Subsequently, F1 flies (separated by sex) were subjected to behavioral tests such as negative geotaxis test, open field test, grooming, and aggression test. They have evaluated the levels of dopamine (DA), serotonin (5-HT), octopamine (OA), tryptophan and tyrosine hydroxylase (TH), acetylcholinesterase, reactive species, and the levels of Fe in the progenitor flies and F1. The Fe levels of F1 flies are directly proportional to what is incorporated during the period of embryonic development; a delay in hatching and a reduction in the number of the hatch of F1 flies exposed during the embryonic period to the 5mM Fe diet were observed, a fact that may be related to the reduction of the cell viability of the ovarian tissue of progenitor flies. The flies exposed to Fe (1 and 5 ​mM) showed an increase in locomotor activity (hyperactivity) and a significantly higher number of repetitive movements. In addition to a high number of aggressive encounters when compared to control flies. An increase was observed in the levels of biogenic amines DA and 5-HT and an increase in TH activity in flies exposed to Fe (1 and 5 ​mM) compared to the control group. It is concluded that the hyperactive-like behavior demonstrated in both sexes by F1 flies exposed to Fe may be associated with a dysregulation in the levels of DA and 5-HT since Fe is a cofactor of TH, which had its activity increased in this study. Therefore, more attention is needed during the embryonic development period for exposure to Fe overload (Poetini, 2021).

A diabetes-like biochemical and behavioural phenotype of Drosophila induced by predator stress

Predation can have both lethal and non-lethal effects on prey. The non-lethal effects of predation can instil changes in prey life history, behaviour, morphology and physiology, causing adaptive evolution. The chronic stress caused by sustained predation on prey is comparable to chronic stress conditions in humans. Conditions like anxiety, depression, and post-traumatic stress syndrome have also been implicated in the development of metabolic disorders such as obesity and diabetes. This study found that predator stress induced during larval development in fruit flies Drosophila melanogaster impairs carbohydrate metabolism by systemic inhibition of Akt protein kinase, which is a central regulator of glucose uptake. However, Drosophila grown with predators survived better under direct spider predation in the adult phase. Administration of metformin and 5-hydroxytryptophan (5-HTP), a precursor of the neurotransmitter serotonin, reversed these effects. These results demonstrate a direct link between predator stress and metabolic impairment, suggesting that a diabetes-like biochemical phenotype may be adaptive in terms of survival and reproductive success. This study provides a novel animal model to explore the mechanisms responsible for the onset of these metabolic disorders, which are highly prevalent in human populations (Krama, 2023).

Ionizing radiation alters functional neurotransmission in Drosophila larvae

Patients undergoing cranial ionizing radiation therapy for brain malignancies are at increased risk of long-term neurocognitive decline, which is poorly understood and currently untreatable. Although the molecular pathogenesis has been intensively researched in many organisms, whether and how ionizing radiation alters functional neurotransmission remains unknown. This is the first study addressing physiological changes in neurotransmission after ionizing radiation exposure. To elucidate the cellular mechanisms of radiation damage, using calcium imaging, the effects were analyzed of ionizing radiation on the neurotransmitter-evoked responses of prothoracicotropic hormone (PTTH)-releasing neurons in Drosophila larvae, which play essential roles in normal larval development. The neurotransmitters dopamine and tyramine decreased intracellular calcium levels of PTTH neurons in a dose-dependent manner. In gamma irradiated third-instar larvae, a dose of 25 Gy increased the sensitivity of PTTH neurons to dopamine and tyramine, and delayed development, possibly in response to abnormal functional neurotransmission. This irradiation level did not affect the viability and arborization of PTTH neurons and successful survival to adulthood. Exposure to a 40-Gy dose of gamma irradiation decreased the neurotransmitter sensitivity, physiological viability and axo-dendritic length of PTTH neurons. These serious damages led to substantial developmental delays and a precipitous reduction in the percentage of larvae that survived to adulthood. These results demonstrate that gamma irradiation alters neurotransmitter-evoked responses, indicating synapses are vulnerable targets of ionizing radiation. The current study provides new insights into ionizing radiation-induced disruption of physiological neurotransmitter signaling, which should be considered in preventive therapeutic interventions to reduce risks of neurological deficits after photon therapy (Zhang, 2023).

Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain

The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. This study developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. The power of CLIn was demonstrated in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. 18 dopaminergic neurons from three distinct clusters were mapped to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. Type II lineages that produce doublesex- and fruitless-expressing neurons were identified, and whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain was examined. Blocking apoptosis in these lineages results in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism (Ren, 2016).

The relationship between neuron classes and lineages is complex in the Drosophila brain, where analogous neurons of a given class may arise from distinct lineages and a single lineage can yield multiple neuron classes. Therefore, a method was developed that would enable mapping and and analysis of neuron classes with respect to lineage identity using intersectional transgenic strategies. Specifically, the neuron class of interest expresses the GAL4 transcriptional activator from a class-specific transgene, while the lineage(s) of interest expresses the KD recombinase from a lineage-specific transgene. The KD recombinase activity triggers production of another recombinase, Cre, under the control of the deadpan (dpn) promoter, which is active in all NBs. Cre recombinase activity then triggers the simultaneous production of the LexA::p65 transcriptional activator and loss of the GAL4 inhibitor, GAL80, in all subsequently born progeny within the lineage(s). The LexA::p65 activates reporter-A expression within lineages of interest via lexAop. Because all other neurons outside lineage(s) of interest express GAL80, GAL4 is only active in neurons of the LexA::p65-expressing lineage(s) and thus can positively mark these neurons by activating expression of a reporter-B under UAS control. One can therefore subdivide any complex set of neurons that express a class-specific GAL4 transgene based on their developmental lineage(s). Consequently, CLIn enables the unambiguous determination of the lineage origins of particular neuron classes, which is essential for understanding the development and organization of the Drosophila brain (Ren, 2016).

The CLIn system unambiguously establishes the correspondence between cell classes and their lineage origins and enables the subdivision of a given neuronal class among certain NB lineages. It also allows interrogation of serially derived neuronal diversity. One can therefore map individual neurons of a given class with respect to their lineage and temporal origins in an effort to unravel the intricate neuron class-lineage relationships in the brain (Ren, 2016).

Revealing diverse cell classes of a lineage, by carefully choosing different GAL4 drivers that each distinguish a particular cell class, will allow better characterization of progeny heterogeneity within a lineage. It is therefore possible to explore how cellular diversity is generated during development. For example, it will be interesting to determine whether a specific cell class develops from one fixed temporal window. Moreover, comparing the cell-class diversity of different lineages will provide insight into the developmental heterogeneity of stem cells (Ren, 2016).

Conversely, for cell classes that originate from multiple lineages, CLIn analysis reveals the distribution of a cell class among different lineages. Vertebrate studies found that neurons of the same lineage origin, compared to neurons of the same class but different lineage origins, are more likely structurally connected via gap junction and have similar network functions. In Drosophila, lineage has been shown to be a developmental and a functional unit. Thus, assigning a cell class to different lineages may reveal the particular function of a neuronal subset within a cell class (Ren, 2016).

Moreover, the CLIn system permits incorporations of additional effectors driven by the GAL4-UAS system or the LexA system to manipulate cell class or lineage, respectively. The toolkit of effectors for different purposes is growing rapidly over recent years. Multiple reporter constructs are available to label specific sub-domains of the cell (dendrite, axon, or synapse). Effectors that affect cell viability could eliminate or immortalize specific neurons or glia. Effectors that alter membrane activity can be used to modulate neural activity. In addition, CLIn enables distinguishing gene's functions in whole lineage including stem and progenitor cells versus only in a subset of lineage progeny by independent gene manipulations via lexAop versus UAS systems (Ren, 2016).

However, the CLIn system requires further improvement to reach its full potential. In particular, the drivers for targeting various NB subsets remain to be fully characterized. Moreover, their targeting efficiency and specificity could vary individually. Engineering drivers based on genes known to be expressed in defined subsets of embryonic NBs may provide an initial complete set of more reliable NB drivers. An additional challenge for the study of type II lineages is how to selectively target INP sublineages. Via the current dpn enhancer, the frequencies of INP1 sublineages are very low compared with that of NB lineages (Ren, 2016).

Type II NBs yield supernumerary neurons plus glia, which are expected to be highly diverse in cell classes. CLIn unambiguously assigned various neuronal classes to common type II lineages. In this study, the majority of progeny remained negative for the drivers employed. Revealing the full spectrum of neuronal heterogeneity within type II lineages requires characterization of additional cell-class drivers (Ren, 2016).

Diverse cell classes could arise from a single INP. Single-cell lineage analysis has shown that one INP can produce multiple morphological classes of neurons most likely pertaining to different functional classes. Temporal mapping by CLIn revealed the birth of both TH-GAL4 and dsx^GAL4 neurons in early windows of larval type II lineages. This lends further support to the production of diverse neuronal classes by common INPs. Examining INP clones labeled by CLIn did validate that the first larval-born INP of the DM6 lineage makes one fru^GAL4 neuron in addition to two TH-GAL4 neurons (Ren, 2016).

Per the limited cell-lineage analysis along the NB axis of type II lineages, sibling INPs produce morphologically similar series of neurons that differ in subsets of terminal neurite elaborations. These phenomena indicate expansion of related neurons across sibling INP sublineages. Assuming production of about 50 sibling INPs and in the absence of apoptosis, one would expect composition of 50 cell units for each neuronal class made by one type II NB. Notably, rescuing apoptotic dsx- or fru-expressing neurons throughout lineage development did restore complements of 50 or so cells in several, but not all, type II lineages. However, most type II lineages yield very few, if any, TH-GAL4 neurons. For instance, the DL1 lineage produces two TH-GAL4 neurons that innervate the upper FB layers. Temporal mapping of the DL1 lineage reveals the existence of multiple (n > 3) morphologically distinct INP clones that contain neurons projecting to the FB upper layers, similar to the DL1 TH-GAL4 neurons. Thus, morphologically similar neurons may belong to different functional classes, highlighting the challenges in sorting out neuronal classes and their interrelationships in the brain (Ren, 2016).

Pioneering studies in C. elegans showed that the acquisition of neurotransmitter identity could be achieved through distinct mechanisms. A shared regulatory signature consisting of three terminal-selector types of transcription factors regulates the terminal identity of all dopaminergic neurons. By contrast, different combinations of terminal selectors act in distinct subsets of glutamatergic neurons to initiate and maintain their glutamatergic identity. In the present study, it was observed that six type II lineages produce 18 dopaminergic neurons but all during early larval neurogenesis. The derivation of TH neurons from multiple neuronal lineages at similar temporal windows argues for their specification by combinations of different lineage-identity genes with common temporal factors (Ren, 2016).

Previous analysis of fru^GAL4 neurons has uncovered 62 discrete MARCM clones in the fly central brain that might arise from an equal number of lineages. Ten clones show dimorphic cell numbers, and 22 clones exhibit dimorphic trajectories. Contrasting the stochastic clonal labeling of only fru^GAL4 neurons, CLIn allows determination among a collection of lineages of whether a given lineage yields any fru^GAL4 neurons. Based on the additional lineage information, two clones (pIP-j and pIP-h) were attributed as being partial clones of another two full-sized clones (pIP-g and pMP-f). Moreover, a more focused approach reveals sexual dimorphism of fru-expressing neurons in all type II NB lineages (Ren, 2016).

The presence of many more dsx- or fru-expressing neurons in male than female brains is proposed to result from selective loss of specific neurons in females through apoptosis. However, blocking apoptosis increased dsx- or fru-expressing neurons in both male and female lineages. This is consistent with a previous report showing that sex-independent apoptosis occur widely in type II lineages. Although the number of apoptosis-blocked female neurons was similar, if not identical, to that of the control male neurons, blocking apoptosis unexpectedly increased the number of male dsx- or fru-expressing neurons such that there were more neurons in the apoptosis-blocked male than female lineages. This unmasks the original potential of the male and female NBs to produce different numbers of dsx- or fru-expressing neurons in type II lineages (Ren, 2016).

Distinct fates have been reported for male and female NBs in the abdominal ganglion of Drosophila CNS. In this study, the male isoform of Dsx, DsxM, promotes additional NB divisions in males relative to females. Similarly, it has been reported that DsxM specifies additional cell divisions in the male, relative to female, central brain NBs that give rise to the pC1 and pC2 clusters. The proliferation of Drosophila intestinal stem cells is also determined by their sexual identity, although this is controlled by genes other than dsx and fru. Consistent with the notion that male and female NBs may possess distinct fates, this study found that male type II lineages contain more neurons committed to express dsx or fru, which possibly results from the greater number of NB divisions in males, as shown in the previous study. Elucidating the underlying molecular mechanisms of sex-specific neuron numbers in the central brain will require additional studies of the sex-dependent production and specification of different dsx- or fru-expressing neurons in the apoptosis-blocked type II NB lineages (Ren, 2016).

Lineage mapping based on morphology provides limited information about neuronal classes. Given the intricate relationship between neuronal classes and cell lineages, CLIn is needed to resolve the detail even in fly brains where invariant neuronal lineages exist. This is critical for fully understanding how cell lineages guide the formation of variant neural circuits with distinct combinations of neuronal classes and types (Ren, 2016).

In mammalian brains, extensive neuronal migration obscures the roles of cell lineages in the global organization of neural networks. However, clonally related neurons preferentially make local connections. Moreover, ample evidence exists for the heterogeneity of mammalian neural stem cells and the control of neuronal identity by spatiotemporal patterning of neural progenitors. Untangling of a further sophisticated brain and its development may absolutely require examination of cell lineages and neuronal classes at the same time. Systems like CLIn with its emphasis on the relationship between cell class and lineage potentially aid greatly in the study of mammalian brain development, anatomy, and function (Ren, 2016).

Electrochemical measurements of optogenetically stimulated quantal amine release from single nerve cell varicosities in Drosophila larvae

The nerve terminals found in the body wall of Drosophila larvae are readily accessible to experimental manipulation. This study used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. A method was developed to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. 22700 +/- 4200 molecules of octopamine were released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse (Majdi, 2015).

Four individually identified paired dopamine neurons signal reward in larval Drosophila

Dopaminergic neurons serve multiple functions, including reinforcement processing during associative learning. It is thus warranted to understand which dopaminergic neurons mediate which function. Larval Drosophila were used, in which only approximately 120 of a total of 10,000 neurons are dopaminergic, as judged by the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. Dopaminergic neurons mediating reinforcement in insect olfactory learning target the mushroom bodies, a higher-order "cortical" brain region. Four previously undescribed paired neurons, the primary protocerebral anterior medial (pPAM) neurons, were discovered. These neurons are TH positive and subdivide the medial lobe of the mushroom body into four distinct subunits. These pPAM neurons are acutely necessary for odor-sugar reward learning and require intact TH function in this process. However, they are dispensable for aversive learning and innate behavior toward the odors and sugars employed. Optogenetical activation of pPAM neurons is sufficient as a reward. Thus, the pPAM neurons convey a likely dopaminergic reward signal. In contrast, DL1 cluster neurons convey a corresponding punishment signal, suggesting a cellular division of labor to convey dopaminergic reward and punishment signals. On the level of individually identified neurons, this uncovers an organizational principle shared with adult Drosophila and mammals. The numerical simplicity and connectomic tractability of the larval nervous system now offers a prospect for studying circuit principles of dopamine function at unprecedented resolution (Rohwedder, 2016).

Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster

This study investigated sexually dimorphic effects of disruptions in dopamine (DA) homeostasis and its relationship to senescence using three different Drosophila melanogaster mutants, Catsup (Catsup26) with elevated DA levels, and pale (ple2), Punch (PuZ22) with depleted DA levels. In all genotypes including controls, DA levels were significantly lower in old (45-50-day-old) flies compared with young (3-5-day-old) in both sexes. Interestingly, females have lower DA content than males at young age whereas this difference is not observed in old age, suggesting that males have a larger decline in DA levels with age. Females, in general, are longer lived compared with males in all genotypes except ple2 mutants with depleted DA levels, and females also demonstrate marked age-related decline in circadian locomotor activity compared with males. Old Catsup26 males with elevated DA levels accumulate significantly lower levels of lipid peroxidation product 4-hydroxy 2-nonenal compared with wild type, ple2 and PuZ22 mutant males. A sexually dimorphic response was also observed in the expression levels of key stress and aging associated transcription factors across genotypes with elevated or depleted DA levels. Taken together, these results reveal a novel sexually dimorphic involvement of DA in senescence characteristics of D. melanogaster (Bednářová, 2017).

Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons

The neurology of male sexuality has been poorly studied owing to difficulties in studying brain circuitry in humans. Dopamine (DA) is essential for both physiological and behavioural responses, including the regulation of sexuality. Previous studies have revealed that alterations in DA synthesis in dopaminergic neurons can induce male-male courtship behaviour, while increasing DA levels in the protocerebral posteriolateral dopaminergic cluster neuron 2ab (PPL2ab) may enhance the intensity of male courtship sustainment in Drosophila. This study reports that changes in the ability of the PPL2ab in the central nervous system (CNS) to produce DA strongly impact male-male courtship in D. melanogaster. Intriguingly, the DA-synthesizing abilities of these neurons appear to affect both the courting activities displayed by male flies and the sex appeal of male flies for other male flies. Moreover, the observed male-male courtship is triggered primarily by target motion, yet chemical cues can replace visual input under dark conditions. This is interesting evidence that courtship responses in male individuals are controlled by PPL2ab neurons in the CNS. This study provides insight for subsequent studies focusing on sexual circuit modulation by PPL2ab neurons (Chen, 2017).

Sleep facilitates memory by blocking dopamine neuron-mediated forgetting

Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. This study shows that this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. These findings integrate psychological and neuroscience research on sleep and forgetting (Berry 2015).

While some memories are long-lasting, most others fade away and are forgotten. Why we forget, has been an intriguing and central question in psychology and neuroscience for more than a century. Even though forgetting is often thought of as a failure or limitation of the brain, recent studies support the view that forgetting is a biologically regulated function of the brain allowing optimal adaptability to an ever-changing environment. In the fruit fly Drosophila, it was recently shown that the very same set of dopamine neurons (DANs) that signal through one receptor to form aversive olfactory memories, also signal through a separate receptor after learning to forget these memories (Berry, 2012). However, it remains unclear whether this dopaminergic forgetting signal is constant and autonomous, or dynamic and regulated (Berry 2015).

From fruit flies to humans, animals routinely alternate between highly active behavioral states and long states of immobility and quiescence called sleep. Despite the obvious disadvantages an inanimate state conveys to survival, sleep has been proposed to have critically important functions, including in memory and cognition. Since the earliest experimental studies of human memory, sleep shortly after learning has been shown to consistently lead to an increase in retention and thus less forgetting of many forms of memory including declarative and emotional memory in mammals and long-term courtship memory in Drosophila. However, there exists controversy as to how sleep benefits memory retention. Many studies in mammals support the idea that sleep benefits memory retention because it is accompanied by specific mechanisms, such as slow wave sleep, rapid eye movement (REM) sleep, and sharp-wave ripple-based memory replay, that increase memory retention by actively consolidating newly formed memories. Alternatively, it was proposed nearly a century ago and recently revisited, that sleep, or long periods of quiet wakefulness, benefit memory retention by muting experience-driven plasticity and new memory formation, thus reducing retroactive interference-based forgetting. In addition, this state of reduced neuronal activity might then allow consolidation to occur more efficiently, referred to as the "opportunistic consolidation" model. Thus, the essence of how exactly sleep benefits memory retention remains debated (Berry 2015).

Previous studies have observed that, after promoting the acquisition of olfactory memories, a small set of DANs that innervate the mushroom body (MB) memory center, intriguingly, display synchronized and ongoing Ca2+-based activity after learning that causes the forgetting of early aversive olfactory memories in Drosophila(Berry, 2012). While this activity occurs as reoccurring bursts, it was noticed that the pattern of activity appeared temporally regulated, occurring in bouts. In order to understand how the DAN-based forgetting signal might be regulated, an in vivo imaging assay was developed allowing simultaneous monitoring of a fly's DAN Ca2+ activity, via GCaMP3.0 expression using TH-gal4 and behavior while walking on a ball supported by air. Focus was placed on two regions of the DAN processes that form synaptic connections to the MBs, referred to as neuropils, one that displays ongoing activity and belongs to the MV1 neuron and an adjacent control region belonging to the V1 neuron, which is relatively inactive. Remarkably, a 1-hr simultaneous recording of locomotion and DAN activity revealed that the MV1 neuropil displayed activity resembling the coarse temporal pattern of locomotor behavior. Ball rotation data was used to cluster time points into either behaviorally active or rest states, and MV1 neuropil activity was found to be robustly elevated during active states, whereas the V1 neuropil activity remained low in both states, but had a slight decrease during active states. Furthermore, the MV1 neuropil Ca2+ signal was strongly correlated with ball rotation, particularly in the lower frequency domains (frequency < 0.002 Hz, or ~1 cycle every 8 min or more, timescales consistent with that of locomotor bout structure). Finally, DAN activity was examined during stable transitions into and out of behaviorally active states ( by aligning transition segments of recordings across all animals. Interestingly, MV1 DAN activity robustly increased upon transition into active states, while, conversely, dropped during rest states. V1 activity remained low and was not significantly regulated with behavioral transitions. Together, these data, along with observations of synchronized activity between MV1 and another DAN, MP1, indicate that the ongoing activity from specific sets of DAN involved in forgetting, including MV1, is regulated with the behavioral state of the animal (Berry 2015).

Given the strong correlation between DAN activity and locomotor activity, tests were performed to see whether DAN activation might promote locomotor activity, that is, whether DAN activity is upstream of locomotor circuits. Two prior studies found no role for these MB innervating DANs in regulating locomotor activity. When the synaptic output from these DANs was blocked with restricted expression in MV1, MP1, and V1 DANs to drive temperature-sensitiveUAS-shi^ts1function, no decreased locomotor activity was seen between temperatures, although the experimental genotype exhibited less activity at high temperature compared to one but not both control genotypes. It was also noted from imaging experiments that locomotor activity occasionally occurs while the MV1 neuron is not active, thus further supporting that locomotor behavior does not require c150-gal4>DAN output. Furthermore, stimulation of these neurons, using UAS-trpA1, did not produce genotype specific and robust increases in locomotor activity. But similar to the blocking experiments, high temperature increased the locomotor activity of the two control genotypes (UAS-trpA1 and c150-gal4 alone). These data indicate that c150-gal4 DAN output is neither necessary nor sufficient to acutely drive locomotor activity. It is therefore concluded that the ongoing signal in MV1 is either downstream of locomotor behavior itself, or is regulated in parallel, by other brain areas that promote arousal and locomotor activity (Berry 2015).

Given that the ongoing activity in MV1 was highest during behaviorally active states, the hypothesis was tested that reducing behavioral activity with increased sleep drive would reduce this ongoing activity. The GABAA agonist, Gaboxadol (or THIP), has been shown to specifically promote deep non-REM sleep in humans, while leaving REM sleep intact; sleep characteristics similar to those occurring during normal homeostatic regulation of sleep. Recently, it was shown that Gaboxadol also induces sleep in Drosophila. To confirm this, attempts were made to induce sleep in Drosophila by feeding flies various doses of Gaboxadol. Shortly after Gaboxadol feeding, long periods of quiescence, (>5 min) conventionally defined as 'sleep' in Drosophila, significantly increased during both day and night in a dose-dependent manner. Next, flies were fed Gaboxadol (0.1 mg/ml) for 1 day and then removed the drug to test whether these effects were reversible. Once again, Gaboxadol treatment increased sleep, occurring as bouts with increased duration, but remarkably, total sleep and bout duration actually decreased after drug removal compared to control fed flies. These data indicate that less sleep is needed in flies given Gaboxadol the prior day, suggestive of a homeostatic response. Finally, to test the arousability of flies fed Gaboxadol, a single mechanical stimulus was delivered every hour for 1 day followed by a day of drug treatment. Interestingly, the average-evoked activity, post-stimulus was significantly reduced with increasing Gaboxadol dosage. These data suggest that having Gaboxadol onboard increases arousal thresholds. Altogether, these data indicate that Gaboxadol, similar to effects on mammals, induces bona fide sleep in Drosophila, with hallmark characteristics that include reversible quiescence, homeostatic regulation, and increased arousal thresholds (Berry 2015).

In order to observe the effects of Gaboxadol on DAN activity, varying concentrations of this sleep agent were perfused across the brain while performing in vivo imaging of MV1 activity and fly body movement was monitored in a recording chamber. Like walking on the ball, ongoing MV1 activity was also regulated with behavioral state in this assay, increasing during bouts of body movement. Remarkably, Gaboxadol perfusion rapidly and robustly attenuated both fly movement and MV1 activity at 0.01 and 0.1 mg/ml. Furthermore, it was found that the quiescent behavioral state and reduced MV1 activity was fully reversible with wash out, thus eliminating pharmacological-induced damage as a cause of decreased physical and DAN activity (Berry 2015).

To rule out non-specific effects of Gaboxadol and extend these results, sleep drive was increased sharply by thermogenetic stimulation of the sleep circuit. Recent studies identified a dorsal fan shaped body (dfsb) circuit in the central brain, specifically represented in the R23E10-gal4 line, which acts as the effector arm of the sleep homeostat (Donlea; 2011 and Donlea; 2014). Consistent with these studies, TrpA1-based stimulation of R23E10-gal4-expressing neurons caused a rapid and robust increase in daytime sleep followed by a negative sleep rebound the day after stimulation, confirming the dfsb circuit's role in homeostatic sleep regulation. In order to measure DAN activity in vivo while using the gal4-uas system to modulate the sleep circuit, a TH-lexA line was developed to express GCaMP^3.0 in the MV1 and V1 DAN neurons, their associated MB neuropil regions, as well as DAN innervation of the anterior inferior medial protocerebrum (PR), a region also exhibiting ongoing activity like MV1 (Berry, 2012). While simultaneously measuring movement and DAN activity, before ('Pre'), during ('Treat'), and after ('Post') stimulation of dfsb neurons was recorded. As predicted, stimulation of the sleep circuit rapidly decreased fly behavioral activity and was accompanied with a robust decrease in MV1 and PR DAN activity, with no change in the control V1 region. Fly behavioral activity was partially restored and ongoing activity in MV1 and PR completely restored to pre-stimulation levels after stimulation of the sleep circuit was ceased. These results, along with those from Gaboxadol administration, indicate that increased sleep drive dramatically reduces the ongoing activity of DANs involved in forgetting (Berry 2015).

Since ongoing MV1 activity is decreased with increasing sleep drive, it was hypothesized that acutely and reversibly increasing sleep drive specifically after learning would reduce DAN-mediated forgetting. To test this, sleep was modulated with Gaboxadol after aversive olfactory conditioning, where populations of flies learn to associate an odor with electric shock. Memory to this association is then tested in a T-maze, giving flies the choice between the trained odor and an unconditioned odor. Since memory from this kind of training decays quickly after training, attempts were made to increase the rate of Gaboxadol consumption and thus the rate of sleep onset, by increasing the hunger of flies via starvation prior to feeding. As was observed previously, flies fed Gaboxadol experienced more sleep than controls, and a 16-hr starvation period increased this effect. Furthermore, it was found that flies removed from Gaboxadol food 1 hr after learning partially returned to control sleep and activity levels by the sixth hour and completely by the eighth hour after learning, indicating that these time points were appropriate for testing memory retrieval. This Gaboxadol feeding protocol led to increased sleep during the period of memory retention. Remarkably, it was found that flies forced to sleep with Gaboxadol treatment after learning exhibited enhanced memory retention at both 6 and 8 hr. Similarly, sleep circuit stimulation after conditioning also rapidly and reversibly induced sleep and enhanced both 3- and 6-hr memory retention. Importantly, simultaneous stimulation of the dfsb sleep circuit and c150-gal4 DANs also led to strong sleep induction. Memory retention, in contrast, was markedly decreased, similar to that observed with stimulation of DANs alone. Therefore, sleep, after learning, loses its protective qualities when DAN signaling is artificially potentiated. This circuit level epistasis experiment indicates that DAN-mediated forgetting is downstream of sleep networks. Together, the data indicate that increased sleep and reduced arousal after learning reduces DAN-mediated forgetting of aversive olfactory memories (Berry 2015).

If the DANs innervating the MB memory center are downstream of arousal circuitry, then they should respond to arousing stimuli. In fact, these neurons have already been shown to respond to many salient stimuli, including odors and electric shock and temperature changes. Since mechanical stimuli have been extensively used to arouse flies for sleep deprivation, airpuffs were delivered to the fly using a protocol shown to induce arousal in flies, while simultaneously recording fly movement and DAN activity. It was found that the DAN processes in all three areas (MV1, PR, V1) of the mushroom body neuropil exhibited robust responses to each airpuff. However, MV1 responsiveness was maintained across stimuli while the other regions showed attenuated responsiveness across stimuli. Importantly, both fly movement and ongoing MV1 activity continued at an elevated level just after stimulation, indicating a stimulus-induced elevation in arousal and MV1 DAN activity (Berry 2015).

Next, it was reasoned that increasing the arousal after learning would accelerate DAN-mediated forgetting. To test this, a population arousal device was developed that allowed delivery of a mechanical stimuli (2-s stimulus every 1 min over 80 min) to flies in population vials after aversive olfactory learning. It was found that mechanical stimuli delivered for the first 80 min after learning significantly aroused populations of flies, leading to an overall increase in activity between each stimulus, with activity levels dropping back to control levels after treatment. Importantly, mechanical stimulation after learning caused a robust decrease in 3-hr memory for wild-type Canton-S flies. However, acquisition and immediate memory retrieval were not disrupted by prior mechanical stimulation, indicating that the stimuli must be delivered after learning to observe its disruptive effects. Remarkably, it was found that blocking neuronal output of c150-gal4DANs specifically during mechanical stimulation blocked the forgetting induced by this treatment. Therefore, these data indicate that increasing arousal after learning accelerates DAN-mediated forgetting (Berry 2015).

The following conclusions are made from the data. First, after learning, the ongoing DA forgetting signal is not constant but instead is regulated with behavioral state. Thus, the forgetting signal does not chronically remove memories at a constant rate. Second, the ongoing forgetting signal is coupled directly to the arousal level of the animal, being suppressed with low levels of arousal such as with the state of sleep and being enhanced by activation of sensory pathways. As a result, forgetting decreases when flies rest or sleep and increases when flies are aroused by external stimuli (Berry 2015).

DA is known to regulate various types of plasticity in mammals. In flies, DA has been shown to elicit presynaptic plasticity within the Kenyon cells of the MB memory center proposed to underlie learning. Additionally, it was previously found that DA after learning regulates forgetting (Berry, 2012), thus implicating a DA-based plasticity mechanism that weakens memories. Synthesizing these previous observations with the current data, it is proposed that the behavioral state-coupled DA signal, discovered in this study, regulates the plasticity of the memory system, making it malleable for memory updating so that memories of current events can be formed and old, unused memories can be forgotten. While it was found previously that different DA receptors underlie learning and forgetting, more work remains to distinguish the molecular cascades involved and the cellular events that underlie these forms of behavioral plasticity (Berry 2015).

These findings add compelling mechanistic evidence to support the model that sleep, which begins with and is accompanied by inactivity or rest, benefits newly formed labile memories by reducing the level of plasticity induced by behavioral activity. Furthermore, as sleep progresses and arousal thresholds increase, DANs become less reactive to stimuli. Thus, the molecular/cellular model is congruent with early psychological models of sleep benefitting memory by muting the retroactive interference that causes forgetting. Nevertheless, the data do not eliminate the possibility that sleep-specific mechanisms exist that enhance memory consolidation, as often proposed from studies with mammalian systems. Mechanistically, the effects of sleep on memory consolidation and forgetting may operate in parallel and independently of one another or more intriguingly; they may operate in serial in a dependent fashion, with reduced forgetting being a prerequisite for sleep-facilitated consolidation, similar to the 'opportunistic consolidation' model proposed by Mednick, 2011 (Berry 2015).

This study has observed that multiple DANs produce the ongoing DA signal, synchronized across the MB memory center and protocerebrum, that leads to forgetting of olfactory memories. It remains to be determined if this network activity is but one segment of a larger and more diffuse DA network that operates on memory types other than olfactory; whether there exist multiple, independent forgetting networks regulated by arousal levels; and whether forgetting of non-olfactory memories occurs through DA-based mechanisms or involves other neuromodulatory transmitters (Berry 2015).

Control of sleep by dopaminergic inputs to the Drosophila mushroom body

The Drosophila mushroom body (MB) is an associative learning network that is important for the control of sleep. Particular intrinsic MB Kenyon cell (KC) classes have been identified that regulate sleep through synaptic activation of particular MB output neurons (MBONs) whose axons convey sleep control signals out of the MB to downstream target regions. Specifically, it was found that sleep-promoting KCs increase sleep by preferentially activating cholinergic sleep-promoting MBONs, while wake-promoting KCs decrease sleep by preferentially activating glutamatergic wake-promoting MBONs. By using a combination of genetic and physiological approaches to identify wake-promoting dopaminergic neurons (DANs) that innervate the MB, it was shown that they activate wake-promoting MBONs. These studies reveal a dopaminergic sleep control mechanism that likely operates by modulation of KC-MBON microcircuits (Sitaraman, 2015b).

This study used a combination of sophisticated cell-specific genetic manipulations with behavioral sleep analysis and optical electrophysiology to identify compartment-specific wake-promoting MB DANs that activate wake-promoting microcircuits. Previous studies have implicated DANs innervating the central complex (CX) - a brain region involved in locomotor control - in regulating sleep, and other non-dopamingeric CX neurons have been implicated in homeostatic control of sleep. In addition, it has recently been shown that manipulations of dopamine signaling in the MB alter sleep, although the precise DANs involved remains unclear. This study has now identified specific wake-promoting MB DANs and shown that they innervate lobe compartments also innervated by wake-promoting KCs and MBONs. Importantly, this study has also shown that dopamine secretion by DANs innervating a particular MB lobe compartment acts through D1 subtype receptors to activate the wake-promoting microcircuit specific to that compartment to a much greater extent than it activates the sleep-promoting microcircuit residing in different compartments. This provides direct physiological evidence for compartment-specific dopamine signaling in the regulation of sleep by the MB, and is consistent with a previous study in the context of learning and memory (Boto, 2014). Future studies are required to determine additional cellular and molecular details of how dopamine signals modulate sleep-regulating microcircuits (Sitaraman, 2015b).

On the basis of recently published studies of MB control of sleep and the results presented in this study, a unified mechanistic model is proposed for homeostatic control of sleep by excitatory microcircuits in the Drosophila MB. Wake-promoting MBON-γ5β'2a/β'2mp/β'2mp_bilateral and sleep-promoting γ2α'1 each receive anatomical inputs from both wake-promoting γm and α'/β' KCs KCs and sleep-promoting γd KCs. However, segregation of sleep control information into separate microcircuits is enforced by greater synaptic weights between γ and γm and α'/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral, and between γm and α/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral, and between γd KCs and MBON-γ2α'1 (Sitaraman, 2015a). Thus it is hypothesize that compartment-specific dopamine signals from MB DANs could potentially determine these differences in synaptic weight. Future studies will test this hypothesis (Sitaraman, 2015b).

Interestingly, other fly behaviors have recently been found to be regulated by sleep-controlling compartment-specific MB microcircuits. For example, the integration of food odor to suppress innate avoidance of CO2 is mediated by MBON-γ5β'2a/β'2mp/β'2mp_bilateral and PAM DANs that innervate the β'2 compartment (Lewis, 2015). Optogenetic activation experiments reveal that wake-promoting γ5β'2a/β'2mp/β'2mp_bilateral mediates innate avoidance, while MBON-γ2α'1 mediates attraction. However, thermogenetic inactivation studies reveal that both MBON-γ5β'2a/β'2mp/β'2mp_bilateral and MBON-γ2α'1 are important for various forms of associative memory formation. These diverse waking behaviors that involve the activity of sleep-regulating neurons raises the interesting question whether such roles are independent, or causally linked, which future studies can address (Sitaraman, 2015b).

Importantly, this study has provided for the first time a cellular and molecular mechanism for for dopaminergic control of sleep through modulation of an associative network. While dopaminergic projections to cerebral cortex are known to be important for regulating sleep and arousal in mammals, underlying cellular and molecular mechanisms remain poorly understood, although D2 subtype dopamine receptors have been implicated in the control of REM sleep. Because of the possible evolutionary relationship between the MB and vertebrate forebrain associative networks (such as mammalian cerebral cortex), these studies thus provide a framework for the design of analogous experiments in genetically tractable vertebrate model systems such as zebrafish and mice (Sitaraman, 2015b)

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain"

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. This study shows in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, novel genetic, pharmacological and optical approaches were used in Drosophila. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H(+) antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, this study found that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects (Freyberg, 2016).

Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. This study has attempted advance understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. A mutant for Drosophila SERT (dSERT) was characterized, and additionally a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), was used whose mechanism of action involves the SERT protein. The results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior and decreased levels in an anxiety-related parameter, centrophobism. It was also shown that 4-MTA increases the negative chemotaxis towards a strong aversive odorant, Benzaldehyde. The neurochemical data suggest that this effect is mediated by dSERT and depends on 4-MTA-increased release of serotonin in the fly brain. The in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum neurochemical, in-silico and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila (Hidalgo, 2017).

The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for MDMA translocation and the induction of substrate efflux

The substituted amphetamine MDMA is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic. Previous, studies has identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. This study performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. The findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser (Sealover, 2016).

Transcriptional changes in specific subsets of Drosophila neurons following inhibition of the serotonin transporter

The transcriptional effects of SSRIs and other serotonergic drugs remain unclear, in part due to the heterogeneity of postsynaptic cells, which may respond differently to changes in serotonergic signaling. Relatively simple model systems such as Drosophila afford more tractable microcircuits in which to investigate these changes in specific cell types. This study focused on the mushroom body, an insect brain structure heavily innervated by serotonin and comprised of multiple different but related subtypes of Kenyon cells. Fluorescence-activated cell sorting of Kenyon cells, followed by either bulk or single-cell RNA sequencing were used to explore the transcriptomic response of these cells to SERT inhibition. The effects of two different Drosophila Serotonin Transporter (dSERT) mutant alleles as well as feeding the SSRI citalopram to adult flies were compared. The genetic architecture associated with one of the mutants contributed to significant artefactual changes in expression. Comparison of differential expression caused by loss of SERT during development versus aged, adult flies, suggests that changes in serotonergic signaling may have relatively stronger effects during development, consistent with behavioral studies in mice. Overall, these experiments revealed limited transcriptomic changes in Kenyon cells, but suggest that different subtypes may respond differently to SERT loss-of-function. Further work exploring the effects of SERT loss-of-function in other circuits may be used help to elucidate how SSRIs differentially affect a variety of different neuronal subtypes both during development and in adults (Bonanno, 2023).

Modulatory action by the serotonergic system: behavior and neurophysiology in Drosophila melanogaster

Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity (Majeed, 2016).

Visual attention in flies-dopamine in the mushroom bodies mediates the after-effect of cueing

Visual environments may simultaneously comprise stimuli of different significance. Often such stimuli require incompatible responses. Selective visual attention allows an animal to respond exclusively to the stimuli at a certain location in the visual field. In the process of establishing its focus of attention the animal can be influenced by external cues. This study characterized the behavioral properties and neural mechanism of cueing in the fly Drosophila melanogaster. A cue can be attractive, repulsive or ineffective depending upon (e.g.) its visual properties and location in the visual field. Dopamine signaling in the brain is required to maintain the effect of cueing once the cue has disappeared. Raising or lowering dopamine at the synapse abolishes this after-effect. Specifically, dopamine is necessary and sufficient in the αβ-lobes of the mushroom bodies. Evidence is provided for an involvement of the αβposterior Kenyon cells (Koenig, 2016).

Operation of a homeostatic sleep switch

In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. This study demonstrates state switching by dFB neurons, identifies dopamine as a neuromodulator that operates the switch, and delineates the switching mechanism. Arousing dopamine causes transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects are transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involves the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that was termed Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreases or increases sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state (Pimentel, 2016).

Recordings were made from dFB neurons (which were marked by R23E10-GAL4 or R23E10-lexA-driven green fluorescent protein (GFP) expression) while head-fixed flies walked or rested on a spherical treadmill. Because inactivity is a necessary correlate but insufficient proof of sleep, the analysis was restricted to awakening, which is defined as a locomotor bout after >5 min of rest, during which the recorded dFB neuron had been persistently spiking. To deliver wake-promoting signals, the optogenetic actuator CsChrimson was expressed under TH-GAL4 control in the majority of dopaminergic neurons, including the PPL1 and PPM3 clusters, whose fan-shaped body (FB)-projecting members have been implicated in sleep control. Illumination at 630 nm, sustained for 1.5 s to release a bolus of dopamine, effectively stimulated locomotion. dFB neurons paused in successful (but not in unsuccessful) trials, and their membrane potentials dipped by 2-13 mV below the baseline during tonic activity. When flies bearing an undriven CsChrimson transgene were photostimulated, neither physiological nor behavioural changes were apparent. The tight correlation between the suppression of dFB neuron spiking and the initiation of movement might, however, merely mirror a causal dopamine effect elsewhere, as TH-GAL4 labels dopaminergic neurons throughout the brain. Because localized dopamine applications to dFB neuron dendrites similarly caused awakening, this possibility is considered remote (Pimentel, 2016).

Flies with enhanced dopaminergic transmission exhibit a short-sleeping phenotype that requires the presence of a D1-like receptor in dFB neurons, suggesting that dopamine acts directly on these cells. dFB-restricted RNA interference (RNAi) confirmed this notion and pinpointed Dop1R2 as the responsible receptor, a conclusion reinforced by analysis of the mutant Dop1R2^MI08664 allele. Previous evidence that Dop1R1, a receptor not involved in regulating baseline sleep, confers responsiveness to dopamine when expressed in the dFB indicates that either D1-like receptor can fulfill the role normally played by Dop1R2. Loss of Dop1R2 increased sleep during the day and the late hours of the night, by prolonging sleep bouts without affecting their frequency. This sleep pattern is consistent with reduced sensitivity to a dopaminergic arousal signal (Pimentel, 2016).

To confirm the identity of the effective transmitter, avoid dopamine release outside the dFB, and reduce the transgene load for subsequent experiments, optogenetic manipulations of the dopaminergic system were replaced with pressure ejections of dopamine onto dFB neuron dendrites. Like optogenetically stimulated secretion, focal application of dopamine hyperpolarized the cells and suppressed their spiking. The inhibitory responses could be blocked at several nodes of an intracellular signalling pathway that connects the activation of dopamine receptors to the opening of potassium conductances: by RNAi-mediated knockdown of Dop1R2; by the inclusion in the patch pipette of pertussis toxin (PTX), which inactivates heterotrimeric G proteins of the Gi/o family; and by replacing intracellular potassium with caesium, which obstructs the pores of G-protein-coupled inward-rectifier channels. Elevating the chloride reversal potential above resting potential left the polarity of the responses unchanged, corroborating that potassium conductances mediate the bulk of dopaminergic inhibition (Pimentel, 2016).

Coupling of Dop1R2 to Gi/o, although documented in a heterologous system, represents a sufficiently unusual transduction mechanism for a predicted D1-like receptor to prompt verification of its behavioural relevance. Like the loss of Dop1R2, temperature-inducible expression of PTX in dFB neurons increased overall sleep time by extending sleep bout length (Pimentel, 2016).

While a single pulse of dopamine transiently hyperpolarized dFB neurons and inhibited their spiking, prolonged dopamine applications (50 ms pulses at 10 Hz, or 20 Hz optogenetic stimulation, both sustained for 2-10 min) switched the cells from electrical excitability (ON) to quiescence (OFF). The switching process required dopamine as well as Dop1R2, but once the switch had been actuated the cells remained in the OFF state-and flies, awake-without a steady supply of transmitter. Input resistances and membrane time constants dropped to 53.3 ± 1.8 and 24.0 ± 1.3% of their initial values (means ± s.e.m.), and depolarizing currents no longer elicited action potentials (15 out of 15 cells). The biophysical properties of single dFB neurons, recorded in the same individual before and after operating the dopamine switch, varied as widely as those in sleep-deprived and rested flies (Pimentel, 2016).

Dopamine-induced changes in input resistance and membrane time constant occurred from similar baselines in all genotypes and followed single-exponential kinetics with time constants of 1.07-1.10 min. The speed of conversion points to post-translational modification and/or translocation of ion channels between intracellular pools and the plasma membrane as the underlying mechanism(s). In 7 out of 15 cases, recordings were held long enough to observe the spontaneous recommencement of spiking, which was accompanied by a rise to baseline of input resistance and membrane time constant, after 7-60 min of quiescence (mean ± s.e.m. = 25.86 ± 7.61 min). The temporary suspension of electrical output is thus part of the normal activity cycle of dFB neurons and not a dead end brought on by the experimental conditions (Pimentel, 2016).

dFB neurons in the ON state expressed two types of potassium current: voltage-dependent A-type (rapidly inactivating) and voltage-independent non-A-type currents. The current-voltage (I-V) relation of iA resembled that of Shaker, the prototypical A-type channel: no current flowed below -50 mV, the approximate voltage threshold of Shaker; above -40 mV, peak currents increased steeply with voltage and inactivated with a time constant of 7.5 ± 2.1 ms (mean ± s.e.m.). Non-A-type currents showed weak outward rectification with a reversal potential of -80 mV, consistent with potassium as the permeant ion, and no inactivation (Pimentel, 2016).

Switching the neurons OFF changed both types of potassium current. iA diminished by one-third, whereas inon-A nearly quadrupled when quantified between resting potential and spike threshold. The weak rectification of inon-A in the ON state vanished in the OFF state, giving way to the linear I-V relationship of an ideal leak conductance. dFB neurons thus upregulate iA in the sleep-promoting ON state. When dopamine switches the cells OFF, voltage-dependent currents are attenuated and leak currents augmented. This seesaw form of regulation should be sensitive to perturbations of the neurons' ion channel inventory: depletion of voltage-gated A-type (KV) channels (which predominate in the ON state) should tip the cells towards the OFF state; conversely, loss of leak channels (which predominate in the OFF state) should favour the ON state. To test these predictions, sleep was examined in flies carrying R23E10-GAL4-driven RNAi transgenes for dFB-restricted interference with individual potassium channel transcripts (Pimentel, 2016).

RNAi-mediated knockdown of two of the five KV channel types of Drosophila (Shaker and Shab) reduced sleep relative to parental controls, while knockdown of the remaining three types had no effect. Biasing the potassium channel repertoire of dFB neurons against A-type conductances thus tilts the neurons' excitable state towards quiescence, causing insomnia, but leaves transient and sustained dopamine responses unaffected. The seemingly counterintuitive conclusion that reducing a potassium current would decrease, not increase, action potential discharge is explained by a requirement for A-type channels in generating repetitive activity of the kind displayed by dFB neurons during sleep. Depleting Shaker from dFB neurons shifted the interspike interval distribution towards longer values, as would be expected if KV channels with slow inactivation kinetics replaced rapidly inactivating Shaker as the principal force opposing the generation of the next spike. These findings identify a potential mechanism for the short-sleeping phenotypes caused by mutations in Shaker, its β subunit Hyperkinetic, or its regulator sleepless (Pimentel, 2016).

Leak conductances are typically formed by two-pore-domain potassium (K2P) channels. dFB-restricted RNAi of one member of the 11-strong family of Drosophila K2P channels, encoded by the CG8713 gene, increased sleep relative to parental controls; interference with the remaining 10 K2P channels had no effect. Recordings from dFB neurons after knockdown of the CG8713 gene product, which this study termed Sandman, revealed undiminished non-A-type currents in the ON state and intact responses to a single pulse of dopamine but a defective OFF switch: during prolonged dopamine applications, inon-A failed to rise, input resistances and membrane time constants remained at their elevated levels, and the neurons continued to fire action potentials (7 out of 7 cells). Blocking vesicle exocytosis in the recorded cell with botulinum neurotoxin C (BoNT/C) similarly disabled the OFF switch. This, combined with the absence of detectable Sandman currents in the ON state, suggests that Sandman is internalized in electrically active cells and recycled to the plasma membrane when dopamine switches the neurons OFF (Pimentel, 2016).

Because dFB neurons lacking Sandman spike persistently even after prolonged dopamine exposure, voltage-gated sodium channels remain functional in the OFF state. The difficulty of driving control cells to action potential threshold in this state must therefore be due to a lengthening of electrotonic distance between sites of current injection and spike generation. This lengthening is an expected consequence of a current leak, which may uncouple the axonal spike generator from somatodendritic synaptic inputs or pacemaker currents when sleep need is low (Pimentel, 2016).

The two kinetically and mechanistically distinct actions of dopamine on dFB neurons-instant, but transient, hyperpolarization and a delayed, but lasting, switch in excitable state-ensure that transitions to vigilance can be both immediate and sustained, providing speedy alarm responses and stable homeostatic control. The key to stability lies in the switching behaviour of dFB neurons, which is driven by dopaminergic input accumulated over time. Unlike bistable neurons, in which two activity regimes coexist for the same set of conductances, dFB neurons switch regimes only when their membrane current densities change. This analysis of how dopamine effects such a change, from activity to silence, has uncovered elements familiar from other modulated systems: simultaneous, antagonistic regulation of multiple conductances; reduction of iA; and modulation of leak currents. Currently little is known about the reverse transition, from silence to activity, except that mutating the Rho-GTPase-activating protein Crossveinless-c locks dFB neurons in the OFF state, resulting in severe insomnia and an inability to correct sleep deficits. Discovering the signals and processes that switch sleep-promoting neurons back ON will hold important clues to the vital function of sleep (Pimentel, 2016).

Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment

Major depressive disorder (MDD) affects millions of patients; however, the pathophysiology is poorly understood. Rodent models have been developed using chronic mild stress or unavoidable punishment (learned helplessness) to induce features of depression, like general inactivity and anhedonia. This study reports a three-day vibration-stress protocol for Drosophila that reduces voluntary behavioural activity. As in many MDD patients, lithium-chloride treatment can suppress this depression-like state in flies. The behavioural changes correlate with reduced serotonin (5-HT) release at the mushroom body (MB) and can be relieved by feeding the antidepressant 5-hydroxy-L-tryptophan or sucrose, which results in elevated 5-HT levels in the brain. This relief is mediated by 5-HT-1A receptors in the α-/β-lobes of the MB, whereas 5-HT-1B receptors in the γ-lobes control behavioural inactivity. The central role of serotonin in modulating stress responses in flies and mammals indicates evolutionary conserved pathways that can provide targets for treatment and strategies to induce resilience (Ries, 2017).

A serotonin-modulated circuit controls sleep architecture to regulate cognitive function independent of total sleep in Drosophila

Both the structure and the amount of sleep are important for brain function. Entry into deep, restorative stages of sleep is time dependent; short sleep bouts selectively eliminate these states. Fragmentation-induced cognitive dysfunction is a feature of many common human sleep pathologies. Whether sleep structure is normally regulated independent of the amount of sleep is unknown. This study shows that in Drosophila melanogaster, activation of a subset of serotonergic neurons fragments sleep without major changes in the total amount of sleep, dramatically reducing long episodes that may correspond to deep sleep states. Disruption of sleep structure results in learning deficits that can be rescued by pharmacologically or genetically consolidating sleep. Two reciprocally connected sets of ellipsoid body neurons were identified that form the heart of a serotonin-modulated circuit that controls sleep architecture. Taken together, these findings define a circuit essential for controlling the structure of sleep independent of its amount (Liu, 2019).

This study describes a circuit that regulates of sleep structure without affecting the total amount of sleep. 5HT acts to enhance the response of 5HT7-GAL4+ neurons to basally active excitatory inputs. 5HT-dependent calcium signals are blocked by TTX, while its ability to increase cAMP is not, supporting the existence of these active excitatory inputs to 5HT7-GAL4+ cells. In contrast, VT-GAL4+ cells do not have basally active excitatory inputs. 5HT modulation of the circuit likely occurs primarily via inputs to 5HT7-GAL4+ neurons since the response of VT038828-GAL4 (VT-GAL4)+ neurons is weaker and lower affinity. Whether there are other, perhaps situationally active, inputs to this circuit is currently unknown (Liu, 2019).

Within the ellipsoid body (EB) the circuit is complex. VT-GAL4+ neurons are functionally connected with the 5HT7-GAL4+ group. VT-GAL4+ neurons provide feedback inhibition to a subset of 5HT7-GAL4+ neurons, which enhances fragmentation, likely via output to non-central complex regions. How inhibition of a subset of the 5HT7-GAL4+ cells acts to modulate the behavioral output of the rest of the population is not yet clear, but it is noted that many of the 5HT7-GAL4+ cells are GABAergic. While all the details of the circuit's complex dynamics remain to be discovered, it is clear that this circuit has a profound and selective effect on sleep architecture (Liu, 2019).

The circuit described in this study is modulated by 5HT, a neurochemical known to be important for regulation of behavioral states in many species. While 5HT in mammals is important in a wide variety of contexts, it was controversial for nearly half a century whether it promoted sleep or wakefulness. In Drosophila, 5HT has only been thought to promote sleep. The current data show that upregulation of serotonergic signaling can also induce sleep fragmentation, suggesting that 5HT's role in sleep in flies exhibits a complexity similar to that of its roles in mammals. The genesis of this apparently conserved complexity may be the extensive involvement of 5HT in non-sleep processes. For an animal in the wild, sleep has inherent risks: predation and loss of opportunities for mating or feeding are just a few. Sleep/wake systems in the brain must control arousal state in collaboration with systems that assess competing needs. 5HT, because it is central to so many critical behavioral circuits, is ideally poised to be an integration point for sleep and the general state of the animal. The diverse, circuit-specific, roles in sleep that 5HT exhibits across phyla may be a result of its ubiquity (Liu, 2019).

The role this study has uncovered for 5HT as a regulator of sleep architecture aligns well with this idea. The daily neuronal activity profile reported by Tric-LUC, a calcium sensor that drives luciferase expression in response to neuronal activity, in sleep fragmentation-generating neurons maps to dawn and dusk, when crepuscular organisms such as fruit flies are most active. Fragmentation of sleep at these times would presumably be beneficial since flies would not enter into deep sleep states at times when they should be feeding and mating. Interestingly, the circuit described in this study accomplishes this feat by increasing P(doze), the probability of falling asleep from a wake state, leaving the scaling of P(wake), a parameter associated with dopamine and arousal, free to be modulated by other factors (e.g., danger from predation, appearance of potential mates). The fact that long sleep bouts can be prevented without putting the animal into a hyperaroused state is advantageous, allowing flexible responsiveness to changing conditions. The involvement of P(Doze), a parameter associated with sleep drive, is also congruent with the sleep-promoting role of 5HT in other brain regions (Liu, 2019).

While controlled sleep fragmentation appears to assist in active period behavior, there is also a need for consolidated sleep. In both mammals and Drosophila, sleep has electrophysiologically distinct substrates with progressively higher arousal thresholds that appear in an ordered fashion during a sleep episode. The deeper sleep stages in mammals, REM and slow wave sleep, are strongly associated with maintenance of cognitive function. Fragmentation of sleep, because it truncates sleep episodes before deeper stages are reached, can result in a selective deprivation of deep sleep stages even when total sleep is not changed. In this study, it was demonstrated that decreasing sleep consolidation, without changing the amount of sleep, can disrupt associative learning. These results suggest that in Drosophila, like in mammals, there are time-dependent changes in the depth of sleep that are important for its beneficial effects. This idea is also supported by modeling and analysis of the structure of fly sleep, which indicate that there are time-dependent changes in the probability of sleep-wake transitions consistent with the existence of deep sleep stages that are only accessed in long sleep episodes (Liu, 2019).

Fragmentation of sleep induced by activation of 5HT inputs to the EB also produced an increase in sleep after the activation was terminated. Excess sleep in the recovery day after a perturbation is a hallmark of a homeostatic process. Homeostatic regulation of total sleep time has been previously demonstrated in Drosophila, but the data suggest that there is also homeostatic regulation of sleep quality. In mammals, individual sleep substates have been demonstrated to be homeostatically regulated- selective deprivation of REM or slow wave sleep, in the absence of loss of total sleep time, drive rebound increases of the deprived stage and mechanical sleep fragmentation has been shown to lead to an increase in total sleep. The ability of the EB circuit in Drosophila to selectively modulate sleep structure, without changing the total amount of sleep, has allowed for the first time the selective probing of the cognitive importance of long sleep bouts and deep sleep stages in the fly. The fact that fragmentation triggers rebound sleep implies that these long sleep bouts may also be important for the general health benefits of sleep (Liu, 2019).

Serotonergic modulation of visual neurons in Drosophila melanogaster

Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila. This study mapped of patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While serotonin receptor expression in L1 neurons was not detected, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila (Sampson, 2020).

A conserved role for sleep in supporting Spatial Learning in Drosophila

Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. This study used the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a 'thermal maze' consisting of a 5X5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold-tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains - Cs and 2U, validating the design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, flies exhibited dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, Spatial Learning was found to requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. These results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging (Melnattur, 2020).

Serotoninergic Modulation of Phototactic Variability Underpins a Bet-Hedging Strategy in Drosophila melanogaster

When organisms' environmental conditions vary unpredictably in time, it can be advantageous for individuals to hedge their phenotypic bets. It has been shown that a bet-hedging strategy possibly underlies the high inter-individual diversity of phototactic choice in Drosophila melanogaster. This study shows that fruit flies from a population living in a boreal and relatively unpredictable climate have more variable phototactic biases than fruit flies from a more stable tropical climate, consistent with bet-hedging theory. This study experimentally showed that phototactic variability of D. melanogaster is regulated by the neurotransmitter serotonin (5-HT), which acts as a suppressor of the variability of phototactic choices. When fed 5-HT precursor, boreal flies exhibited lower variability, and they were insensitive to 5-HT inhibitor. The opposite pattern was seen in the tropical flies. Thus, the reduction of 5-HT in fruit flies' brains may be the mechanistic basis of an adaptive bet-hedging strategy in a less predictable boreal climate (Krams, 2021).

Biological functions of alpha2-adrenergic-like octopamine receptor in Drosophila melanogaster

Octopamine regulates various physiological phenomena including memory, sleep, grooming and aggression in insects. In Drosophila, four types of octopamine receptors have been identified: Oamb, Oct/TyrR, OctβR and Octα2R. Among these receptors, Octalpha2R was recently discovered and pharmacologically characterized. However, the effects of the receptor on biological functions are still unknown. This study showed that Octα2R regulated several behaviors related to octopamine signaling. Octα2R hypomorphic mutant flies showed a significant decrease in locomotor activity. Octα2R expressed in the pars intercerebralis, which is a brain region projected by octopaminergic neurons, is involved in control of the locomotor activity. Besides, Octα2R hypomorphic mutants increased time and frequency of grooming and inhibited starvation-induced hyperactivity. These results indicated that Octα2R expressed in the central nervous system is responsible for the involvement in physiological functions (Nakagawa, 2022).

Maintenance of quiescent oocytes by noradrenergic signals

All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. This paper reports that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles. This study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes (Kim, 2021).

Autism-like behaviors regulated by the serotonin receptor 5-HT2B in the dorsal fan-shaped body neurons of Drosophila melanogaster

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and repetitive stereotyped behaviors. Previous studies have reported an association of serotonin or 5-hydroxytryptamine (5-HT) with ASD, but the specific receptors and neurons by which serotonin modulates autistic behaviors have not been fully elucidated. RNAi-mediated knockdown was done to destroy the function of tryptophan hydroxylase (Trh) and all the five serotonin receptors. Given that ubiquitous knockdown of 5-HT2B showed significant defects in social behaviors, the CRISPR/Cas9 system was used to knock out the 5-HT2B receptor gene. Social space assays and grooming assays were the major methods used to understand the role of serotonin and related specific receptors in autism-like behaviors of Drosophila melanogaster. A close relationship was identified between serotonin and autism-like behaviors reflected by increased social space distance and high-frequency repetitive behavior in Drosophila. The binary expression system was further utilized to knock down all the five 5-HT receptors; the 5-HT2B receptor was observed to act as the main receptor responsible for the normal social space and repetitive behavior in Drosophila for the specific serotonin receptors underlying the regulation of these two behaviors. These data also showed that neurons in the dorsal fan-shaped body (dFB), which expressed 5-HT2B, were functionally essential for the social behaviors of Drosophila. Collectively, these data suggest that serotonin levels and the 5-HT2B receptor are closely related to the social interaction and repetitive behavior of Drosophila. Of all the 5 serotonin receptors, 5-HT2B receptor in dFB neurons is mainly responsible for serotonin-mediated regulation of autism-like behaviors (Cao, 2022).

Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development

The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. This study explored this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. This developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, this study showed that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. These effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development (Deliu, 2022).

An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency

NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. This study used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which was experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease (Hope, 2022).

SSRI antidepressants differentially modulate serotonin reuptake and release in Drosophila

Selective serotonin reuptake inhibitor (SSRI) antidepressants are commonly prescribed treatments for depression, but their effects on serotonin reuptake and release are not well understood. Drosophila expresses the serotonin transporter (dSERT), the major target of SSRIs, but real-time serotonin changes after SSRIs have not been characterized in this model. Various doses of fluoxetine (Prozac), escitalopram (Lexapro), citalopram (Celexa), and paroxetine (Paxil), were applied to ventral nerve cord (VNC) tissue and optogenetically-stimulated serotonin release was measured with fast-scan cyclic voltammetry (FSCV). Fluoxetine increased reuptake from 1-100 μM, but serotonin concentration only increased at 100 μM. Thus, fluoxetine occupies dSERT and slows clearance, but does not affect concentration. Escitalopram and paroxetine increased serotonin concentrations at all doses, but escitalopram increased reuptake more. Citalopram showed lower concentration changes and faster reuptake profiles compared to escitalopram, so the racemic mixture of citalopram does not change reuptake as much as the S-isomer. Dose response curves were constructed to compare dSERT affinities and paroxetine showed the highest affinity and fluoxetine the lowest. These data demonstrate SSRI mechanisms are complex, with separate effects on reuptake or release (Dunham, 2022).

Development under predation risk increases serotonin-signaling, variability of turning behavior and survival in adult fruit flies Drosophila melanogaster

The development of high-throughput behavioral assays, where numerous individual animals can be analyzed in various experimental conditions, has facilitated the study of animal personality. Previous research showed that isogenic Drosophila melanogaster flies exhibit striking individual non-heritable locomotor handedness. The variability of this trait, i.e., the predictability of left-right turn biases, varies across genotypes and under the influence of neural activity in specific circuits. This suggests that the brain can dynamically regulate the extent of animal personality. It has been recently shown that predators can induce changes in prey phenotypes via lethal or non-lethal effects affecting the serotonergic signaling system. In this study, we tested whether fruit flies grown with predators exhibit higher variability/lower predictability in their turning behavior and higher survival than those grown with no predators in their environment. These predictions were confirmed, and it was found that both effects were blocked when flies were fed an inhibitor (αMW) of serotonin synthesis. The results of this study demonstrate a negative association between the unpredictability of turning behavior of fruit flies and the hunting success of their predators. It was also shown that the neurotransmitter serotonin controls predator-induced changes in the turning variability of fruit flies, regulating the dynamic control of behavioral predictability (Krama, 2023).

Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila

Kohlschutter-Tonz syndrome (KTS) is a rare genetic disorder with neurological dysfunctions including seizure and intellectual impairment. Mutations at the Rogdi locus have been linked to development of KTS, yet the underlying mechanisms remain elusive. This study demonstrates that a Drosophila homolog of Rogdi acts as a novel sleep-promoting factor by supporting a specific subset of gamma-aminobutyric acid (GABA) transmission. Rogdi mutant flies displayed insomnia-like behaviors accompanied by sleep fragmentation and delay in sleep initiation. The sleep suppression phenotypes were rescued by sustaining GABAergic transmission primarily via metabotropic GABA receptors or by blocking wake-promoting dopaminergic pathways. Transgenic rescue further mapped GABAergic neurons as a cell-autonomous locus important for Rogdi-dependent sleep, implying metabotropic GABA transmission upstream of the dopaminergic inhibition of sleep. Consistently, an agonist specific to metabotropic but not ionotropic GABA receptors titrated the wake-promoting effects of dopaminergic neuron excitation. Taken together, these data provide the first genetic evidence that implicates Rogdi in sleep regulation via GABAergic control of dopaminergic signaling. Given the strong relevance of GABA to epilepsy, it is proposed that similar mechanisms might underlie the neural pathogenesis of Rogdi-associated KTS (Kim, 2017).

Neurological disorders caused by single-gene mutations are important genetic models to understand how individual genes execute their roles to support the development and function of the brain, as in the case of Kohlschutter-Tönz syndrome (KTS). KTS patients display developmental delays and psychomotor regression. The most prominent symptoms include amelogenesis imperfecta, early-onset seizures, and intellectual disabilities. Linkage analyses followed by genomic sequencing have revealed that most, if not all, KTS patients have homozygous nonsense, frameshift deletion, or splicing site mutations at the Rogdi locus. ROGDI protein expression is not detectable in affected individuals, indicating that the loss of Rogdi function is responsible for the pathogenesis of KTS. In wild-type human tissues, Rogdi transcripts are ubiquitously expressed while the highest enrichment is evident in adult brain and spinal cord. This observation is consistent with the neurological phenotypes observed in KTS patients (Kim, 2017).

Given that Rogdi homologs are relatively well conserved in higher eukaryotes, animal models may facilitate understanding of Rogdi-dependent neural processes. In fact, Rogdi was initially identified in a Drosophila genetic screen as a memory-relevant gene and was thus named after one of Pavlov’s dogs. Sequence analyses of Rogdi homologs revealed a putative leucine zipper (ZIP) motif, which could mediate the dimerization of DNA-binding basic ZIP (bZIP) transcription factors. Interestingly, ROGDI proteins localize to the nuclear envelope in cultured human cells, although they lack basic amino acid residues that are typically located at the N-terminus of the ZIP domain and are required for the DNA-binding and nuclear localization of the bZIP transcription factors. Nonetheless, few or no studies have demonstrated the biological activity of Rogdi and genetic models for Rogdi homologs have not been reported yet. Therefore, how Rogdi exerts its physiological roles particularly in the central nervous system and how its mutation leads to the development of KTS are largely unknown (Kim, 2017).

In the course of genetic studies to elucidate genes and regulatory pathways involved in sleep behaviors, novel sleep mutant alleles were identified in the Drosophila Rogdi gene. This study employed the sleep-promoting effects of Rogdi as a readout of its neural function and demonstrated that Rogdi acts cell-autonomously in GABAergic neurons to enhance metabotropic GABA transmission and thereby sustain sleep. In addition, dopaminergic rescue of Rogdi mutant sleep revealed a novel sleep-regulatory mechanism that functionally links a specific subset of sleep-promoting GABAergic neurons to a wake-promoting dopaminergic pathway. Since epilepsy, a well penetrated phenotype in KTS patients, implicates GABAergic transmission, the current findings provide an important genetic clue to understanding the molecular and neural pathogenesis of KTS (Kim, 2017).

Modeling of neurological diseases and disease-relevant genes has greatly advanced understanding of the fundamental principles that underlie disease pathogenesis as well as brain function. This study has established the first genetic model of the KTS-associated disease gene Rogdi to demonstrate that Rogdi functions as a novel sleep-promoting factor in GABAergic neurons by promoting GABA transmission. While GABA-dependent sleep regulation via ionotropic GABA receptors have been well documented in Drosophila, the data suggest that GABAergic transmission via metabotropic GABA receptors might be primarily compromised by Rogdi mutation. Furthermore, the wake-promoting DA pathway was identified as a neural locus downstream of Rogdi-dependent GABA signaling given that Rogdi mutant sleep could be rescued by pharmacological or genetic manipulation of dopaminergic transmission. This sleep-regulatory pathway was further supported by the observation that wake-promoting effects of TH-expressing dopaminergic neurons could be selectively titrated by an agonist of metabotropic GABA receptors. Rogdi thus defines a novel pathway coupling these two neurotransmitters to promote baseline sleep in Drosophila as exemplified in other behavioral paradigms across species. On the other hand, Rogdi-dependent GABA transmission might have inhibitory effects on a sleep-promoting neural pathway for sleep homeostasis to suppress recovery sleep after sleep loss (Kim, 2017).

What is the molecular basis by which Rogdi supports GABAergic transmission and promotes sleep? A possible role of ROGDI as a transcription factor has been suggested by the nuclear localization of human ROGDI protein, particularly in the nuclear envelope of blood mononuclear cells and dermal fibroblasts, and by the conservation of a putatively dimerizing leucine zipper (ZIP) motif among ROGDI homologs. Several lines of evidence, however, argue against this possibility. bZIP transcription factors possess basic residues followed by their ZIP domains whereas ROGDI protein lacks the canonical motif (i.e., basic residues) for DNA-binding activity and nuclear localization. Drosophila ROGDI actually displays its subcellular distribution in both nucleus and cytoplasm of cultured cells or adult fly neurons although the exclusive nuclear localization might not be a prerequisite for transcriptional activities. The crystal structure of human ROGDI protein showed that, unlike other bZIP transcription factors, human ROGDI protein exists as a monomer containing two structurally distinguishable domains (designated as α and β domains, respectively). The α domain exhibits an α-helical bundle that consists of H1, H2, H3, and H6 helices. In fact, the ZIP-like motif in the α domain appears to mediate their intramolecular interactions, contributing to the overall structure and stability of a monomeric ROGDI protein. Based on sequence homology between Drosophila and human ROGDI proteins, it is predicted that Rogdi[del] allele removes the majority of the first helix including the repeated leucine residues in the α domain and the first three strands in the β domain, explaining the instability of ROGDI^del proteins. A smaller but comparable deletion of the ZIP-like motif has been reported in a KTS patient with a splicing mutation in human Rogdi gene. In addition, a functional study in cervical cancer cell lines demonstrated Rogdi effects on cell cycle progression and radio-sensitivity. However, further investigations will be required to understand how these cellular phenotypes could be linked to the molecular and neural function of ROGDI protein (Kim, 2017).

What will be the relevance of these findings to KTS pathogenesis? Genetic heterogeneity has been reported among KTS patients, indicating that Rogdi-independent genetic mutations could contribute to KTS pathogenesis. A recent study indeed showed that familial mutations in a sodium-citrate transporter gene SLC13A5 are the second genetic cause of KTS. The pathogenic phenotypes commonly found in Rogdi- and SLC13A5-associated KTS gives rise to the intriguing possibility that these two genes might work together to control the intracellular levels of citrate. This idea is further supported by the relevance of citrate metabolism to neurological phenotypes in KTS patients. Neurons are energetically dependent on astrocytes because neurons lack pyruvate carboxylase, an enzyme that converts pyruvate to oxaloacetate in the citric acid cycle. SLC13A5 plays an important role in the transport of glial citrate into neurons to supplement the neuronal citric acid cycle and thereby supply cellular energy. Furthermore, citrate, an intermediate in the citric acid cycle, acts as a precursor of α-ketoglutarate, which can be metabolized to glutamate and GABA, implicating SLC13A5 in the biogenesis of GABA. Consistently, anti-epileptic drugs that elevate GABAergic transmission rescued the seizure phenotypes in SLC13A-associated KTS patients. In addition, this study showed that the pharmacological enhancement of GABAergic transmission by oral administration of GABA-T or GAT inhibitors was sufficient to rescue the short sleep phenotypes in Rogdi mutant flies (Kim, 2017).

These genetic studies strongly implicate Rogdi function in GABAergic transmission, providing the first clue to understanding the neurological phenotypes observed in KTS patients. Molecular and neural deficits selectively caused by Rogdi mutation might explain why seizures in Rogdi-associated KTS are often resistant to anti-epileptic drugs. Future studies should thus address if Rogdi mutant flies display seizure-like behaviors similarly as in KTS patients and if Rogdi-dependent neural relay of GABAergic transmission controls seizure susceptibility in parallel with baseline sleep. In addition, it will be important to determine whether sleep deficiencies are also observed in KTS patients and whether reduced GABAergic transmission in Rogdi- and, possibly, SLC13A5-associated KTS patients is responsible for their neural dysfunctions, including early-onset seizures. Taken together, this genetic model would constitute an important platform for elucidating the molecular and neural pathogenesis underlying KTS and hint towards a precise development of a therapeutic strategy for KTS in the future (Kim, 2017).

LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila

Amino-acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis and energy metabolism. In addition, the existence of bidirectional relationships between extracellular content, transport systems and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino-acid transport and sleep/wake regulation remains elusive. To address this question, this study used Drosophila melanogaster and investigated the role of LAT1 (Large neutral Amino-acid Transporter 1) transporters. This study shows that the two Drosophila LAT1-like transporters: JhI-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, amino-acid transport efficiency was assessed by L-DOPA feeding. Downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate TOR (Target Of Rapamycin) signaling, the role of this amino-acid sensing pathway in dopaminergic neurons was investigated. Consistently, it is reported that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1 mediated amino-acid transport in dopaminergic neurons, is playing a significant role in sleep/wake regulation, and is providing several entry points to elucidate the role of nutrients such as amino-acids in sleep/wake regulation (Aboudhiaf, 2018).

Emerging evidence suggests bidirectional relationships between extracellular space content and vigilance states, emphasizing the so far little explored sleep-regulatory role of the transmembrane transport of ions and small molecules. Sleep and wakefulness have a pervasive impact on brain cellular activities linked to neurotransmission, neuronal plasticity, neurotransmitter synthesis, nutrient supply, and waste elimination, relying on the efficient and precise coordination of transport systems. Investigating how transporters and underlying molecular and cellular mechanisms are involved in these mutual interactions requires the targeting of individual genes in specific cell types. This strategy is highly amenable to the Drosophila model. Among the large array of cellular transporters present in the genome and conserved between insects and mammals, the well-characterized large neutral amino acid transporters are particularly relevant given their role in neurotransmitter synthesis and recycling, in the regulation of synaptic strength, in protein synthesis and energy metabolism. In addition, the de novo synthesis of brain monoamines associated with wakefulness and neuromodulation, such as serotonin, dopamine, and noradrenaline, is dependent on large neutral essential amino acids provided by the blood (Aboudhiaf, 2018).

The SLC7A5 (or large neutral amino-acid transporter, light chain or LAT1) and SLC7A8 (LAT2) amino acid transporters are present in most cell types and appear to play a prominent role in the Na+-independent transport of large branched and aromatic neutral amino acids. These transporters belong to the heterodimeric amino acid transporters (HAT) family and require co-expression of the CD98hc /4F2hc (SLC3A2) heavy chain, to which they can be covalently linked by a di-sulfur bridge. The heavy subunit does not appear to confer transport-specific properties, nor to be confined to HAT transporter function. In the mammalian brain, LAT1 is highly expressed in the cells of the blood-brain-barrier and is thought to play a critical role in providing the central nervous system with essential amino acids such as phenylalanine, tyrosine, leucine, and tryptophan, which are nutrients and precursors for monoamine synthesis. The uptake of leucine through LAT1 is a major activator signal for target of rapamycin complex 1 (TORC1), a cellular pathway dependent on the TOR kinase that controls protein synthesis, brain excitability, and plasticity. Reciprocally, inhibition of mTOR by rapamycin has been shown to significantly reduce the activity and the mRNA expression of LAT1. Despite a few pieces of evidence, it remains to be investigated whether LAT1, LAT2, or other SLC7A transporters are also localized in neurons, and whether their function is linked to sleep and wake. In Drosophila, the HAT family of transporters is represented by one heavy chain, CD98hc, and five light chains: juvenile hormone inducible-21 (JHI-21), minidiscs (MND), genderblind (GB), CG9413 protein, and CG1607 protein. The specific activity of these transporters cannot be easily predicted from their sequences and requires functional testing. JHI-21 and MND can transport leucine, are inhibited by BCH (2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and, at least for JHI-21, require CD98hc to become functional, thus classifying them as LAT1-LAT2 homologs. Both genes are expressed at high levels in the central nervous system and have been shown to play important neurophysiological functions. JHI-21 and GB regulate glutamatergic synaptic strength, primarily through the regulation of extracellular glutamate levels, and are, respectively, required in motor neurons and in glial cells. In a recent report, it has also been shown that MND is required to activate brain insulin-producing neurons in response to circulating leucine. Interestingly those same insulin-producing neurons are connected to wakefulness promoting circuits. This study used the molecular-genetic tools of Drosophila to investigate the potential impact of JhI-21 and Mnd downregulation in neuronal subsets on sleep/wake regulation (Aboudhiaf, 2018).

Evidence is provided that the LAT-1 like transporters JhI-21 and Mnd are required in adult fly dopaminergic neurons to achieve adequate sleep/wake regulation. The results demonstrate that a downregulation of these transporters in dopaminergic neurons results in a decrease in wakefulness, under baseline conditions but also in conditions that increase dopaminergic transmission. This implies that the activity of dopaminergic neurons and/or their ability to release neurotransmitter requires JhI-21 and Mnd. These two amino acid transporters are the closest drosophila LAT1 homologs based on sequence and functional data, suggesting that some amino acid availability plays a critical role in dopaminergic neuronal function. Supporting this hypothesis, this study finds that downregulating the TOR pathway in dopaminergic neurons results in a decrease in wakefulness (Aboudhiaf, 2018).

As in mammals, dopaminergic transmission plays a major role in Drosophila wakefulness and has been suggested to constitute a core ancestral regulator of arousal and sleep entry across invertebrates. Drosophila and mammals share homologs for genes playing a central role in dopamine synthesis, reuptake, and signaling. This includes two D1-like receptors and one D2-like dopamine receptor. Among those, the D1-like receptor Dop1R1 (dDA1) plays a prominent role in sleep/wake regulation. Dop1R1 mutant flies display high daily sleep, longer sleep bout duration, and normal waking activity, a phenotype that closely resembles the one obtained in this studu by downregulating JhI-21 or Mnd in dopaminergic neurons. The effect of Dop1R1 on sleep/wake depends on its expression in the dorsal fan-shaped body (dFB), a key sleep-wake regulatory structure, and on the release of dopamine by a very limited set of dopaminergic neurons projecting to the dFB and located in the PPL1 and PPM3 cluster. Thus, it is possible that Mnd and JhI-21 are required in those specific neurons to achieve normal sleep-wake regulation. Expressing Mnd and JhI-21 UAS-RNAi constructs in PPL1 and PPM3 neurons failed to produce an abnormal sleep/wake phenotype. This lack of effect may be attributable to the limited efficiency of the UAS-RNAi constructs. Alternatively, the inhibition of JhI-21 and Mnd may affect multiple dopamine dependent microcircuits throughout the brain and thus cannot be easily replicated by a more specific manipulation (Aboudhiaf, 2018).

What function could JHI-21 and MND fulfill in dopaminergic neurons? At the glutamatergic neuromuscular junction, JHI-21 appears to regulate the clustering of post-synaptic glutamate receptors. JHI-21 does not appear to trigger directly the release of glutamate in this context, but possibly mediates the entry of amino acids such as leucine to activate molecular pathways controlling glutamatergic physiology. Although abnormal sleep/wake regulation was not observed when JhI-21 was inhibited in glutamatergic neurons, the results indicate that JhI-21 could play a role in dopaminergic physiology similar to the one hypothesized for the neuromuscular junction. The synthesis of brain monoamines depends on the supply of essential amino acids that are provided by food intake, thus requiring efficient cellular transport systems. Although dopaminergic neuron function is impaired when JhI-21 expression is downregulated, no evidence was found of reduced brain dopamine levels under baseline conditions. Dopamine levels were increased in the mutant flies fed with the dopamine precursor L-DOPA, an amino acid that has been shown to be transported by LAT1, further suggesting that JhI-21 expression is not critical for dopamine synthesis. In this experiment, dopamine synthesis could also take place ectopically in dopa-decarboxylase (ddc) expressing serotoninergic neurons, in which the UAS-JhI-21-RNAi construct was not expressed. Dopamine content was still increased, although moderately, in flies in which the UAS-JhI-21-RNAi construct was targeted to both dopaminergic and serotoninergic neurons using the ddc-Gal4 driver. The lack of impact of JhI21 downregulation on L-DOPA-induced increase in dopamine synthesis could be due to functional redundancy between JhI-21 and Mnd, or could be a consequence of the partial inhibition provided by the UAS-RNAi constructs. In contrast to those findings on tissue content, this study clearly observed that JhI-21 downregulation in dopaminergic neurons reduced the sensitivity to L-DOPA as measured with sleep loss, and significantly attenuated the sleep loss phenotype of the TH-GaL4 > UAS-TrpA1 hyperdopaminergic condition. This raises the possibility that JhI-21 mediates the entry of amino acids critical for the physiology of dopaminergic neurons, such as leucine that could activate TOR-dependent processes influencing protein synthesis and energy metabolism, but is not directly involved in dopamine synthesis through the transport of precursors such as tyrosine or phenylalanine. The phenotype observed in flies where both Rheb and JhI-21 are manipulated indicates that JhI-21 could be required for TOR pathway activation. This hypothetical model is conceivable since in mammals enriched intracellular leucine levels via LAT1 transport activate mTORC1. Alternatively, TOR signaling could modulate JHI-21 function and affect LAT1-like-dependent transport mechanisms required for neuronal activity. Elucidating the relationships between TOR signaling and LAT1-like transporters, and understanding how they play a critical role in dopaminergic neurons function will require further work (Aboudhiaf, 2018).

This study showed that the MND and JHI-21 transporters are broadly expressed in the brain. However, targeting the JhI-21 and Mnd UAS-RNAi constructs to most nondopaminergic cell types, including the wake-promoting octopaminergic neurons, failed to affect sleep-wake regulation. This lack of effect may be attributable to the efficiency of genetic tools or alternatively to the more stringent requirement for these transporters in dopaminergic neurons. Further studies are warranted to further evaluate this question. Interestingly, accumulating pieces of evidence support the existence of a dynamic regulation for JhI-21 and Mnd: JhI-21 is modulated during larval development in close correlation with behavioral changes and in adult after long-term memory conditioning, whereas Mnd is differentially expressed after sleep deprivation. The expression of LAT1 transporters in mammalian dopaminergic neurons has not been investigated yet; however, a report using pharmacological methods suggested that such transporters could modulate neuronal activity. The fact that sleep deprivation induces changes in TOR signaling in the brain opens the possibility that LAT1 could be modulated by sleep/wake in mammals. Of note, sleep deprivation could induce changes in LAT1 expression at the blood-brain barrier (Aboudhiaf, 2018).

In conclusion, this study reveals the role of LAT1-like transporters in the function of dopaminergic neurons, adding one more element to the array of cellular and molecular events affecting sleep/wake regulation. Since these transporters are known to be dynamically regulated by physiological cellular states, they provide an entry point to elucidate the role of nutrient in sleep/wake regulation (Aboudhiaf, 2018).

Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake- and sleep-promoting cell types. The large and small ventrolateral neurons (l-LN(v)s and s-LN(v)s) have been identified as wake-promoting neurons within the clock neuron network. The l-LN(v)s are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LN(v)s and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. This study tested this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. Dopamine application indeed increases cAMP levels and depolarizes the l-LN(v)s, but surprisingly, it does not result in increased firing rates. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l- and s-LN(v)s, but not of Dop1R2, abolished the depolarization of l-LN(v)s in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LN(v)s. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LN(v)s does not affect sleep in males. Unexpectedly, a moderate decrease of daytime sleep was found with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LN(v)s do not utilize Dop1R2 receptors and the s-LN(v)s also respond to dopamine, it is concluded that the s-LN(v)s are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LN(v)s is not required for daytime arousal, but likely promotes nighttime sleep via the s-LN(v)s (Fernandez-Chiappe, 2020).

Protocerebral Bridge Neurons That Regulate Sleep in Drosophila melanogaster

The central complex is one of the major brain regions that control sleep in Drosophila. However, the circuitry details of sleep regulation have not been elucidated yet. This study shows a novel sleep-regulating neuronal circuit in the protocerebral bridge (PB) of the central complex. Activation of the PB interneurons labeled by the R59E08-Gal4 and the PB columnar neurons with R52B10-Gal4 promoted sleep and wakefulness, respectively. A targeted GFP reconstitution across synaptic partners (t-GRASP) analysis demonstrated synaptic contact between these two groups of sleep-regulating PB neurons. Furthermore, it was found that activation of a pair of dopaminergic (DA) neurons projecting to the PB (T1 DA neurons) decreased sleep. The wake-promoting T1 DA neurons and the sleep-promoting PB interneurons formed close associations. Dopamine 2-like receptor (Dop2R) knockdown in the sleep-promoting PB interneurons increased sleep. These results indicated that the neuronal circuit in the PB, regulated by dopamine signaling, mediates sleep-wakefulness (Tomita, 2021).

Consolidation of Sleep-Dependent Appetitive Memory Is Mediated by a Sweet-Sensing Circuit

Sleep is a universally conserved physiological state which contributes toward basic organismal functions, including cognitive operations such as learning and memory. Intriguingly, organisms can sometimes form memory even without sleep, such that Drosophila display sleep-dependent and sleep-independent memory in an olfactory appetitive training paradigm. Sleep-dependent memory can be elicited by the perception of sweet taste, and this study now shows that a mixed-sex population of flies maintained on sorbitol, a tasteless but nutritive substance, do not require sleep for memory consolidation. Consistent with this, silencing sugar-sensing gustatory receptor neurons in fed flies triggers a switch to sleep-independent memory consolidation, whereas activating sugar-sensing gustatory receptor neurons results in the formation of sleep-dependent memory in starved flies. Sleep-dependent and sleep-independent memory relies on distinct subsets of reward signaling protocerebral anterior medial dopaminergic neurons (PAM DANs) such that PAM-β'2mp DANs mediate memory in fed flies whereas PAM-α1 DANs are required in starved flies. Correspondingly, a feeding-dependent calcium increase was observed in PAM-β'2mp DANs, but not in PAM-α1 DANs. Following training, the presence of sweet sugars recruits PAM-β'2mp DANs, whereas tasteless medium increases calcium in PAM-α1 DANs. Together, this work identifies mechanistic underpinnings of sleep-dependent memory consolidation, in particular demonstrating a role for the processing of sweet taste reward signals (Chouhan, 2022).

Flies can form sleep-dependent and sleep-independent memory in an appetitive conditioning paradigm, but how they switch between these distinct forms of memory consolidation remains unexplored. This study found that Gr64f GRNs-mediated sweet taste signal is essential for sleep-dependent memory. Sweet taste activates a specific subset of PAM DANs, PAM-β'2mp, for the consolidation of sleep-dependent memory. In contrast, in the absence of sweet stimulus, sleep becomes dispensable for memory consolidation and PAM-α1 DANs are recruited to form long-term memory. These findings indicate that the presence of reward can modulate the role of sleep in memory consolidation (Chouhan, 2022).

Taste serves as an important modality in feeding decisions in Drosophila as perceived sweetness is used to estimate the carbohydrate content of food. Flies prefer sweet sugars over substances with a higher nutritive value indicating that taste determines initial feeding preferences. Sweetness drives sugar ingestion during odor-reward pairing that is essential to form appetitive memories. Also, sweet taste tempers food-seeking behavior as flies on a sweet but non-nutritive medium show lower activity than starved flies. Therefore, the presence of sweet taste modifies behavior by providing a sensation of food (Chouhan, 2022).

Flies kept on food vials require sleep to form appetitive memories (Chouhan, 2021). As in the case of the other behaviors mentioned above, sweet taste appears to be sufficient to signal the presence of food and thereby trigger sleep-dependent memory formation. Flies kept on sucralose, a sweet but non-nutritive sugar, show an enhancement in post-training sleep and require sleep to consolidate long-term memory. In contrast, flies on sorbitol, a tasteless but nutritive substance, form sleep-independent memory. Also, disrupting the activity of sweet-sensing Gr64f+ neurons results in the formation of sleep-independent memory in fed flies, while stimulating Gr64f+ neurons in starved flies induces a switch to sleep-dependent memory consolidation. While sweet taste may induce sleep-dependent memory, it is also possible that processes driving sleep-dependent and sleep-independent memory consolidation interact so as to be mutually exclusive. For instance, as starvation suppresses sleep, it may trigger sleep-independent memory, while sweet taste could drive sleep-dependent memory consolidation by releasing starvation-induced suppression of sleep. Neural connections between the two relevant circuits (described below) could mediate such interactions (Chouhan, 2022).

Sleep is typically thought to be induced under conditions of higher energy consumption; however, the data indicate that, at least for sleep-dependent memory consolidation, sweet taste rather than caloric intake serves as a signal for sleep. Sweet taste is not an accurate reporter for the nutritional value of food. Indeed, flies switch their preference from non-nutritive sweeteners to tasteless nutritive sugars over time in a two-choice assay. Therefore, sweet taste can only provide a short-term impression of the presence of food, which might be sufficient to induce sleep-dependent consolidation as flies form sleep-dependent or sleep-independent memory in the first 4 h after training (Chouhan, 2022).

The reward signal from PAM DANs during training is essential to form odor-reward associations. MB neurons are tiled by individual DAN terminals and the dendrites of corresponding MBONs to form 15 anatomically and functionally distinct compartments. PAM DANs largely innervate distinct compartments of the horizontal MB lobes. As per a previous study, a recurrent circuit that couples PAM-α1 DANs with the corresponding MBON in the α1 MB compartment mediates appetitive memory consolidation in starved flies. However, the current study found that PAM-α1 DANs are dispensable for memory consolidation in fed flies. Instead, PAM DANs that innervate the β'2mp compartment of the MB are essential for long-term memory in fed flies. Consistent with the findings that distinct PAM DANs mediate memory consolidation in fed versus starved states, the activity of PAM-β'2mp DANs was higher in trained and fed flies, while calcium in PAM-α1 DANs was significantly higher in trained and starved flies. These results complement previous work in rats, humans, and flies demonstrating that discrete neural circuits mediate sleep-dependent and sleep-independent memory consolidation (Chouhan, 2022).

PAM-β'2mp DANs signal reward to form 'safety-memory' of the unpaired odor during spaced aversive conditioning. Intriguingly, these neurons connect to MBONs implicated in appetitive memory consolidation under fed and starved conditions. PAM-β'2mp DANs receive input from MBON-γ2α'1 neurons, which are required for memory under fed conditions, and through axo-axonic projections they connect to the MBON-γ1Pedc neurons that are essential for memory under starved conditions. It is proposed that MBON-γ2α'1 neurons engage PAM-β'2mp DAN-mediated reward signaling to establish a sleep-dependent memory trace. By contrast, an axo-axonal connection from MBON-γ1Pedc neurons may modulate neurotransmission in PAM-β'2mp DANs such that it allows PAM-α1 DANs to form sleep-independent memory. Future work can investigate how these discrete neural circuits interact to form sleep-dependent or sleep-independent memories (Chouhan, 2022).

Sweet taste is perceived by a set of seven transmembrane receptors on the proboscis and tarsal segments of the leg. The taste information is transmitted by GRNs that terminate in the suboesophageal ganglion. Second-order neurons of the gustatory system send projections from the suboesophageal ganglion to the central brain for higher-order processing of taste information. A subset of these projections terminates in the superior protocerebrum that also contains processes from DANs. Consistent with a connection between sweet-sensing neurons and DANs, an exposure to sucrose evokes a robust calcium response in PAM DANs. Therefore, sweet taste may signal reward by stimulating PAM DANs. Consistently, a significant calcium/GFP increase in PAM-β'2mp DANs was found in fed flies compared with starved flies. Also, flies kept on arabinose or sucralose after training demonstrate a significant increase in PAM-β'2mp DAN activity compared with flies moved to sorbitol following training. It is suggested that the Gr64f+ GRNs mediated sweet taste signal acts in conjunction with the feedback from MBON-γ2α'1 neurons to recruit PAM-β'2mp DANs to form sleep-dependent memory (Chouhan, 2022).

Rewarding experiences modulate behavior to enhance survival. These experiences are preferentially replayed during sleep for better retention. Indeed, memory consolidation involves neuronal reactivation in the mesolimbic dopaminergic reward system during sleep. Conversely, sleep loss modifies reactivity in the reward centers of the brain such that it induces reward-seeking behavior. This work suggests that the processing of sweet taste reward signals determines the necessity for sleep in memory consolidation. A general role for reward in determining the need for sleep is open to investigation (Chouhan, 2022).

Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis

Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

Octopamine functions as a neuromodulator: regulation of reward signaling, synaptic and behaviorial plasticity, activity, sleep, aggression, starvation resistance, appetite, metabolic traits, life span, ovulation and the neuromuscular junction

Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling

Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. As a highly conserved neurobehavioral state, sleep has been described in species ranging from jellyfish to humans. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. PAM-DANs were found to be GABA responsive and required GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM (protocerebral anterior medial) neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, this study has identified and characterized a dopamine modulated sleep microcircuit within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave way for understanding how flies balance sleep, wakefulness and arousal (Driscoll, 2021).

The mushroom body lobes are tiled by discrete anatomic compartments defined by the axons of a specific subset of DANs and the dendrites of one or two mushroom body output neurons (MBONs). This anatomical arrangement positions DANs to strategically convey positive and negative reinforced information by changing the synaptic weight of KC-MBONs in producing aversive and appetitive responses (Driscoll, 2021).

While, the most in-depth analysis of these synapses and distinct DAN-KC-MBON connectivity and behavioral output comes from studies of olfactory conditioning, there is evidence that these synapses play a critical role in innate behaviors like feeding and sleep. Although, role of DA on sleep has been extensively investigated in Drosophila, the commonly used TH-Gal4 driver line labels most dopamine neuron clusters, but is absent from the several PAM clusters that projects to MB (Driscoll, 2021).

This study specifically probed PAM subsets that project to γ5, γ4, and β'2 MB compartments. This study focused on this subset because KCs and MBONs downstream of these PAM neurons can be neuroanatomically resolved and have been shown to be required for wakefulness. Further, KCs and MBONs that form the γ5, γ4, and β'2 synaptic compartments alter their spontaneous neural activity in response to sleep need (induced by mechanical sleep-deprivation). The ability to use cell-specific split-GAL4 tools provides opportunity to resolve the precise circuit mechanisms by which PAM neurons regulate wakefulness (Driscoll, 2021).

GABA signaling also modulates sleep and wake microcircuits within MB. The key source of GABA in the MB is anterior paired lateral neurons, APL and dorsal paired medial neurons (DPM), which are electrically coupled and increase sleep by GABAergic inhibition of wake-promoting KCs. In the context of associative learning, there is strong evidence for interactions between KCs, APL, DPM and DANs but it is not clear if GABA and dopamine signaling represent opposing inputs to the KCs and MBONs in the regulation of sleep. This study found that the excitability of PAM DANs involved in wakefulness is blocked by sleep-promoting GABA signaling and mediated by ionotropic receptor subtype GABAA-Rdl (Driscoll, 2021).

A recent study showed that GABA inhibitory input to the presynaptic terminals of the PAM neurons regulates appetitive memory and that this interaction is mediated by GABA-B3 receptors that are clustered in PAM boutons localized to PAM-γ5 and -α1 compartments. These data are consistent with the findings that PAM-γ5 are GABA responsive and that multiple receptors are critical to this interaction. Since, no role was found for GABA-B3 in PAM mediated sleep regulation, it is likely that PAM γ5, γ4, and β'2 express multiple GABA receptors which are differentially recruited in sleep and learning. How and what regulates the expression of these receptors in PAM subsets presents a potential mechanism of presynaptic gating to MB core circuits. Transcriptomic analysis of PAM neurons reveals extremely high levels of Rdl expression followed by GABA-B3. Among the PAM subsets mean TPM or transcripts per million of Rdl receptor in PAM γ5, γ4, and β'2 are much higher as compared to other PAM subsets (Driscoll, 2021).

Simple connection query search of the recently released hemibrain data85 reveals there is significant bidirectional connectivity between APL, DPM, and PAM neurons. Further, a recent study showed that APL neurons express the inhibitory D2R receptor55. APL mediated GABAergic inhibition of the PAM neurons was recently shown to control the intensity and specificity of olfactory appetitive memory but previous results show that blocking GABA release from APL neurons only modestly affects sleep phenotypes (Driscoll, 2021).

While, the role of APL in GABA signaling to PAM γ5, γ4, and β'2 cannot be completely ruled out, other inputs to wake-regulating PAM DANs could also be GABAergic and critical for promoting sleep. A recent study using EM dataset of a Full Adult Female Fly Brain (FAFB) mapped the inputs and outputs of the PAMγ5 DANs and identified that this cell type is highly heterogenous and in addition to recurrent feedback from MBON01 γ5β'2a, it receives extensive input from other MBONs, sub-esophageal output neurons (SEZONs) and lateral horn output neurons86. The EM data also reveals that octopaminergic neurons synapse onto PAM γ5, γ4, and β'2 DANs. Whether, these inputs play a role in wakefulness is unknown but suggests that the PAMγ5 could serve as a key link between sensory inputs, wake-promoting octopamine signal and core sleep regulating circuitry within the MB. Each of these inputs could modulate PAM-DAN activity and dopamine release in regulating wakefulness via the MB (Driscoll, 2021).

In addition to probing the release and activity of these PAM-DANs the dopamine receptors and their location within the MB in signaling wakefulness were also explored. To this end validated RNAi lines were expressed in subsets of KCs and MBONs; DopR1 and DopR2 were found to be critical in mediating the wakefulness signal via KCs and γ5β'2 MBONs. Knocking down the receptor consistently increased total sleep and bout length. Furthermore, specific manipulations of DopR receptors within the MB did not directly alter locomotor activity as observed by manipulation of these receptors in CX. Although, loss-of-function mutations of D1 dopamine receptor DopR are shown to enhance repetitive air puff startle-induced arousal and increase sleep. Expression and restoration of DopR in the mutant background specifically in the central complex rescues the startle response, while, the sleep phenotype is rescued via a broad MB driver. The current data extends these findings by showing that the DopR receptors regulate sleep via the MB γ5 and β'2 compartment. Although, targeted RNAi experiments show that DopR's are required for sleep regulation by KCs and MBONs, the lack of a sleep phenotype in DopR2 mutant could be a result of global loss of receptor in the mutant as opposed to targeted loss of receptor function within MB. Dopamine signals wakefulness by activation of wake-promoting neurons of MB via DopR1 and DopR2 and within. the central complex, neurons of dFB are inhibited by dopamine via DopR2. Hence, DopR2 has opposing effects within MB and CX (Driscoll, 2021).

In vitro characterization indicates that DopR's signal through distinct G-proteins, with DopR1 via Gαs to stimulate cAMP production and DopR2 coupling to Gαq via increased calcium. These receptors are thought to have differential sensitivity to dopamine and could be potentially recruited by varying DA release or DAN activity. In the context of sleep regulation, this work reveals that both DopR1 and DopR2 induce wakefulness via the γ5 β'2 MB compartment but not γ4 compartment. Although, chronic activation of PAM γ4 induces wakefulness, the glutamatergic MBON γ4 < γ1,2 projects to multiple compartments and could potentially activate or inhibit MBONs and PAMs projecting to γ1 and γ2 compartment. The interaction between compartments is not well understood in the context of sleep and wake regulation and requires further investigation to better understand the role of DopR2 in regulating the γ4 compartment. The neuroanatomical specificity obtained from split-Gal4 lines combined with EM data has paved way for more detailed analysis of the role of dopamine signaling to MB in the context of sleep and other behaviors (Driscoll, 2021).

The sleep-regulating PAM DANs and associated KCs and MBONs identified in this study are also involved in mediating satiety, novelty, caffeine induced arousal, punishment and reward associated experiences suggesting that the activity of these neurons is tuned to several wake and arousal associated behaviors. This is further supported by the EM connectome data showing that MB receives extensive gustatory, auditory and visual input in addition to olfactory input (Driscoll, 2021).

Current models of sleep regulation rely on two main processes, the circadian clock and the sleep homeostat and don't completely account for multiple external and internal factors that influence wakefulness. The ability to sleep, however, is influenced by motivational or cognitive stimuli. It is therefore envisioned that sleep, wakefulness and arousal within MB are not located in distinct circuits, but rather mediated by distinct processes within a common circuit (Driscoll, 2021).

GABA transmission from mAL interneurons regulates aggression in Drosophila males

Aggression is known to be regulated by pheromonal information in many species. But how central brain neurons processing this information modulate aggression is poorly understood. Using the fruit fly model of Drosophila melanogaster, this study systematically characterize the role of a group of sexually dimorphic GABAergic central brain neurons, popularly known as mAL, in aggression regulation. The mAL neurons are known to be activated by male and female pheromones. This report shows that mAL activation robustly increases aggression, whereas its inactivation decreases aggression and increases intermale courtship, a behavior considered reciprocal to aggression. GABA neurotransmission from mAL is crucial for this behavior regulation. Exploiting the genetic toolkit of the fruit fly model, a small group of approximately three to five GABA(+) central brain neurons were found with anatomical similarities to mAL. Activation of the mAL resembling group of neurons is necessary for increasing intermale aggression. Overall, these findings demonstrate how changes in activity of GABA(+) central brain neurons processing pheromonal information, such as mAL in Drosophila melanogaster, directly modulate the social behavior of aggression in male-male pairings (Sengupta, 2022).

A conditional GABAergic synaptic vesicle marker for Drosophila

Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others. Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, this study developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT. 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons. This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver. A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles (Certel, 2022).

Serotonin distinctly controls behavioral states in restrained and freely moving Drosophila

When trapped in a physical restraint, animals must select an escape strategy to increase their chances of survival. After falling into an inescapable trap, they react with stereotypical behaviors that differ from those displayed in escapable situations. Such behaviors involve either a wriggling response to unlock the trap or feigning death to fend off a predator attack. The neural mechanisms that regulate animal behaviors have been well characterized for escapable situations but not for inescapable traps. This study reports that restrained vinegar flies exhibit alternating flailing and immobility to free themselves from the trap. Optogenetics and intersectional genetic approaches were used to show that, while broader serotonin activation promotes immobility, serotonergic cells in the ventral nerve cord (VNC) regulate immobility states majorly via 5-HT7 receptors. Restrained and freely moving locomotor states are controlled by distinct mechanisms. Taken together, this study has identified serotonergic switches of the VNC that promote environment-specific adaptive behaviors (Gowda, 2023).

Serotonergic control of feeding microstructure in Drosophila

To survive, animals maintain energy homeostasis by seeking out food. Compared to freely feeding animals, food-deprived animals may choose different strategies to balance both energy and nutrition demands, per the metabolic state of the animal. Serotonin mediates internal states, modifies existing neural circuits, and regulates animal feeding behavior, including in humans and fruit flies. However, an in-depth study on the neuromodulatory effects of serotonin on feeding microstructure has been held back for several technical reasons. Firstly, most feeding assays lack the precision of manipulating neuronal activity only when animals start feeding, which does not separate neuronal effects on feeding from foraging and locomotion. Secondly, despite the availability of optogenetic tools, feeding in adult fruit flies has primarily been studied using thermogenetic systems, which are confounded with heat. Thirdly, most feeding assays have used food intake as a measurement, which has a low temporal resolution to dissect feeding at the microstructure level. To circumvent these problems, OptoPAD assay, which provides the precision of optogenetics to control neural activity contingent on the ongoing feeding behavior, was utilized. Manipulating the serotonin circuit optogenetically affects multiple feeding parameters state-dependently. Food-deprived flies with optogenetically activated and suppressed serotonin systems feed with shorter and longer sip durations and longer and shorter inter-sip intervals, respectively. It was further shown that serotonin suppresses and enhances feeding via 5-HT1B and 5-HT7 receptors, respectively (Banu, 2022).

Reserpine and PCPA reduce heat tolerance in Drosophila melanogaster

Drosophila melanogaster is a model organism to study molecular mechanisms and the role of the genes and proteins involved in thermal nociception. Monoamines (i.e. dopamine) have been involved in temperature preference behavior in D. melanogaster. Therefore, this study investigated whether the monoamines, particularly dopamine and serotonin, participate in the response to thermal nociceptive stimuli in D. melanogaster. Flies were treated with reserpine (an inhibitor of vesicular monoamines transporter, 3-300 μM), 3-Iodo-L-tyrosine (3-I-T, an inhibitor of tyrosine hydroxylase, 16.28-65.13 mM), and para-Chloro-DL-phenylalanine (PCPA, an inhibitor of tryptophan hydroxylase, 20-80 mM); then, the flies were subjected to tests of thermal tolerance and avoidance of noxious heat. Climbing behavior was used as a test to evaluate locomotor activity. Reserpine reduces the thermal tolerance profile of the D. melanogaster, as well as the avoidance of noxious heat and locomotor activity depending on the concentration. PCPA, but not 3-I-T, decreased heat tolerance and avoidance of noxious heat. These data suggest that monoamines, particularly serotonin, are associated with the impaired avoidance of noxious heat which could be related to the reduction of heat tolerance in D. melanogaster (Bressan, 2023).

Sex peptide regulates female receptivity through serotoninergic neurons in Drosophila

The courtship ritual is a dynamic interplay between males and females. Courtship successfully leading to copulation is determined by the intention of both parties which is conveyed by complex action sequences. In Drosophila, the neural mechanisms controlling the female's willingness to mate, or sexual receptivity, have only recently become the focus of investigations. This study reports that pre-mating sexual receptivity in females requires activity within a subset of serotonergic projection neurons (SPNs), which positively regulate courtship success. Of interest, a male-derived sex peptide, SP, which was transferred to females during copulation acted to inhibit the activity of SPN and suppressed receptivity. Downstream of 5-HT, subsets of 5-HT7 receptor neurons played critical roles in SP-induced suppression of sexual receptivity. Together, this study reveals a complex serotonin signaling system in the central brain of Drosophila which manages the female's desire to mate (Yang, 2023).

A higher brain circuit for immediate integration of conflicting sensory information in Drosophila

Animals continuously evaluate sensory information to decide on their next action. Different sensory cues, however, often demand opposing behavioral responses. How does the brain process conflicting sensory information during decision making? This study shows that flies use neural substrates attributed to odor learning and memory, including the mushroom body (MB), for immediate sensory integration and modulation of innate behavior. Drosophila melanogaster must integrate contradictory sensory information during feeding on fermenting fruit that releases both food odor and the innately aversive odor CO2. Using this framework, this study examined the neural basis for this integration. A local circuit consisting of specific glutamatergic output and PAM dopaminergic input neurons with overlapping innervation in the MB-β'2 lobe region was identified, which integrates food odor and suppresses innate avoidance. Activation of food odor-responsive dopaminergic neurons reduces innate avoidance mediated by CO2-responsive MB output neurons. The study hypothesizes that the MB, in addition to its long recognized role in learning and memory, serves as the insect's brain center for immediate sensory integration during instantaneous decision making (Lewis, 2015).

Serotonergic modulation enables pathway-specific plasticity in a developing sensory circuit in Drosophila

How experiences during development cause long-lasting changes in sensory circuits and affect behavior in mature animals are poorly understood. This study establish a novel system for mechanistic analysis of the plasticity of developing neural circuits by showing that sensory experience during development alters nociceptive behavior and circuit physiology in Drosophila larvae. Despite the convergence of nociceptive and mechanosensory inputs on common second-order neurons (SONs), developmental noxious input modifies transmission from nociceptors to their SONs, but not from mechanosensors to the same SONs, which suggests striking sensory pathway specificity. These SONs activate serotonergic neurons to inhibit nociceptor-to-SON transmission; stimulation of nociceptors during development sensitizes nociceptor presynapses to this feedback inhibition. The results demonstrate that, unlike associative learning, which involves inputs from two sensory pathways, sensory pathway-specific plasticity in the Drosophila nociceptive circuit is in part established through feedback modulation. This study elucidates a novel mechanism that enables pathway-specific plasticity in sensory systems (Kaneko, 2017).

A subset of serotonergic neurons evokes hunger in adult Drosophila

Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. Although progress is being made, how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. This study uses Drosophila melanogaster to identify neurons encoding hunger. A small group of neurons was found that, when activated, induces a fed fly to eat as though it is starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. The neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend the understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit (Albin, 2015).

Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger

Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. This study identified a dopamine circuit that encodes protein-specific hunger in Drosophila. The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis (Liu, 2017).

Beyond satisfying caloric needs, animals must also ingest nutrients that cannot be biosynthesized. The 'specific appetite' hypothesis posits that nutrient-specific hunger drives homeostatic consumption of substances such as protein and salt. Protein is an indispensable macronutrient and is particularly required in anabolic states, such as infancy and pregnancy. Recent studies in Drosophila have identified signaling mechanisms regulating protein hunger, particularly in the context of postmating effects. However, specific circuits encoding protein hunger remain unknown (Liu, 2017).

Yeast is an ethologically relevant protein food source for Drosophila, containing a negligible amount of sugars. Studies in fruit flies demonstrated that mated females have greater drive for protein and amino acid intake. This study used this potent physiological drive for protein in mated females as an entry point to investigate the neural basis of protein hunger. Yeast-feeding in mated females were assayed after conditional silencing of different neuromodulatory cell groups. Dopamine (DA) neurons were specifically required for yeast consumption and preference. To identify the specific DA neurons mediating this effect, two restricted Gal4 drivers (TH-C-Gal4 and TH-D-Gal4) containing regulatory sequences of the tyrosine hydroxylase gene (TH) were used, and it was found that conditional silencing of the neurons labeled with either driver led to significantly reduced protein preference. These drivers label largely nonoverlapping DA cells, aside from two cells in each protocerebral posterior medial 2 (PPM2) subgroup (Liu, 2017).

To isolate these PPM2 neurons, an intersectional approach (TH-C-FLP with TH-D-Gal4, was used where FLP encodes flippase) to drive expression of FRT-stop-FRT-mCD8-GFP (a membrane tethered green fluorescent protein). Two PPM2 neurons in each hemisphere were identifed that project ventrally to the 'wedge' neuropil. On the basis of this projection pattern, these two PPM2 neurons were named DA-WED cells. Next, using the FLP-induced intersectional GAL80/GAL4 repression (FINGR) system [TH-D-Gal4, TH-C-FLP, tub-FRT-Gal80-FRT (WED1-Gal4)] to express dendritic versus terminal markers, it was found that the wedge area contains the dendritic field, whereas the two dorsally bifurcating branches contain presynaptic terminals. Intersection between TH-C-Gal4 and another restricted driver TH-F3-Gal4 (15) (WED2-Gal4) also revealed the two DA-WED neurons. These cells were specifically inactivated with the inward-rectifying potassium channel Kir2.1, and substantial inhibition was found of yeast preference and consumption. General hunger, thirst, and salt appetite were not affected when DA-WED neurons were silenced (Liu, 2017).

It was next asked whether the DA-WED neurons play a general role in homeostatic regulation of protein intake, beyond mated females. Although male flies did not prefer yeast at baseline, they exhibited significant protein preference and consumption after yeast deprivation for 8 days, which was fully suppressed if an amino acid mix was provided during the deprivation period. Inactivating the DA-WED neurons significantly reduced yeast preference and consumption in protein-deprived male flies. Conversely, conditional activation of these cells with the heat-activated cation channel dTrpA1 induced yeast preference and consumption in males. Similar data were obtained for virgin females. Conditional silencing or activation of DA-WED neurons reduced or increased protein consumption, respectively, over a range of internal protein-hunger states in both mated female and male flies, it was decided to focus on male flies for subsequent experiments to avoid postmating effects. Reducing DA levels in DA-WED neurons by knocking down the neuronal specific isoform of TH in these cells decreased yeast intake in protein-starved male flies (Liu, 2017).

To investigate whether the activity of the DA-WED neurons correlates with protein need, perforated patch-clamp recordings were performed from these neurons. After yeast deprivation, the spontaneous action potential (AP) firing rate of DA-WED neurons increased about fourfold compared to controls. Moreover, evoked AP firing rates were higher at all measured depolarizing currents after yeast deprivation. Protein starvation did not significantly alter the resting membrane potential or input resistance of these cells. Cytosolic Ca2+ concentrations in these cells (but not in nearby PPM3 DA neurons) was substantially increased after yeast deprivation when monitored with the CaLexA (calcium-dependent nuclear import of LexA) activity reporter system. This increase in intracellular Ca2+ concentration was suppressed when an amino acid mix was provided in the protein-deficient diet. This effect was specific for protein, as general starvation did not affect CaLexA signal in the DA-WED neurons. Similar data were obtained with GCaMP imaging, as an alternative method to measure cytosolic Ca2+ levels (Liu, 2017).

What component of protein serves to signal protein satiety to the DA-WED neurons? It was hypothesized that a specific amino acid may play this role. Flies were first subjected to protein deprivation, but provided individual amino acids in the diet, and then monitored the activity of DA-WED neurons using CaLexA. Tryptophan and glutamine (Gln) supplementation suppressed the enhanced activity of DA-WED neurons induced by protein deprivation, and proline and glutamate supplementation showed a trend toward this effect. Next, whether supplementation of these four individual amino acids modulated homeostatic regulation of yeast intake was tested. Yeast intake was significantly inhibited when Gln was added back to a protein-deficient diet. Conversely, an amino acid mix lacking only Gln failed to suppress the increase in DA-WED activity and was less effective at inhibiting protein consumption after yeast deprivation. It was also found that Gln levels in the hemolymph were significantly reduced after protein deprivation. Together, these data suggest that Gln in particular may be important for signaling protein deficiency (Liu, 2017).

Despite strong preference for sucrose at baseline, yeast deprivation caused flies to decrease their consumption of this nutrient. Because DA-WED neurons are activated after substantial protein deprivation, it was hypothesized that they also play a direct role in reducing sucrose intake under these conditions. Silencing DA-WED neurons with Kir2.1 significantly enhanced sugar intake after protein deprivation, whereas activating these neurons reduced sucrose consumption (Liu, 2017).

The mechanisms underlying the opposing effects of DA-WED neurons on sucrose and yeast intake was investigated. There are four DA receptors in Drosophila encoded by DopR1, DopR2, D2R, and DopEcR. Both DopR1 and DopR2 mutants exhibited a reduced preference for yeast after protein deprivation in a two-choice assay, which could reflect either a reduced preference for protein or a greater preference for sucrose. Thus, focus was placed on these two receptors as potential targets acting downstream of the DA-WED neurons. In one-choice assays, DopR2 mutants exhibited significantly reduced yeast intake, whereas DopR1 mutants displayed a robust increase in sucrose feeding. Mutations in DopR2 and DopR1 also suppressed the increase in yeast consumption and decrease in sucrose feeding triggered by activating DA-WED neurons, respectively. These findings were confirmed by pan-neuronal knockdown of DopR1 or DopR2 (nsyb-Gal4>UAS-DopR1-miR or nsyb-Gal4>UAS-DopR2-miR), which yielded similar results. It was therefore hypothesized that DopR2 and DopR1 act in discrete neuronal targets of the DA-WED cells to regulate protein and sugar feeding, respectively (Liu, 2017).

To identify putative candidate circuits acting downstream of the DA-WED cells, a GRASP (GFP reconstitution across synaptic partners)-based screen was performed with Rubin Gal4 drivers derived from DopR2 and DopR1 enhancer sequences to assess connectivity with the DA-WED neurons. Among DopR2-derived driver lines, only the R70G12-Gal4 line exhibited GRASP signal at the DA-WED terminal branches (specifically, the medial branch). Moreover, knockdown of DopR2 with R70G12-Gal4 significantly reduced yeast intake after protein starvation, without affecting general hunger or sucrose consumption in yeast-deprived animals. This reduction in protein appetite in R70G12-Gal4>UAS-DopR2-miR flies was observed over a range of protein-hunger states in both mated female and male flies. To identify the specific cells in this driver that contact the DA-WED neurons, a multicolor flip out (MCFO) experiment was performed. A neuron whose cell body is located in the posterior medial protocerebrum and sends its projections first to the fan-shaped body (FB), before extending more anteriorly to the lateral accessory lobe (LAL) area was identified where the GRASP signal was observed. These neurons were named FB-LAL cells based on this projection pattern. An independent driver R75B10-Gal4 was identified that also labeled the FB-LAL neuron and displayed GRASP signal at the medial branch of the DA-WED neurons. Knockdown of DopR2 with R75B10-Gal4 decreased yeast intake in protein-starved flies. As expected, intersection between R70G12-Gal4 and R75B10-Gal4 drivers (R70G12-Gal4, R75B10-LexA>LexAop-FLP, UAS-FRT-stop-FRT-mCD8-GFP) revealed two pairs of FB-LAL neurons. Silencing these cells reduced yeast intake in protein-starved flies, whereas activating these cells induced elevated yeast consumption in male flies. Activation of DA-WED neurons expressing ATP (adenosine 5'-triphosphate)-gated P2X2 receptors induced a substantial increase in GCaMP signal in the cell bodies of downstream FB-LAL neurons (Liu, 2017).

By contrast, among the DopR1-derived lines, the R72B03-Gal4 driver demonstrated GRASP signal at the DA-WED lateral branches. Knockdown of DopR1 with the R72B03-Gal4 driver triggered an increase in sucrose intake in protein-deprived flies, without affecting general hunger or yeast intake after protein starvation. MCFO analysis using R72B03-Gal4 revealed a small subset of neurons in the posterior lateral protocerebrum (PLP) that send their projections locally to the ventrolateral and superior neuropils, so these cells were named PLP neurons. PLP neurons were also labeled by another driver line R32A06-Gal4. Similar to R72B03-Gal4, R32A06-Gal4 exhibited GRASP signal at the lateral branch of DA-WED neurons. MCFO analysis with the new R32A06-Gal4 driver revealed cells whose cell body location and projection pattern match that seen with the PLP neuron identified in the R72B03-Gal4 driver. Knockdown of DopR1 with R32A06-Gal4 increased sucrose intake in protein-starved flies. Silencing or activating these PLP neurons with R72B03-Gal4 or R32A06-Gal4 led to a reduction or an increase, respectively, of sucrose intake (Liu, 2017).

Following substantial deprivation of an essential nutrient, animals must engage in persistent behavior aimed at replenishing it. Plasticity of relevant neural circuits may underlie the persistent drive for motivated behaviors. To address whether the DA-WED neurons undergo protein hunger-dependent plastic changes, the morphology of their terminal branches was assessed using mCD8-GFP and a GFP-tagged version of the synaptic vesicle-associated protein synaptotagmin (Syt-GFP). The medial, but not the lateral, branches were significantly elongated following protein deprivation, which was suppressed when Gln was selectively restored in the protein-deficient diet. Similar data were obtained for the number and total volume of Syt-GFP+ puncta. To address whether the number of active zones also changed in the medial branches of DA-WED neurons after protein starvation, a truncated fragment of the active-zone marker Bruchpilot (Brp-short) was expressed in these cells. The number of BRP+ puncta in the medial, but not lateral, branch was significantly elevated when flies were protein-deprived, and this increase showed a trend toward being inhibited by Gln in the protein-deficient diet. GRASP was used to determine whether protein starvation altered the connectivity between the DA-WED cells and their downstream targets. GRASP signal between the medial branch of the DA-WED neurons and the FB-LAL cells, but not the lateral branch and PLP neurons, was substantially elevated with protein starvation, and this increase was again suppressed if Gln was provided in the protein-deficient diet (Liu, 2017).

To characterize the functional consequences of the plastic changes in the DA-WED neurons, sharp intracellular current-clamp recordings of the FB-LAL cells were performed to measure frequency and amplitude of postsynaptic potentials (PSPs). Protein deprivation induced a substantial increase in the frequency of PSPs in the FB-LAL neurons. Moreover, examination of the distribution of PSP amplitudes revealed the presence of high-amplitude PSPs solely in yeast-deprived animals. This population of high-amplitude PSPs likely reflects evoked responses seen only with protein deprivation and suggests that downstream signaling is markedly enhanced after protein starvation. Knockdown of DopR2 in FB-LAL cells significantly reduced the number of high-amplitude PSPs in yeast-deprived animals, consistent with a model wherein the DA-WED neurons signal via DopR2 on the FB-LAL cells to promote protein consumption (Liu, 2017).

Whether the plastic changes in the medial branches of the DA-WED cells led to a change in feeding behavior was examined. dTrpA1 was used to activate the DA-WED cells and yeast or sucrose consumption was assessed at different time points after cessation of heat treatment. dTrpA1 activation of the DA-WED neurons resulted in a prolonged increase in yeast consumption lasting at least 6 hours and induced persistent plastic changes in the medial, but not lateral, branches as revealed by Syt-GFP staining. In contrast, although sucrose intake was reduced immediately after dTrpA1 activation, this response did not persist and was no longer present within 1 hour. After 24 hours, both yeast consumption and terminal morphology of DA-WED neurons were indistinguishable from control flies. Together, these findings demonstrate branch-specific plastic changes of the DA-WED neurons in response to substantial protein deprivation, providing a mechanism for the persistent hunger for protein, but not sugar, under these conditions (Liu, 2017).

Appropriate regulation of food consumption is essential for the survival of organisms that must navigate environments with variable and uncertain food availability and quality. A number of studies have investigated how animals reject diets devoid of essential amino acids and how monoaminergic and TOR-S6K (target of rapamycin-S6 kinase) signaling in Drosophila regulates mating-induced protein feeding. However, little is known about the circuit mechanisms mediating the homeostatic regulation of protein intake. This study suggests that protein hunger is encoded by the DA-WED neurons, providing a path toward dissecting these mechanisms. The findings suggest that glutamine, directly or indirectly, regulates the activity of these neurons, thus modulating behavioral responses to protein deprivation (Liu, 2017).

The data suggest that the DA-WED neurons play a crucial role in homeostatic control of protein intake, functioning on multiple time scales to restore protein balance. Shortly after substantial protein deprivation, the DA-WED neurons act to mediate the behavioral switch between consumption of a food source preferred at baseline (sucrose) and the deprived nutrient (protein), by activating a dedicated 'protein' feeding circuit, while simultaneously inhibiting a 'sugar' feeding circuit. Over a longer time frame, the DA-WED cells undergo branch-specific plastic changes that underlie the selective and persistent hunger for protein under these conditions. In the wild, these actions may correspond to promoting greater selectivity for protein in an initial foraging response, followed by maintenance of protein consumption after the protein food source has been identified (Liu, 2017).

Given that branch-specific plasticity has generally been described in postsynaptic dendrites, the finding that the presynaptic terminals of the DA-WED neurons exhibit this phenomenon is highly unusual. The mechanisms underlying this process are currently unclear but may depend on extrinsic neuromodulatory influences that differentially regulate potential for plasticity of the distinct presynaptic terminals of the DA-WED cells. Further characterization of these and related circuit mechanisms should help delineate the fundamental principles governing protein-specific hunger. A better understanding of how animals choose to consume protein may also have implications for the treatment of obesity (Liu, 2017).

A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila

Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. This study dissected the function of the key regulator of serotonin signaling - the serotonin transporter - in innate olfactory attraction to ethanol in Drosophila melanogaster. A mutated version of the serotonin transporter was generated that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. Four serotonergic neurons were identified that inhibit the olfactory attraction to ethanol, and two additional neurons were identified that counteract this inhibition by strengthening olfactory information. These results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior (Xu, 2016).

Olfactory attraction to ethanol is regulated by two sets of serotonergic neurons. Two neurons -- the serotonergic CSD neurons -- counteract the inhibitory function of a second set of four serotonergic neurons. The enhancement of sensory input by altered serotonin signaling of the CSD neurons is stronger than more internal information that might influence the behavioral outcome. The observation that strong sensory information overrules internal conditions unravels a hierarchy of information that governs behavioral output (Xu, 2016).

Prolonged serotonin signaling in approximately 83% of the serotonergic neurons using the Trh-Gal4 driver results in normal degree of attraction to 5% EtOH containing food odors. At the first glance, these finding suggest that dSert in these broad set of serotonergic neurons is not involved in the regulation of ethanol attraction. This apparently normal behavior is shared by other Sert knock out animals. For example Sert knock out mice develop normal conditioned place preference -- a model for the cocaine reward -- despite the fact that cocaine binds to Sert. However, breaking down the serotonergic networks underlying the regulation of the attraction to ethanol revealed that a dysregulation of serotonergic neurons can phenocopy normal behavior and that the identified serotonergic neurons under normal circumstance indeed regulate the olfactory attraction. The results show that compensation can occur on network level. The serotonergic neurons underlying the gating of olfactory attraction to ethanol innervate distinct brain regions normally involved in the regulation of olfactory responses. The attraction promoting serotonergic CSD neuron receives synaptic input from the ipsilateral antennal lobes from a broad set of glomeruli, collecting and exchanging olfactory information with the mushroom bodies and lateral horn and releasing information to the contralateral antennal lobe regions (Xu, 2016).

The second set of inhibitory serotonergic neurons includes one neuron each from the IP and LP1 clusters that suppresses innate attraction to ethanol odor. The neuron of the IP cluster projects into the superior medial protocerebrum (smpr), posterior lateral protocerebrum (plpr), and the optic system. The smpr receives olfactory output both directly from the antenna lobes and indirectly from lobes of the LH. The LP1 neuron innervates the ventral lateral protocerebrum (vlpr), a region implicated as a multimodal integration center of sensory stimuli including olfactory information. This multimodal nature of this brain region is further supported by recent finding that a pair of similar serotonergic neurons is involved in aggression and mediating the hunger state of the fly. The fact that the alteration of serotonin signaling in the CSD neuron bypasses the inhibitory effect of the Sert3-Gal4-dependent neurons further supports that the inhibitory effect of those neurons is not due to loss of olfactory perception. Combined with the observation that activation of the Sert3-Gal4-dependent neurons alone does not cause attraction or aversion, these results show that these neurons regulate the execution of a function rather than being involved in the initial step of odor information processing or execution of the approach behavior. This is consistent with a role for prolonged serotonin signaling in response inhibition. This suppression of innate olfactory attraction to ethanol can be overruled by strengthening the constant sensory input to the olfactory system. The consequence of the failing inhibitory mechanism is that the likelihood of exposure to food odors containing ethanol increases, and the opportunity to ingest ethanol might increase in turn (Xu, 2016).

The observation that strong sensory information overrules internal conditions also suggests that under normal circumstances both mechanisms require a tight temporal control. That timing matters is further substantiated by the observations that short term systemic application of a serotonin precursor and short term activation of the Sert3-Gal4 dependent subset of serotonergic neurons using opto-genetics reduce ethanol attraction, but long term intervention of serotonin signaling by dSertDN expression under the control of the Trh-Gal4 driver did not interfere with ethanol attraction. However the regulation of innate ethanol odor attraction is even more complex since other neurotransmitter systems are also involved in regulating the approach to an olfactory stimulus. Previous work has shownn that olfactory ethanol attraction depends on the reinforcement of the olfactory information by a subset of octopaminergic/tyraminergic neurons and the aversion depends on the activation of a subset of dopaminergic neurons acting as a negative reinforcer. This study shows that the maintenance of olfactory information by the serotonergic CSD neurons promote the approach to the ethanol food odor mixture. In the presence of prolonged external olfactory information, the inhibitory information of an internal serotonergic reference system is overruled. The regulation of behavioral outcome requires a tight temporal regulation between the positive and negative reinforcing, inhibiting and prolonging mechanisms depending on the location of the fly relative to the ethanol containing food source and its internal condition. It will be interesting to see where the four mechanisms intersect with each other, how they are orchestrated and whether they are sufficient to regulate the attraction to ethanol. Furthermore, given the evolutionarily conserved nature of the serotonin transporter and the wide expression of serotonin, the serotonergic gating mechanism might delineate a basic principle for other behaviors and organisms as well (Xu, 2016).

Dynamics of memory-guided choice behavior in Drosophila

Memory retrieval requires both accuracy and speed. Olfactory learning of the fruit fly Drosophila melanogaster serves as a powerful model system to identify molecular and neuronal substrates of memory and memory-guided behavior. The behavioral expression of olfactory memory has traditionally been tested as a conditioned odor response in a simple T-maze, which measures the result, but not the speed, of odor choice. This study developed multiplexed T-mazes that allow video recording of the choice behavior. Automatic fly counting in each arm of the maze visualizes choice dynamics. Using this setup, it was shown that the transient blockade of serotonergic neurons slows down the choice, while leaving the eventual choice intact. In contrast, activation of the same neurons impairs the eventual performance leaving the choice speed unchanged. This new apparatus contributes to elucidating how the speed and the accuracy of memory retrieval are implemented in the fly brain (Ichinose, 2016).

Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila

Learned and adaptive behaviors rely on neural circuits that flexibly couple the same sensory input to alternative output pathways. This study shows that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, depending on the state and experience of the fly. Using functional synaptic imaging and electrophysiology, it was shown that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, the dopaminergic neurons function as an interconnected network, encoding information about both an animal's external context and internal state to coordinate synaptic plasticity throughout the mushroom body. These data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity (Cohn, 2015).

Neural circuits for long-term water-reward memory processing in thirsty Drosophila

The intake of water is important for the survival of all animals and drinking water can be used as a reward in thirsty animals. This study found that thirsty Drosophila melanogaster can associate drinking water with an odour to form a protein-synthesis-dependent water-reward long-term memory (LTM). Furthermore, the reinforcement of LTM requires water-responsive dopaminergic neurons projecting to the restricted region of mushroom body (MB) β' lobe, which are different from the neurons required for the reinforcement of learning and short-term memory (STM). Synaptic output from α'β' neurons is required for consolidation, whereas the output from γ and αβ neurons is required for the retrieval of LTM. Finally, two types of MB efferent neurons retrieve LTM from γ and αβ neurons by releasing glutamate and acetylcholine, respectively. These results therefore cast light on the cellular and molecular mechanisms responsible for processing water-reward LTM in Drosophila (Shyu, 2017).

Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila

The functional organization of central motor circuits underlying feeding behaviors is not well understood. This study combined electrophysiological and genetic approaches to investigate the regulatory networks upstream of the motor program underlying food intake in the Drosophila larval central nervous system. The serotonergic network of the CNS was found to be capable of setting the motor rhythm frequency of pharyngeal pumping. Pharmacological experiments verified that modulation of the feeding motor pattern is based on the release of serotonin. Classical lesion and laser based cell ablation indicated that the serotonergic neurons in the subesophageal zone represent a redundant network for motor control of larval food intake (Schoofs, 2017).

Unveiling the dual role of the dopaminergic system on locomotion and the innate value for an aversive olfactory stimulus in Drosophila

The communication between sensory systems and the specific brain centers that process this information is crucial to develop adequate behavioral responses. Modulatory systems, including dopaminergic circuits, regulate this communication to finely tune the behavioral response associated to any given stimulus. For instance, the Mushroom Body (MB), an insect brain integration center that receives and processes several sensory stimuli and organizes the execution of motor programs, communicates with MB output neurons (MBONs) to develop behavioral responses associated to olfactory stimuli. This communication is modulated by dopaminergic neural systems. This study shows that silencing dopaminergic neurons increases the aversive response observed in adult flies exposed to Benzaldehyde (Bz) or octanol. The contribution was studied of two dopaminergic clusters that innervate different zones of MB, Protocerebral anterior medial (PAM) and Protocerebral posterior lateral 1 (PPL1), on the innate value to the aversive stimulus and the associated locomotor behavior. In order to do this, the synaptic transmission of these neural clusters was manipulated through the expression of Tetanus toxin, Kir2.1 and Transient receptor potential cation channel A1 (TrpA1) channels. The results show that neurons in PPL1 and PAM differentially modulate the innate value to Bz in adult flies. On the other hand, blocking neurotransmission or genetic silencing of PAM neurons results in decreased locomotor behavior in flies, an effect not observed when silencing PPL1. These results suggest that as in mammals, specific dopaminergic pathways differentially modulate locomotor behavior and the innate value for an odorant, a limbic-like response in Drosophila (Fuenzalida-Uribe, 2017).

The prominin-like Gene Expressed in a Subset of Dopaminergic Neurons Regulates Locomotion in Drosophila

CD133, also known as prominin-1, was first identified as a biomarker of mammalian cancer and neural stem cells. Previous studies have shown that the prominin-like (promL) gene, an orthologue of mammalian CD133 in Drosophila, plays a role in glucose and lipid metabolism, body growth, and longevity. Because locomotion is required for food sourcing and ultimately the regulation of metabolism, this study examined the function of promL in Drosophila locomotion. Both promL mutants and pan-neuronal promL inhibition flies displayed reduced spontaneous locomotor activity. As dopamine is known to modulate locomotion, the effects of promL inhibition on the dopamine concentration and mRNA expression levels of tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc), the enzymes that are responsible for dopamine biosynthesis, were examined in the heads of flies. Compared with those in control flies, the levels of dopamine and the mRNAs encoding TH and Ddc were lower in promL mutant and pan-neuronal promL inhibition flies. In addition, an immunostaining analysis revealed that, compared with control flies, promL mutant and pan-neuronal promL inhibition flies had lower levels of the TH protein in protocerebral anterior medial (PAM) neurons, a subset of dopaminergic neurons. Inhibition of promL in these PAM neurons reduced the locomotor activity of the flies. Overall, these findings indicate that promL expressed in PAM dopaminergic neurons regulates locomotion by controlling dopamine synthesis in Drosophila (Ryu, 2022).

Recurrent architecture for adaptive regulation of learning in the insect brain

Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. This study provides a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. Afferent sensory pathways and a large population of neurons were discovered that provide feedback from mushroom body output neurons and link distinct memory systems (aversive and appetitive). This was combined with functional studies of DANs and their presynaptic partners and with comprehensive circuit modeling. It was found that DANs compare convergent feedback from aversive and appetitive systems, which enables the computation of integrated predictions that may improve future learning. Computational modeling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. This study provides the most detailed view to date of biological circuit motifs that support associative learning (Eschbach, 2020).

To behave adaptively in an ever-changing environment, animals must be able to learn new associations between sensory cues (conditioned stimuli, CS) and rewards or punishments (aversive and appetitive unconditioned stimuli, US), and continuously update previous memories, depending on their relevance and reliability (Eschbach, 2020).

Modulatory neurons (for example, DANs) convey information about rewards and punishments, and provide the teaching signals for updating the valence associated with CS in learning circuits across the animal kingdom (for example, the vertebrate basal ganglia or the insect mushroom body, MB). The co-occurrence of CS and modulatory neuron activity tuned only to the received US can support simple associative memory formation. To account for more complex behavioral phenomena, theories have been developed in which learning can be regulated by previously formed associations. According to reinforcement learning theories, learning is driven by errors between predicted and actual US (prediction errors) which are thought to be represented by the activity of DANs. Indeed, in many model organisms, the responses of modulatory neurons have been shown to be adaptive, including monkeys, rodents and insects. Despite recent progress the basic principles by which DAN activity is adaptively regulated and teaching signals are computed are not well understood (Eschbach, 2020).

A prerequisite for the adaptive regulation of modulatory neuron activity is convergence of afferent pathways that convey information about received US with feedback pathways that convey information about previous experiences. A comprehensive synaptic-resolution connectivity map of the feedback circuits that regulate modulatory neurons would provide a basis for understanding how learning is adaptively regulated by prior learning, but it has previously been out of reach (Eschbach, 2020).

Insects, especially in their larval stages, have small and compact brains that have recently become amenable to large-scale electron microscopy (EM) circuit mapping. Both adult and larval insect stages possess a brain center that is essential for associative learning, the MB. The MB contains neurons called Kenyon cells (KCs) that sparsely encode CS, MB modulatory neurons (collectively called MB input neurons, MBINs) that provide the teaching signals and MB output neurons (MBONs) whose activity represents learnt valences of stimuli. In the Drosophila larva, most modulatory neurons are DANs, some are octopaminergic neurons (OANs) and some have unidentified neurotransmitters (simply called MBINs). Modulatory neurons and MBONs project axon terminals and dendrites, respectively, onto the KC axons in a tiled manner, defining MB compartments, in both adult and larval Drosophila. In adult Drosophila, it has been shown that coactivation of KCs and DANs reduces the strength of the KC-MBON synapse in that compartment. Different compartments have been implicated in the formation of distinct types of memories, for example, aversive and appetitive, or short and long term. However, despite a good understanding of the structure and function of the core components of the MB in both adult and larval Drosophila, the circuits presynaptic to modulatory neurons that regulate their activity have remained relatively uncharacterized (Eschbach, 2020).

This study therefore reconstructed all neurons presynaptic to all modulatory neurons in an EM volume that spans the entire nervous system of a first instar Drosophila larva, in which all the core components of the MB were previously reconstructed. This study also determined which individual modulatory neurons are activated by punishments and reconstructed their afferent US pathways from nociceptive and mechanosensory neurons. The neurotransmitter profiles of some of the neurons in the network were characterized and some of the identified structural connections were functionally confirmed. Finally, a model was developed of the circuit constrained by the connectome, the neurotransmitter data and the functional data, and it was used to explore the computational advantages offered by the recently discovered architectural motifs for performing distinct learning tasks (Eschbach, 2020).

Modulatory neurons (for example, DANs) are key components of higher-order circuits for adaptive behavioral control, and they provide teaching signals that drive memory formation and updating. This study provides a synaptic-resolution connectivity map of a recurrent neural network that regulates the activity of modulatory neurons in a higher-order learning center, the Drosophila larval MB. Some of the recently identified structural pathways were functionally tested, and a model of the circuit was developed to explore the roles of these motifs in different learning tasks (Eschbach, 2020).

A large population was discovered of 61 feedback neuron pairs that provide one- and/or two-step feedback from the MBONs to modulatory neurons. Strikingly, it was found that many modulatory neurons receive more than 50% of their total dendritic input from feedback pathways. These results suggest that prior memories as represented by the pattern of MBON activity can strongly influence modulatory neuron activity (Eschbach, 2020).

Learning and memory systems in vertebrates and insects are often organized into distinct compartments implicated in forming distinct types of memories (for example, aversive and appetitive or short and long term). Interestingly, it was found that the majority of the discovered feedback pathways link distinct memory systems, suggesting that the MB functions as an interconnected ensemble during learning. Thus, prior memories formed about an odor in one compartment can influence the formation and updating of distinct types of memories about that odor in other compartments (Eschbach, 2020).

In adult Drosophila, functional connections between some MBONs and DANs have been reported, and some have been shown to play a role in short-term memory formation, long-term memory consolidation, extinction and reconsolidation, or in synchronizing DAN ensemble activity in a context-dependent manner. In some cases, direct MBON-to-DAN connections have been demonstrated. Although direct connections from several MBONs onto DANs exist in the larva, this study found that indirect connections via the feedback neurons account for a much larger fraction of a modulatory neuron's dendritic input than direct MBON synapses. This suggests that adaptive DAN responses may be largely driven by such indirect feedback (Eschbach, 2020).

Some of the one-step within-compartment feedback motifs that were found are analogous to the feedback motifs so far described for the DANs in the vertebrate midbrain. Although the diversity and the inputs of striatal feedback neurons have not yet been fully explored, in the future it will be interesting to determine whether many of the striatal feedback neurons also link distinct memory systems (Eschbach, 2020).

The use of internal predictions can dramatically increase the flexibility of a learning system. This study reveals candidate circuit motifs that could compute integrated predicted value signals across appetitive and aversive memory systems. A prominent motif that was identified is convergence of excitatory and inhibitory connections from MBONs from compartments of opposite valence onto DANs. In naive animals, odor-evoked MBON excitation in all compartments is thought to be similar. However, associative learning selectively depresses conditioned odor drive to MBONs in compartments where modulatory neuron activation has been paired with the odor. It is proposed that by comparing the conditioned odor-evoked MBON excitation in compartments of opposite valence via cross-compartment feedback connections, modulatory neurons compute an integrated predicted value signal across appetitive and aversive domains (Eschbach, 2020).

Convergence of feedback and US pathways could allow the computation of prediction errors An important aspect of reinforcement learning theories is the idea that modulatory neurons compare predicted and actual US (to compute so-called prediction errors) and drive memory formation or extinction depending on the sign of the prediction error. Although Drosophila modulatory neurons have not yet been directly shown to represent prediction errors, adult and larval Drosophila are capable of extinction, and the current study reveals candidate motifs that could support the comparison of expected and actual US. Modulatory neurons were found to receive convergent input from feedback pathways from MBONs and from US pathways. Modulatory neurons could therefore potentially compute prediction errors by comparing inhibitory drive from the feedback pathways with the excitatory drive from the US pathways, or vice versa. Consistent with this idea, some DANs were observed in this model that are inhibited by US alone and activated by CS+ alone, or vice versa (Eschbach, 2020).

This study also reveals that US pathways and feedback pathways converge at two levels: not only at the modulatory neurons themselves, but also at the FB2Ns. Actual and expected outcomes could therefore also be compared by FB2Ns. A recent study in the mouse ventral tegmental area has found that some pre-DAN neurons encoded only actual or only expected reward, whereas others encoded both variables. Thus, both in vertebrates and in insects, comparing predicted and actual outcomes may be a complex computation involving multiple levels of integration that eventually converge onto an ensemble of modulatory neurons (Eschbach, 2020).

An assumption in many reinforcement learning models is that all modulatory neurons receive a global scalar reward prediction error signal. The current study was able to analyze the comprehensive set of inputs of every individual uniquely identifiable modulatory neuron in a learning center. This revealed that each modulatory neuron receives a unique set of feedback inputs that could enable each neuron to compute a unique set of features. Consistent with this, a diversity of adaptive response types in the modulatory neurons was observed in this model This suggests that instead of computing a single global reward prediction error that is distributed to all modulatory neurons, the network uses a range of distinct compartmentalized and distributed teaching signals (Eschbach, 2020).

The connectivity and modeling studies revealed two architectural features of the circuit that provide input to the modulatory neurons that increase its performance and flexibility on learning tasks. The first is the multilevel feedback architecture that includes not only the previously known direct MBON feedback, but also multiple levels of indirect feedback. The second is the extensive set of cross-compartment connecti,.ons. Modeling suggests that these motifs support improved performance on complex tasks that require the computation of variables such as predictions, prediction errors and context (Eschbach, 2020).

In summary, this study presents a complete circuit diagram of a recurrent network that computes teaching signals in a biological system, providing insights into the architectural motifs that increase its computational power and flexibility. The connectome-constrained model provides numerous predictions that can be tested in the future in a tractable model organism, for which genetic tools can be generated to monitor and manipulate individual neurons. The connectome, together with the functional and modeling studies, therefore provides exciting opportunities for elucidating the biological implementation of reinforcement learning algorithms (Eschbach, 2020).

Dopamine Modulates Drosophila Gut Physiology, Providing New Insights for Future Gastrointestinal Pharmacotherapy

Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, Drosophila was used as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males' gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies' gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. These findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency (El Kholy, 2021).

Serotonergic neurons translate taste detection into internal nutrient regulation

The nervous and endocrine systems coordinately monitor and regulate nutrient availability to maintain energy homeostasis. Sensory detection of food regulates internal nutrient availability in a manner that anticipates food intake, but sensory pathways that promote anticipatory physiological changes remain unclear. This study identified serotonergic (5-HT) neurons as critical mediators that transform gustatory detection by sensory neurons into the activation of insulin-producing cells and enteric neurons in Drosophila. One class of 5-HT neurons responds to gustatory detection of sugars, excites insulin-producing cells, and limits consumption, suggesting that they anticipate increased nutrient levels and prevent overconsumption. A second class of 5-HT neurons responds to gustatory detection of bitter compounds and activates enteric neurons to promote gastric motility, likely to stimulate digestion and increase circulating nutrients upon food rejection. These studies demonstrate that 5-HT neurons relay acute gustatory detection to divergent pathways for longer-term stabilization of circulating nutrients (Yao, 2022).

The nervous and endocrine systems coordinately monitor and regulate nutrient availability to maintain energy homeostasis. Sensory detection of food regulates internal nutrient availability in a manner that anticipates food intake, but sensory pathways that promote anticipatory physiological changes remain unclear. This study identified serotonergic (5-HT) neurons as critical mediators that transform gustatory detection by sensory neurons into the activation of insulin-producing cells and enteric neurons in Drosophila. One class of 5-HT neurons responds to gustatory detection of sugars, excites insulin-producing cells, and limits consumption, suggesting that they anticipate increased nutrient levels and prevent overconsumption. A second class of 5-HT neurons responds to gustatory detection of bitter compounds and activates enteric neurons to promote gastric motility, likely to stimulate digestion and increase circulating nutrients upon food rejection. These studies demonstrate that 5-HT neurons relay acute gustatory detection to divergent pathways for longer-term stabilization of circulating nutrients (Yao, 2022).

This study identified two classes of 5-HT neurons that play distinct roles in gustatory processing and function independently to promote nutrient homeostasis. Sugar-SELs, located in the lateral subesophageal ganglion (SEL) respond to sugar gustatory detection, promote insulin-producing cell (IPC) activity, and reduce feeding drive, suggesting that they prevent overconsumption in nutrient rich environments. Bitter-SELs respond to bitter taste detection and promote crop contractions, likely to utilize stored food upon food rejection. Thus, 5-HT neurons coordinate endocrine and digestive function in anticipation of altered food intake (Yao, 2022).

5-HT profoundly modulates appetite and feeding across animal species. In humans and rodents, the global effect of brain 5-HT signaling is suppression of food intake; however, the involvement of multiple brain regions (including the hypothalamus, solitary tract nucleus, and parabrachial nuclei) and multiple 5-HT receptors (e.g., 5-HT1B, 5-HT2C, 5-HT6) underscores the complex nature of 5-HT modulation of appetite and feeding. Studies in invertebrates have likewise demonstrated multiple, sometimes opposite, roles for 5-HT neurons in regulating feeding. For instance, in Drosophila adults, activating all 5-HT neurons suppresses food intake whereas activating a smaller yet diverse subset promotes food intake, suggesting heterogeneity in 5-HT feeding regulation. With the exception of a few cases where specific 5-HT neurons that influence feeding have been identified—for example, 5-HT neurons that promote pharyngeal pumping to enhance ingestion in C. elegans and Drosophila larvae—the diversity of 5-HT neurons that contributes to feeding regulation and nutrient homeostasis remains largely uncharacterized (Yao, 2022).

This study identified multiple classes of 5-HT neurons that are activated by gustatory detection and signal to the endocrine and digestive systems to influence nutrient availability. These studies reveal 5-HT neurons that have different projection patterns, relay sugar and bitter gustatory information to different downstream targets, and regulate internal nutrient availability by distinct mechanisms. These studies provide insight into the multifaceted roles of 5-HT neurons in gustatory processing, feeding regulation, and nutrient homeostasis, highlighting the importance of understanding the myriad functions of 5-HT at the neural circuit level (Yao, 2022).

Food-derived sensory cues are used as anticipatory signals to regulate endocrine function and feeding drive. Work in mammals has demonstrated that there are two phases of insulin release in response to food consumption, a pre-absorptive phase in response to sensory detection of food and a post-absorptive phase in response to elevated blood glucose levels. The pre-absorptive phase (cephalic phase) is triggered by sensory detection prior to food consumption and nutrient absorption. The neural circuit that underlies pre-absorptive insulin release, however, is not fully understood (Yao, 2022).

The current findings suggest that insulin release in anticipation of food consumption is a process shared in flies and mammals, perhaps indicating an effective strategy to promote rapid nutrient storage during feeding. Using in vivo calcium imaging, this study found that fly IPCs are rapidly excited by sugar gustatory detection independent of consumption. The findings that sugar-SELs respond to sugar taste detection and activate IPCs suggest a neural circuit mechanism for pre-absorptive insulin release independent of ingestion. Moreover, as gustatory sensory neurons detect both nutritive and non-nutritive sugars and are inputs to sugar-SELs and IPCs, it is anticipated that sugar-SELs, IPCs, and pre-absorptive insulin release are also activated by non-nutritive sugars, but this requires further investigation. The finding that fly IPCs are activated by sugar taste detection contrasts with previous whole-brain imaging studies, likely indicating signal detection limits. In addition to identifying sugar taste responses in IPCs, this study also identified the sugar-SELs as a specific neural pathway mediating the preparatory insulin response (Yao, 2022).

Knocking down 5-HT2A in IPCs reduces but does not abolish gustatory-induced IPC activity and does not impact consumption. One caveat of this approach is that RNAi reduces gene expression and may not produce complete loss-of-function phenotypes. The IPCs are activated by many nutritional state signals, including multiple pathways that report circulating sugars and signals from the intestine and fat body. The current work shows that external nutrients in the form of sugar taste detection also activate this important hub and identifies sugar-SELs as a defined pathway conveying the sugar taste signal. As IPCs release multiple peptides, the activation of IPCs by sugar-SELs may coordinate widespread changes in metabolism and behavior. Thus, this study has identified a specific class of 5-HT neurons that participates in the preparatory insulin response and the reduction of feeding drive in response to sugar gustatory detection, shedding light on the neural circuit mechanisms that anticipate sugar consumption (Yao, 2022).

A surprising finding from this study is that bitter-SELs, which respond to bitter gustatory detection, promote contractions of the crop food storage organ. While there is evidence that intestinal bitter detection modulates gastrointestinal physiology, the regulation of gastrointestinal function by bitter gustatory detection is less examined. Activation of bitter gustatory neurons, bitter-SELs, and 5-HT7 neurons all promote crop contractions, although rates differ, possibly based on optogenetic activation strength or propagation of activity to the crop (Yao, 2022).

Why do bitter compounds promote crop contractions? It was reasoned that because bitter compounds are feeding deterrents, frequent encounters with bitter compounds may prevent food intake, leading to depletion of internal nutrients. Under such conditions, bitter-SELs may promote crop motility to utilize food reserves in anticipation of limited food intake. It is therefore proposed that bitter gustatory compounds may have an unappreciated role in predicting food scarcity and stimulating digestion as a preparatory response (Yao, 2022).

Previous studies in Drosophila have demonstrated that bitter taste detection elicits inhibition of proboscis extension and suppression of consumption. In addition, detection of bitter compounds drives avoidance behavior and increased locomotion, likely to promote departure to new areas. The current findings suggest an additional role for bitter taste detection in promoting mobilization of food stores to increase circulating nutrients. These actions are aligned in mitigating the impact of a potentially toxic food source by limiting consumption, promoting relocation, and maintaining internal nutrient levels. Whereas bitter taste detection rapidly leads to consumption inhibition and increased locomotion, whether bitter taste detection activates crop contractions acutely or only under specific conditions of food deprivation is not resolved. In addition, although increased crop contractions were observed upon activation of bitter-SELs, it remains to be examined whether this impacts internal circulating nutrients. Bitter-SELs may also influence additional aspects of feeding behavior not tested in this study, including modulating consumption under different physiological states or when different feeding assays or different bitter compounds are used (Yao, 2022).

In addition to the hypocerebral ganglion (HCG), bitter-SELs project to diverse targets, suggesting that they may carry out other functions besides enteric modulation. For example, bitter-SELs broadly arborize on the dorsal surface of the VNC. Thus, bitter-SELs may also set the 5-HT tone in the VNC or secrete 5-HT into the hemolymph to modulate target tissues in a paracrine or endocrine fashion (Yao, 2022).

This study found that 5-HT neurons are critical nodes in the circuits that transform gustatory detection into changes in endocrine and digestive function. Although the timescale of activation of sugar-SELs and bitter-SELs and the dynamics of 5-HT release requires further investigation, 5-HT receptors are metabotropic receptors ideally suited for transforming transient neural signals into more sustained cellular responses. In this regard, neuromodulatory circuits are prime candidates for eliciting preparatory responses that require the transformation of neural signals across time scales. This work thus sheds light on neural circuit mechanisms that translate external sensory cues into preparatory physiological responses and suggests that neuromodulators such as 5-HT may contribute to anticipatory mechanisms in other animals (Yao, 2022).

Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state

Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. This study shows that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyramine(honoka) receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. It was further shown that the level of tyramine-beta-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH (Schutzler, 2019).

AANAT1 functions in astrocytes to regulate sleep homeostasis

How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. This study found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and it was found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation. Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight sleep deprivation. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep (Davla, 2020).

Characteristic features of sleep are conserved among species, and from humans to insects sleep is influenced by neural circuits involving monoamines such as serotonin and dopamine. Glial cells are known to take up and metabolize monoamines and they have been increasingly implicated in mechanisms of baseline and homeostatic sleep regulation in mammals and flies, but it remains unknown whether and how glia might influence monoaminergic control of sleep. Sleep is regulated by circadian clocks and a homeostatic drive to compensate for prolonged wakefulness, and growing evidence suggests that neural mechanisms controlling homeostatic sleep can be discriminated from those controlling baseline sleep. In Drosophila, mutants of arylalkylamine N-acetyltransferase 1 (AANAT1^lo) were reported to have normal baseline amounts of sleep and motor activity, but increased recovery sleep ('rebound') following deprivation. AANAT1 corresponds to speck, a long-known mutation characterized by a darkly pigmented region at the wing hinge. AANAT1 can acetylate and inactivate monoamines in vitro, but the role of AANAT1 in vivo remains poorly understood (Davla, 2020).

This paper reports that AANAT1 is expressed in astrocytes and subsets of neurons in the adult Drosophila brain, with levels in astrocytes first rising then declining markedly overnight. In sleep deprived AANAT1 mutant flies, heightened recovery sleep is accompanied by increased serotonin and dopamine levels in the brain. With cell type selective AANAT1 knockdown, this study found that AANAT1 functions in astrocytes, but not neurons, to limit the amount of recovery sleep that flies take in response to sleep deprivation (SD). These findings identify a critical role for astrocytes in the regulation of monoamine bioavailability and calibration of the response to sleep need (Davla, 2020).

These findings illustrate a newly discovered role for astrocytes in the control of monoamine bioavailability and homeostatic sleep drive, where they are specifically engaged to catabolize monoamines whose levels are elevated by overnight SD. Drosophila astrocytes also express the enzyme Ebony, which couples dopamine to N-β-alanine, and a receptor for octopamine and tyramine, reinforcing how they are well-equipped to metabolize monoamines, and to monitor and respond to monoaminergic neuronal activity. Neither gene expression studies nor RNA sequencing databases provide evidence for monoamine-synthesizing enzymes in Drosophila astrocytes, so it appears likely that monoamines inactivated by AANAT1 in astrocytes are brought into these cells by an unidentified transporter. Astrocytes are particularly well-suited for regulating sleep in this way because they have ramified processes that infiltrate neuropil regions to lie in close proximity to synapses. SD can seemingly reduce the degree of contact between astrocytes and neurons in the fly brain, and so it is possible that these structural changes could influence monoamine uptake and inactivation by astrocytes (Davla, 2020).

In neurons, AANAT1 may function to limit sleep consolidation at night, but evidence for this came from only one of the two RNAi lines used in this study and was not observed in AANAT1^lo mutants. Further studies are needed to characterize sleep-control functions of AANAT1 in neurons, if any, to understand better how cellular context can impact AANAT1 function in sleep regulation. In light of this, it is noted that loss of the related enzyme aanat2 in zebrafish larvae decreases baseline sleep, which could be attributed to a loss of melatonin since the AANAT1 product N-acetylserotonin is an intermediate in the synthesis of melatonin in vertebrates. Clearly, the appropriate balance and cellular context of AANAT activity is critical for the regulation of sleep, and this study shows in Drosophila that astrocytes are an important contributor to this balance. Interestingly, astrocytes in rodents express the monoamine transporters and receptors for dopamine and serotonin, raising the possibility that astrocytes in mammals might also participate in mechanisms of sleep regulation involving monoaminergic neural signaling (Davla, 2020).

Endocrine signals fine-tune daily activity patterns in Drosophila

Animals need to balance competitive behaviors to maintain internal homeostasis. The underlying mechanisms are complex but typically involve neuroendocrine signaling. Using Drosophila, this study systematically manipulated signaling between energy-mobilizing endocrine cells producing adipokinetic hormone (AKH), octopaminergic neurons, and the energy-storing fat body to assess whether this neuroendocrine axis involved in starvation-induced hyperactivity also balances activity levels under ad libitum access to food. The results suggest that AKH signals via two divergent pathways that are mutually competitive in terms of activity and rest. AKH increases activity via the octopaminergic system during the day, while it prevents high activity levels during the night by signaling to the fat body. This regulation involves feedback signaling from octopaminergic neurons to AKH-producing cells (APCs). APCs are known to integrate a multitude of metabolic and endocrine signals. The results add a new facet to the versatile regulatory functions of APCs by showing that their output contributes to shape the daily activity pattern under ad libitum access to food (Pauls, 2021).

cAMP signals in Drosophila motor neurons are confined to single synaptic boutons

The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. This study suggests a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, this study demonstrates the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, evidence is provided that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons (Maiellaro, 2016).

The cyclic AMP (cAMP) pathway plays fundamental roles in the nervous system, where it is prominently involved in synaptic plasticity and memory formation. Previous studies in vertebrate and invertebrate models have shown that cAMP can propagate from dendrites to the cell body of neurons, in line with the properties of a small diffusible molecule. However, a local mode of action for cAMP has also been proposed, whereby cAMP signals are localized to the periphery of neurons, namely, dendrites, creating a cAMP microdomain. While the existence of cAMP microdomains in neuronal dendrites is disputed based on the experimental and theoretical data, very little is known about possible cAMP compartmentation in axons and how this may exert local effects at the presynaptic site. In particular, it is unclear how biochemical signals may spread from presynaptic boutons through the axon (Maiellaro, 2016).

To investigate this question in vivo, the neuromuscular junction (NMJ) of Drosophila melanogaster was used, that displays different forms of synaptic plasticity, many of which are dependent on cAMP signaling. Both structural and functional properties of larval neuromuscular synapses are heterogeneous, varying between boutons belonging to the same motor neuron. How such site-specific synaptic differentiation may be achieved at high spatial resolution is currently unknown, though it is tempting to speculate that local cAMP signals play a role. Thus, the developing Drosophila NMJ is a powerful model to investigate the role of cAMP in synaptic plasticity under physiological conditions (Maiellaro, 2016).

This study focused on glutamatergic type Ib motor neurons, which are structurally regulated via G-protein-coupled receptors (GPCRs) for octopamine. Stimulation of these receptors has been shown to induce synaptic bouton outgrowth via a cAMP- and CREB-dependent pathway. Therefore, this study set out to measure spatiotemporal patterns of octopamine-induced cAMP signals in these neurons. To this end, a FRET (Forster resonance energy transfer)-based sensor for cAMP (Epac1-camps) was expressed that has previously been used to image cAMP levels in central Drosophila neurons. The results reveal that cAMP signals are confined to their initiation site, the individual synaptic bouton, and suggest a highly efficient local mechanism for controlling site-specific synaptic plasticity (Maiellaro, 2016).

This study genetically expressed the cAMP sensor, Epac1-camps, at the Drosophila NMJ to monitor and quantify spatiotemporal cAMP dynamics induced by octopamine stimulation. The results reveal an unexpectedly high degree of cAMP compartmentalization in motor neurons, which may serve as a basis for local synaptic plasticity, with cAMP FRET signals being ultimately limited to single synaptic boutons. Three cAMP signaling compartments were identified within the motor neuron: boutons, axon, and cell body. For each cellular compartment, the particular mechanism responsible for the segregation of cAMP increases is described. Specifically, this study found that boutons constitute the most reactive compartment of the motor neuron in terms of cAMP accumulation. The results demonstrate that the production of cAMP is heterogeneous among boutons, with stronger responses to octopamine in large boutons rather than in smaller ones, possibly related to the increased synaptic strength measured at large boutons. Moreover, the activity and the specific localization of PDEs within the synaptic bouton prevent the propagation of cAMP to the cell body and its diffusion from one bouton to the next (Maiellaro, 2016).

In contrast to the boutons, octopamine receptors seem to be absent or inaccessible in the axon, and PDEs were not detected. Accordingly, cAMP FRET signals recorded in the axon differ from those in boutons in terms of amplitude and kinetics. Hence, the axon emerges as the second independent, but not isolated, cAMP signaling compartment. Interestingly, and in contrast to dendrites, cAMP did not propagate along the axon. It remains to be clarified whether this may relate to functional differences between axons and dendrites, between species, or between specific neuron types. Finally, the cell body was determined as the third cAMP signaling compartment within the motor neuron. The cell body has a very low sensitivity toward octopamine, and it was demonstrated that cAMP FRET signals generated in the cell body are not affected by the cell body's physical isolation from the axon. High PDE activity in the cell body contributes to this local suppression of cAMP signaling and may prevent spillover activation of cAMP effectors in the cell body and in the nucleus (Maiellaro, 2016).

Evidence of cAMP microdomains restricted to single boutons provides a biophysical basis for the local control of synaptic plasticity. Spatially constrained cAMP changes help to establish differences in morphology and synaptic content of boutons, suggesting that local cAMP, bouton structure, and synapse formation are intimately linked. The observed confinement of cAMP supports the notion that individual synaptic boutons may represent largely autonomous signaling units, which can receive and integrate signals independently of the other (Maiellaro, 2016).

The concept that cAMP could act as a local messenger was postulated almost 40 years ago. The existence of cAMP microdomains has been demonstrated in other cell types (e.g., cardiomyocytes). However, in neurons, there are contradicting experimental lines of evidence and simulations concerning the existence of cAMP microdomains. The current data clearly show that cAMP can act as a local messenger upon physiological stimulation of a neuron. The detailed spatiotemporal analysis of the dynamics of cAMP reveals that this messenger can be restricted at the micrometer level to induce highly localized physiological responses (Maiellaro, 2016).

Maintenance of quiescent oocytes by noradrenergic signals

All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. This study reports that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles.This study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes (Kim, 2021).

Octopaminergic/tyraminergic Tdc2 neurons regulate biased sperm usage in female Drosophila melanogaster

In polyandrous internally fertilizing species, a multiply-mated female can use stored sperm from different males in a biased manner to fertilize her eggs. The female's ability to assess sperm quality and compatibility is essential for her reproductive success, and represents an important aspect of postcopulatory sexual selection. In Drosophila melanogaster, previous studies demonstrated that the female nervous system plays an active role in influencing progeny paternity proportion, and suggested a role for octopaminergic/tyraminergic Tdc2 neurons in this process. This study reports that inhibiting Tdc2 neuronal activity causes females to produce a higher-than-normal proportion of first-male progeny. This difference is not due to differences in sperm storage or release, but instead is attributable to the suppression of second-male sperm usage bias that normally occurs in control females. It was further shown that a subset of Tdc2 neurons innervating the female reproductive tract is largely responsible for the progeny proportion phenotype that is observed when Tdc2 neurons are inhibited globally. On the contrary, overactivation of Tdc2 neurons does not further affect sperm storage and release or progeny proportion. These results suggest that octopaminergic/tyraminergic signaling allows a multiply-mated female to bias sperm usage, and identify a new role for the female nervous system in postcopulatory sexual selection (Chen, 2022).

Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster

Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. This study reports that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca(2+) signaling. Moreover, Matrix metalloproteinase-2 was identified as a downstream component of the octopamine-Ca(2+) signaling to induce GSC increase. This study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling (Yoshinari, 2020).

This study reports that the mating-induced GSC increase in female D. melanogaster is regulated by octopaminergic neurons directly projecting to the ovary. From in vivo and ex vivo experiments, the following model is proposed to explain the mating-induced GSC increase. After mating, the male seminal fluid SP is transferred into the female uterus, stimulating SPR-positive neurons. As the liganded SPR silences the neuronal activity of SPR-positive sensory neurons (SPSNs), the acetylcholine released from SPSNs is suppressed. As SPSNs and dsx+ Tdc2+ neurons are directly connected, this suppression directly modulates dsx+ Tdc2+ neuronal activity. Because this study has shown that nAChRs in dsx+ Tdc2+ neurons exhibit an inhibitory effect with an unknown mechanism, the inactivation of nAChRs in the absence of acetylcholine results in the activation of dsx+ Tdc2+ neurons in mated females. As a consequence, octopamine is released from dsx+ Tdc2+ neurons, received by Oamb, induces [Ca2+]i in the escort cells, and finally activates the Mmp2 enzymatic activity. The activity of Mmp2 positively regulates the Dpp-mediated niche signaling, thereby leading to mating-induced GSC increase. (Yoshinari, 2020).

The proposed model is that the OA from dsx+ Tdc2+ neurons is directly received by the escort and follicle cells in the germarium. This model is supported by two observations. First, mating-induced GSC increase is impaired by Oamb RNAi using a GAL4 driver that is active specifically in the germarium cells but not mature follicle cells. Second, OA treatment evokes [Ca2+]i elevation in these germarium cells in an Oamb-dependent manner. However, this study did not address whether the escort cells and/or the follicle cells in the germarium express Oamb, as no clear GAL4 expression was observed in two independent Oamb-T2A-GAL4 drivers. It is surmised that this may be due to lower amounts of Oamb transcript in the germarium (Yoshinari, 2020).

It was shown that the activation of the ovary-projecting dsx+ Tdc2+ neurons is necessary and sufficient to induce GSC increase. However, from an anatomical point of view, the dsx+ Tdc2+ neurons project to the distal half of the ovary but not to the germarium. Considering the model described above, this disagreement can be attributed to the characteristic volume transmission of monoamine neurotransmitters. In other words, neurotransmitters act at a distance well beyond their release sites from cells or synapses. Therefore, the OA secreted from the terminals of dsx+ Tdc2+ neurons could reach the germarium located at the most proximal part of the ovary (Yoshinari, 2020).

Several previous studies have revealed that OA signaling has a pivotal role in reproductive tissues other than germarium, such as mature follicle cells, oviduct, and ovarian muscle, to promote ovulation, oviduct remodeling, and ovarian-muscle contraction, respectively. Therefore, it is likely that the dsx+ Tdc2+ neurons orchestrate multiple different events during oogenesis in response to mating stimulus. Because a mated female needs to activate oogenesis to continuously produce eggs in concert with sperm availability, it is reasonable that the ovary-projecting neurons switch on the activity of the entire process of reproduction (Yoshinari, 2020).

Based on the present study and several previous studies, the OA-Oamb-Ca2+-Mmp2 axis is required for GSC increase and follicle rupture, both of which are induced by mating stimuli in D. melanogaster. In both cases, Mmp2 enzymatic activity is likely to be essential, as the overexpression of Timp encoding a protein inhibitor of Mmp2 suppresses GSC increase, as well as follicle rupture. Mmp2 in mature follicle cells cleaves and downregulates Viking/collagen VI. In fact, several previous studies have revealed that Viking/collagen VI is required for GSC maintenance in female D. melanogaster. However, no significant change was observed in Viking/Collagen VI levels in the germarium between the control and Mmp2 RNAi flies. Therefore, it is concluded that Viking/collagen VI is not a substrate of Mmp2 in the regulation of mating-induced GSC increase. Besides Viking/Collagen VI, Dally-like (Dlp) is another basement membrane protein associated with extracellular matrix and known as the Mmp2 substrate. Interestingly, dlp is expressed in the escort cells. Moreover, Dlp controls the distribution of Dpp and Wnts, both of which significantly affect GSC self-renewal and differentiation. Future research should decipher the exact substrate by which Mmp2 controls Dpp and/or Wnts to modulate GSC behavior in response to mating stimulus (Yoshinari, 2020).

Another remaining question to be addressed is how Mmp2 function is regulated in GSC increase. Ecdysteroid biosynthesis and signaling in the ovary are necessary but not sufficient for the OA-Oamb-Ca2+-mediated GSC increase and follicle rupture. This study found that in the regulation of mating-induced GSC increase, ecdysteroid signaling acts downstream of Ca2+ signaling. On the other hand, in the follicle rupture process, ecdysteroid signaling either acts downstream, upstream, or both, of Ca2+ signaling. Further, the precise action of ecdysteroid has yet to be elucidated. The Mmp2-GFP fusion protein level in the follicle cells is not changed in the loss-of-Ecdysone receptor-function flies, implying that ecdysteroid signaling might regulate Mmp2 enzymatic activity by an unknown mechanism. Considering the involvement of both the OA-Oamb-Ca2+-Mmp2 axis and ecdysteroid biosynthesis, it is very likely that the Mmp2 enzymatic activity is also regulated by the same, unknown mechanism not only in the mature follicle cells to control follicle rupture, but also in the germarium to control mating-induced GSC increase (Yoshinari, 2020).

In many animals, reproduction involves significant behavioral and physiological shifts in response to mating. In female D. melanogaster, several post-mating responses are coordinated by SPSNs and their downstream afferent neuronal circuit, including Stato-Acoustic Ganglion neurons, the ventral abdominal lateral Myoinhibitory peptide neurons, and the efferent dsx+ Tdc2+ neurons. GRASP analysis indicates a direct synaptic connection between cholinergic SPSNs and OAergic neurons. Moreover, this study demonstrated that nAChRs in dsx+ Tdc2+ neurons are responsible for the suppression of their neuronal activity in virgin females. However, nAChRs are the cation channels leading to depolarization upon acetylcholine binding, and therefore usually activate neurons. How is the opposite role of nAChRs in dsx+ Tdc2+ neuronal activity achieved? One possibility is that acetylcholine-nAChR signaling does not evoke a simple depolarization but rather generates a virgin-specific temporal spike pattern in dsx+ Tdc2+ neurons. Interestingly, recent studies demonstrated that the pattern, instead of the frequency, of neuronal firing is significant in adjusting the neuronal activity of clock neurons in D. melanogaster. The firing pattern relies on control of ionic flux by the modulation of Ca2+-activated potassium channel and Na+/K+ ATPase activity. Because whether mating changes the firing pattern of dsx+ Tdc2+ neurons remains to be examined, the neuronal activity in SPSNs and the dsx+ Tdc2+ neuronal circuit between virgin and mated females are future research areas (Yoshinari, 2020).

In the last decades, there is growing evidence that GSCs and their niche are influenced by multiple humoral factors. Based on the data from the current study and previous studies, there are at least four crucial humoral factors for regulating the increase and/or maintenance of D. melanogaster female GSCs, including DILPs, ecdysteroids, Neuropeptide F (NPF), and OA. Notably, all of these come from different sources: DILPs are from the insulin-producing cells located in the pars intercerebralis of the central brain; ecdysteroids from the ovary; NPF from the midgut; and OA from the neurons located in the abdominal ganglion. In addition to these identified humoral factors, recent studies also imply that adiponectin and unknown adipocyte-derived factor(s) are essential for GSC maintenance. These data clearly indicate that D. melanogaster female GSCs are systemically regulated by interorgan communication involving multiple organs. The additional interorgan communication mechanisms that ensure the faithful coupling of the increase and maintenance of GSC to the organism's external and physiological environments are essential to be investigated in future studies (Yoshinari, 2020).

To modulate the increase and maintenance of GSC, ecdysteroids are received by both GSCs and niche cells, whereas DILPs, NPF, and OA are received by niche cells. A major signal transduction mechanism of each of these humoral factors have been well characterized, namely phosphoinositide 3-kinase pathway for DILPs-InR signaling, EcR/Ultraspiracle-mediated pathway for ecdysteroid signaling, cAMP pathway for NPF-NPFR signaling, and Ca2+ pathway for OA-Oamb signaling. However, it remains unclear whether and how each of these signaling pathways control the production and secretion of the niche signal, as well as its distribution and transduction. In addition, it is important to understand whether and how the multiple system signals are integrated to control the mating-induced increase and maintenance of GSCs (Yoshinari, 2020).

In recent years, many studies have revealed that not only local niche signals but also systemic and neuronal factors play indispensable roles in regulating GSC behavior. In D. melanogaster, ecdysteroid signaling is essential for the proliferation and maintenance of GSCs and neural stem cells. This study has identified the ovary-projecting OAergic neurons as new regulators of stem cell homeostasis. Both steroid hormones and OA-like monoamines, such as noradrenaline, are also involved in stem cell regulation in mammals. For example, the mammalian steroid hormone, estrogen, is important in regulating cell division and/or maintenance of hematopoietic stem cells, mammary stem cell, neural stem cells, and hematopoietic stem cells. Moreover, noradrenergic neurons, which directly project to the bone marrow, regulate the remodeling of hematopoietic stem cells niche. Therefore, the steroid hormone- and noradrenergic nerve-dependent control of stem cell homeostasis are likely conserved across animal species. In this regard, the D. melanogaster reproductive system will further serve as a powerful model to unravel the conserved systemic and neuronal regulatory mechanisms for stem cell homeostasis in animals (Yoshinari, 2020).

Identification of a dopamine pathway that regulates sleep and arousal in Drosophila

Sleep is required to maintain physiological functions, including memory, and is regulated by monoamines across species. Enhancement of dopamine signals by a mutation in the dopamine transporter (DAT) decreases sleep, but the underlying dopamine circuit responsible for this remains unknown. This study found that the D1 dopamine receptor (DA1, also known as DopR) in the dorsal fan-shaped body (dFSB) mediates the arousal effect of dopamine in Drosophila. The short sleep phenotype of the DAT mutant was completely rescued by an additional mutation in the DA1 gene, but expression of wild-type DA1 in the dFSB restored the short sleep phenotype. Anatomical and physiological connections were found between dopamine neurons and the dFSB neuron. Finally, mosaic analysis with a repressive marker found that a single dopamine neuron projecting to the FSB activates arousal. These results suggest that a local dopamine pathway regulates sleep (Ueno, 2012).

Neurons in the dFSB are involved in dopaminergic sleep regulation in Drosophila an the PPM3-FSB dopamine pathway, which is distinct from that required for memory formation, regulates arousal. A previous study found that the rescue of DA1 mutants outside of the mushroom body using a pan-neuronal GAL4 driver, elav-GAL4, coupled with the mushroom body suppressor MB-GAL80 in DA1^dumb1 mutants can recover methamphetamine sensitivity. This suggests that dopamine regulates arousal outside of the mushroom body. Previous findings have shown that DA1 in the PDF neurons (lateral ventral neurons) regulates sleep-wake arousal and that DA1 in the ellipsoid body regulates stress-induced arousal (Ueno, 2012).

A previous study identified PDF neurons that mediate the buffering effects of light on dopamine-induced arousal. However, the current ablation experiments showed that dopamine can elicit strong arousal effects without PDF neurons. The previous report used heterozygous DA1 mutant flies in a wild-type background for most experiments. However, this study used homozygous (null) DA1 mutants, as we found that the heterozygous DA1 mutants crossed with DAT^fmn showed an almost equivalent short sleep phenotype. Thus, one possible explanation for the difference between the previous study and this one is that heterozygous expression of DA1 in the FSB is sufficient to elicit the arousal effects of dopamine, which is also regulated partly through PDF neurons. Given that DA1 expression in the dFSB neurons alone is sufficient for the majority of the wake-promoting effects of dopamine, the arousal regulating dopamine pathway appears to converge at the FSB (Ueno, 2012).

Activation of the dopamine neurons required for aversive memory formation had little effect on sleep. On the other hand, TrpA1 stimulation in a single FSB-projecting PPM3 dopamine neuron was able to reduce sleep. As the magnitude of the decrease in sleep by the activation of the single PPM3 neuron was smaller than that of most dopamine neurons with TH-GAL4, it is possible that other dopamine neurons, which were not labeled in MARCM screening, also affect arousal. For example, in the PPL1 cluster, at least five types of projections to the mushroom body and FSB-projecting neurons have been described (Ueno, 2012).

However, in a MARCM experiment, many of the labeled neurons in the PPL1 cluster were seen to project to the alpha lobe of the mushroom body; it is possible that the contribution of other PPL1 neurons were not fully determine. Alternatively, combinatorial activation of the FSB-projecting PPM3 neurons and other dopamine neurons may have a synergistic effect. Further MARCM experiments using various clone induction protocols may help to formulate a more comprehensive characterization of single dopamine neurons. In addition, it is also possible that dopamine neurons that are not labeled by TH-GAL4 also have an arousal effect. It was noticed that TH-GAL4–induced GFP expression and tyrosine hydroxylase staining do not overlap completely, and some clusters, such as the PAM cluster, are covered only partially by TH-Gal4 (Ueno, 2012).

The association between sleep and memory in Drosophila has recently been described in various reports. Other short sleep flies, hyperkinetic and calcineurin knockdown, also suffer from impaired memory. Sleep deprivation has been shown to impair aversive olfactory memory and learned phototaxis suppression. Conversely, memory formation increased sleep duration in normal flies, and this has not been observed in learning-deficient mutants. In the Drosophila brain, the expression of synaptic component proteins decreased during sleep and increased during waking, suggesting that sleep is required for the maintenance of synapses. In contrast, the current data imply a functional dissociation between sleep and memory circuits by different dopamine neurons. These results will provide clues to uncover a possible physiological relationship between them, for example, by the activation of only one to examine the causal relationship (Ueno, 2012).

The FSB has been reported to have a role in visual memory processing, but its involvement in other behaviors is not yet fully understood. A recent report showed that activation of dFSB neurons induces sleep. This suggests that the dopamine signal regulates sleep via control of the neural properties of the dFSB neurons through DA1. In mammals, dopamine signaling via the D1-type dopamine receptor is thought to increase firing probability. However, dopamine signaling via the D1-like receptor DA1 in flies results in the inhibition of neural activity. Although the previous report suggested that the inhibition is independent of protein kinase A, activation of adenylyl cyclase with Gs* in dFSB decreased sleep levels. Taken together, these findings indicate that DA1 activation in the dFSB inhibits neural activity and results in the promotion of wakefulness. It has been reported that sleep induction through the activation of the dFSB neurons promotes memory consolidation after courtship conditioning. However, this study found that DA1 expression in the FSB itself had little effect on aversive olfactory memory. The contradiction between these findings might be a result of the difference in the memory tasks. It is also possible that functional interaction between sleep and memory is implemented downstream of the FSB. Further studies are required to elucidate the causal relationship between sleep and memory (Ueno, 2012).

Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits

Arousal is fundamental to many behaviors, but whether it is unitary or whether there are different types of behavior-specific arousal has not been clear. In Drosophila, dopamine promotes sleep-wake arousal. However, there is conflicting evidence regarding its influence on environmentally stimulated arousal. This study shows that loss-of-function mutations in the D1 dopamine receptor DopR enhance repetitive startle-induced arousal while decreasing sleep-wake arousal (i.e., increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose effects in each case are opposite to, and abrogated by, the DopR mutation. Selective restoration of DopR function in the central complex rescues the enhanced stimulated arousal but not the increased sleep phenotype of DopR mutants. These data provide evidence for at least two different forms of arousal, which are independently regulated by dopamine in opposite directions, via distinct neural circuits (Lebestky, 2009).

'Arousal', a state characterized by increased activity, sensitivity to sensory stimuli, and certain patterns of brain activity, accompanies many different behaviors, including circadian rhythms, escape, aggression, courtship, and emotional responses in higher vertebrates. A key unanswered question is whether arousal is a unidimensional, generalized state. Biogenic amines, such as dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine, as well as cholinergic systems, have all been implicated in arousal in numerous behavioral settings. However, it is not clear whether these different neuromodulators act on a common 'generalized arousal' pathway or rather control distinct arousal pathways or circuits that independently regulate different behaviors. Resolving this issue requires identifying the receptors and circuits on which these neuromodulators act, in different behavioral settings of arousal (Lebestky, 2009).

Most studies of arousal in Drosophila have focused on locomotor activity reflecting sleep-wake transitions, a form of 'endogenously generated' arousal. Several lines of evidence point to a role for DA in enhancing this form of arousal in Drosophila. Drug-feeding experiments, as well as genetic silencing of dopaminergic neurons, have indicated that DA promotes waking during the subjective night phase of the circadian cycle. Similar conclusions were drawn from studying mutations in the Drosophila DA transporter (dDAT). Consistent with these data, overexpression of the vesicular monoamine transporter (dVMAT-A), promoted hyperactivity in this species, as did activation of DA neurons in quiescent flies (Lebestky, 2009).

Evidence regarding the nature of DA effects on 'exogenously generated' or environmentally stimulated arousal, such as that elicited by startle, is less consistent. Classical genetic studies and quantitative trait locus (QTL) analyses have suggested that differences in DA levels may underlie genetic variation in startle-induced locomotor activity. Fmn (dDAT; Dopamine transporter) mutants displayed hyperactivity in response to mechanical shocks, implying a positive-acting role for DA in controlling environmentally induced arousal. In contrast, other data imply a negative-acting role for DA in controlling stimulated arousal. Mutants in Tyr-1, which exhibit a reduction in dopamine levels, show an increase in stimulated but not spontaneous levels of locomotor activity. Genetic inhibition of tyrosine hydroxylase-expressing neurons caused hyperactivity in response to mechanical startle. Finally, transient activation of DA neurons in hyperactive flies inhibited locomotion. Whether these differing results reflect differences in behavioral assays, the involvement of different types of DA receptors, or an 'inverted U'-like dosage sensitivity to DA, is unclear (Lebestky, 2009).

This investigation has developed a novel behavioral paradigm for environmentally stimulated arousal, using repetitive mechanical startle as a stimulus, and a screen was carried out for mutations that potentiate this response. One such mutation is a hypomorphic allele of the D1 receptor ortholog, DopR. This same mutation caused decreased spontaneous activity during the night phase of the circadian cycle, due to increased rest bout duration. In both assays, cocaine influenced behavior in the opposite direction as the DopR mutation, and the effect of cocaine was abolished in DopR mutant flies, supporting the idea that DA inversely regulates these two forms of arousal. Genetic rescue experiments, using Gal4 drivers with restricted CNS expression, indicate that these independent and opposite influences of DopR are exerted in different neural circuits. These data suggest the existence of different types of arousal states mediated by distinct neural circuits in Drosophila, which can be oppositely regulated by DA acting via the same receptor subtype (Lebestky, 2009).

Previous studies of arousal in Drosophila have focused on sleep-wake transitions, a form of 'endogenous' arousal. This study has introduced and characterized a quantitative behavioral assay for repetitive startle-induced hyperactivity, which displays properties consistent with an environmentally triggered ('exogenous') arousal state. A screen was conducted for mutations affecting this behavior, the phenotype of one such mutation (DopR) was analyzed, and the neural substrates of its action was mapped by cell-specific genetic rescue experiments. The results reveal that DopR independently regulates Repetitive Startle-induced Hyperactivity (ReSH) and sleep in opposite directions by acting on distinct neural substrates. Negative regulation of the ReSH response requires DopR function in the ellipsoid body (EB) of the central complex (CC), while positive regulation of waking reflects a function in other populations of neurons, including PDF-expressing circadian pacemaker cells. Both of these functions, moreover, are independent of the function of DopR in learning and memory, which is required in the mushroom body. These data suggest that ReSH behavior and sleep-wake transitions reflect distinct forms of arousal that are genetically, anatomically, and behaviorally separable. This conclusion is consistent with earlier suggestions, based on classical genetic studies, that spontaneous and environmentally stimulated locomotor activity reflect 'distinct behavioral systems' in Drosophila (Lebestky, 2009).

Several lines of evidence suggest that ReSH behavior represents a form of environmentally stimulated arousal. First, hyperactivity is an evolutionarily conserved expression of increased arousal. Although not all arousal is necessarily expressed as hyperactivity, electrophysiological studies indicate that mechanical startle, the type of stimulus used in this study, evokes increases in 20-30 Hz and 80-90 Hz brain activity, which have been suggested to reflect a neural correlate of arousal in flies. Second, ReSH does not immediately dissipate following termination of the stimulus, as would be expected for a simple reflexive stimulus-response behavior, but rather persists for an extended period of time, suggesting that it reflects a change in internal state. Third, this state, like arousal, is scalable: more puffs, or more intense puffs, produce a stronger and/or longer-lasting state of hyperactivity. Fourth, this state exhibits sensitization: even after overt locomotor activity has recovered to prepuff levels, flies remain hypersensitive to a single puff for several minutes. Fifth, this sensitization state generalizes to a startle stimulus of at least one other sensory modality (olfactory). In Aplysia, sensitization of the gill/siphon withdrawal reflex has been likened to behavioral arousal. Taken together, these features strongly suggest that ReSH represents an example of environmentally stimulated ('exogenous') arousal in Drosophila (Lebestky, 2009).

DopR mutant flies exhibited longer rest periods during their subjective night phase, suggesting that DopR normally promotes sleep-wake transitions. These data are consistent with earlier studies indicating that DA promotes arousal by inhibiting sleep (Andretic, 2005, Kume, 2005; Wu, 2008). In contrast, prior evidence regarding the role of DA in startle-induced arousal is conflicting. Some studies have suggested that DA negatively regulates locomotor reactivity to environmental stimuli, consistent with the current observations, while others have suggested that it positively regulates this response. Even within the same study, light-stimulated activation of TH⁺ neurons produced opposite effects on locomotion, depending on the prestimulus level of locomotor activity (Lebestky, 2009 and references therein).

This study has found that DA and DopR negatively regulate environmentally stimulated arousal: the DopR mutation enhanced the ReSH response, while cocaine suppressed it. Furthermore, the effect of cocaine in the ReSH assay was eliminated in the DopR mutant but could be rescued by Gal4-driven DopR expression, confirming that the effect of the drug is mediated by DA. Taken together, these results reconcile apparently conflicting data on the role of DA in 'arousal' in Drosophila by identifying two different forms of arousal -- repetitive startle-induced arousal and sleep-wake arousal -- that are regulated by DA in an inverse manner (Lebestky, 2009).

The finding that DopR negatively regulates one form of environmentally stimulated arousal leaves open the question of whether this is true for all types of exogenous arousing stimuli. The 'sign' of the influence of DA on exogenously generated arousal states may vary depending on the type or strength of the stimulus used, the initial state of the system prior to exposure to the arousing stimulus, or the precise neural circuitry that is engaged. Future studies using arousing stimuli of different sensory modalities or associated with different behaviors should shed light on this question (Lebestky, 2009).

Several lines of evidence suggest that endogenous DopR likely acts in the ellipsoid body (EB) of the central complex (CC) to regulate repetitive startle-induced arousal. First, multiple Gal4 lines that drive expression in the EB rescued the ReSH phenotype of DopR mutants. Second, endogenous DopR is expressed in EB neurons, including those in which the rescuing Gal4 drivers are expressed. Third, the domain of DopR expression in the EB overlaps the varicosities of TH⁺ fibers. In an independent study of dopaminergic inputs required for regulating EtOH-stimulated hyperactivity TH⁺ neurons were identified that are a likely source of these projections to the EB. Fourth, rescue of the ReSH phenotype is associated with re-expression of DopR in EB neurons. Finally, rescue is observed using conditional DopR expression in adults. Taken together, these data argue that rescue of the ReSH phenotype by the Gal4 lines tested reflects their common expression in the EB and that this is a normal site of DopR action in adult flies (Lebestky, 2009).

A requirement for DopR in the EB in regulating ReSH behavior is consistent with the fact that the CC is involved in the control of walking activity. However, the mushroom body has also been implicated in the control of locomotor behavior, and DopR is strongly expressed in this structure as well. Rescue data argue against the MB and in favor of the CC as a neural substrate for the ReSH phenotype of DopR mutants. Unexpectedly, the nocturnal hypoactivity phenotype of DopR mutants was not rescued by restoration of DopR expression to the CC. Thus, not all locomotor activity phenotypes of the DopR mutant necessarily reflect a function for the gene in the CC (Lebestky, 2009).

Interestingly, Gal4 line c547 expresses in R2/R4m neurons of the EB, while lines 189y and c761 express in R3 neurons, yet both rescued the ReSH phenotype of DopR mutants. Similar results have been obtained in experiments to rescue the deficit in ethanol-induced behavior exhibited by the DopR mutant. Double-labeling experiments suggest that endogenous DopR is expressed in all of these EB neuronal subpopulations. Perhaps the receptor functions in parallel or in series in R4m and R3 neurons, so that restoration of DopR expression in either population can rescue the ReSH phenotype. Whether these DopR-expressing EB subpopulations are synaptically interconnected is an interesting question for future investigation (Lebestky, 2009).

Despite its power as a system for studying neural development, function, and behavior, Drosophila has not been extensively used in affective neuroscience, in part due to uncertainty about whether this insect exhibits emotion-like states or behaviors. Increased arousal is a key component of many emotional or affective behaviors. The data presented in this study indicate that Drosophila can express a persistent arousal state in response to repetitive stress. ReSH behavior exhibits several features that distinguish it from simple, reflexive stimulus-response behaviors: scalability, persistence following stimulus termination, and sensitization. In addition, the observation that mechanical trauma promotes release from Drosophila of an odorant that repels other flies suggests that the arousal state underlying ReSH behavior may have a negative 'affective valence' as well. These considerations, taken together with the fact that ReSH is influenced by genetic and pharmacologic manipulations of DA, a biogenic amine implicated in emotional behavior in humans, support the idea that the ReSH response may represent a primitive 'emotion-like' behavior in Drosophila (Lebestky, 2009).

The phenotype of DopR flies is reminiscent of attention-deficit hyperactivity disorder (ADHD), an affective disorder linked to dopamine, whose symptoms include hyper-reactivity to environmental stimuli. If humans, like flies, have distinct circuits for different forms of arousal, then the current data suggest that ADHD may specifically involve dopaminergic dysfunction in those circuits mediating environmentally stimulated, rather than endogenous (sleep-wake), arousal. Given that DA negatively regulates environmentally stimulated arousal circuits in Drosophila, such a view would be consistent with the fact that treatment with drugs that increase synaptic levels of DA, such as methylphenidate (ritalin), can ameliorate symptoms of ADHD (Lebestky, 2009).

In further support of this suggestion, in mammals, dopamine D1 receptors in the prefrontal cortex (PFC) have been proposed to negatively regulate activity, while D1 receptors in the nucleus accumbens are thought to promote sleep-wake transitions. Numerous studies have linked dopaminergic dysfunction in the PFC to ADHD. While most research has focused on the role of the PFC in attention and cognition, rather than in environmentally stimulated arousal per se, dysfunction of PFC circuits mediating phasic DA release has been invoked to explain behavioral hypersensitivity to environmental stimuli in ADHD This view of ADHD as a disorder of circuits mediating environmentally stimulated arousal suggests that further study of such circuits in humans and in vertebrate animal models, as well as in Drosophila, may improve understanding of this disorder and ultimately lead to improved therapeutics (Lebestky, 2009).

Caffeine promotes wakefulness via dopamine signaling in Drosophila

Caffeine is the most widely-consumed psychoactive drug in the world, but the understanding of how caffeine affects brains is relatively incomplete. Most studies focus on effects of caffeine on adenosine receptors, but there is evidence for other, more complex mechanisms. In the fruit fly Drosophila melanogaster, which shows a robust diurnal pattern of sleep/wake activity, caffeine reduces nighttime sleep behavior independently of the one known adenosine receptor. This study shows that dopamine is required for the wake-promoting effect of caffeine in the fly, and that caffeine likely acts presynaptically to increase dopamine signaling. A cluster of neurons, the paired anterior medial (PAM) cluster of dopaminergic neurons, were identified as the ones relevant for the caffeine response. PAM neurons show increased activity following caffeine administration, and promote wake when activated. Also, inhibition of these neurons abrogates sleep suppression by caffeine. While previous studies have focused on adenosine-receptor mediated mechanisms for caffeine action, this study identifies a role for dopaminergic neurons in the arousal-promoting effect of caffeine (Nall, 2016).

Identified serotonin-releasing neurons induce behavioral quiescence and suppress mating in Drosophila

Animals show different levels of activity that are reflected in sensory responsiveness and endogenously generated behaviors. Biogenic amineshave been determined to be causal factors for these states of arousal. It is well established that, in Drosophila, dopamine and octopamine promote increased arousal. However, little is known about factors that regulate arousal negatively and induce states of quiescence. Moreover, it remains unclear whether global, diffuse modulatory systems comprehensively affecting brain activity determine general states of arousal. Alternatively, individual aminergic neurons might selectively modulate the animals' activity in a distinct behavioral context. This study shows that artificially activating large populations of serotonin-releasing neurons induces behavioral quiescence and inhibits feeding and mating. The study systematically narrows down a role of serotonin in inhibiting endogenously generated locomotor activity to neurons located in the posterior medial protocerebrum. Neurons of this cell cluster were identified that suppress mating, but not feeding behavior. These results suggest that serotonin does not uniformly act as global, negative modulator of general arousal. Rather, distinct serotoninergic neurons can act as inhibitory modulators of specific behaviors (Pooryasin, 2015).

Pre- and postsynaptic role of Dopamine D2 Receptor DD2R in Drosophila olfactory associative learning

Dopaminergic neurons in Drosophila play critical roles in diverse brain functions such as motor control, arousal, learning, and memory. Using genetic and behavioral approaches, it has been firmly established that proper dopamine signaling is required for olfactory classical conditioning (e.g., aversive and appetitive learning). Dopamine mediates its functions through interaction with its receptors. There are two different types of dopamine receptors in Drosophila: 1) Dopamine 1-like, including Dopamine 1-like receptor 1 and Dopamine 1-like receptor 2 (DDR2, the subject of this report) and 2) Dopamine 2-like receptor. Currently, no study has attempted to characterize the role of DD2R in Drosophila learning and memory. Using a DD2R-RNAi transgenic line, this study has examined the role of DD2R, expressed in dopamine neurons (i.e., the presynaptic DD2R autoreceptor), in larval olfactory learning. The function of postsynaptic DD2R expressed in mushroom body (MB) was also studied as MB is the center for Drosophila learning, with a function analogous to that of the mammalian hippocampus. These results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning. The data confirm, for the first time, that DD2R plays an important role in Drosophila olfactory learning (Qi, 2014).

Dopamine (DA) is an important neurotransmitter mediating a variety of brain functions including locomotion, reward, awareness, learning and memory, and cognition. Genetic and pharmacological studies revealed that the dopaminergic system in the fruit fly Drosophila melanogaster plays multiple roles in motor function and associative learning. Using the sophisticated genetic tools available for the fruit fly, it has been firmly established that release of dopamine is required for associative learning in Drosophila adults and larvae. Dopaminergic neural circuits mediating olfactory learning have been also characterized in the fruit fly brain (Qi, 2014).

DA mediates its physiological functions through interaction with its receptors. Analysis of the primary structure of the DA receptors revealed that those receptors belong to the G-protein coupled receptor (GPCR) family. Generally, DA receptors can be divided into two families in vertebrates. The D1-like receptor family stimulates cAMP production by activation of the receptor-coupled Gs subunit of G proteins. The D2-like receptor family belongs to the pertussis toxin (PTX)-sensitive G protein (i.e., Gi and Go)-coupled receptor (GPCR) superfamily. Therefore, actions of D2-like receptor members have been characterized as inhibitory. In relation to regulation of DA signaling, DA neurons were found to possess receptors for their own transmitter, dopamine, at their synaptic nerve terminals. These DA autoreceptors (autoR) function as self-inhibitory regulators (Qi, 2014).

In Drosophila, there are four DA receptors (dDA1, DAMB, DopEcR, and DD2R) that have been cloned and characterized. Two DA receptors (dDA1, DAMB) were cloned first and appear to be members of the D1-like receptor family on the basis of their ability to stimulate adenylyl cyclase (AC) in a heterologous expression system. In contrast, only one Drosophila DA receptor DD2R gene has been identified. Functional expression of the DD2R gene in HEK293 cells indicated that DA caused a marked decrease in forskolin-induced cAMP level, indicating that DD2R belongs to the inhibitory D2-like receptor family. Interestingly, two recent studies confirmed the existence of DA autoreceptors in Drosophila (Qi, 2014).

Olfactory associative learning in adult flies requires expression of Drosophila D1 receptor dDA1 in the mushroom body, the anatomical center for learning and memory. The dDA1 mutant dumb showed impaired appetitive learning as well as aversive learning. These impaired learning behaviors were fully rescued by expression of the wild-type dDA1 transgene in MB neurons in mutant flies, further confirming the role of Drosophila D1-like receptors in learning. However, no previous study has attempted to characterize the role of D2-like DD2R in Drosophila learning and memory. Interestingly, there was one study showing that a D2 agonist eticlopride did not disrupt visual learning (e.g., T maze assay) in adult flies (Qi, 2014).

Drosophila larvae carrying DD2R-RNAi transgene were used to examine the role of D2-like receptors in associative learning. Two different types of tissue-specific drivers were used to examine both presynaptic D2 autoreceptors and postsynaptic D2 receptors. Dopaminergic-specific driver TH-Gal4 was used to induce DD2R-RNAi expression in DA neurons. Since the target of dopaminergic innervation is the mushroom body (MB), the center for learning and memory in Drosophila, MB-specific drivers (201Y-Gal4, 30Y-Gal4) were used to down-regulate postsynaptic DD2R combined with DD2R-RNAi transgene. The results showed that both presynaptic DD2R autoreceptors and postsynaptic receptors are required for aversive and appetitive olfactory learning in Drosophila larvae (Qi, 2014).

The Drosophila D2 receptor DD2R has been shown to play an important role in locomotion, aggression, and neuroprotection. Interestingly, no study has shown whether Drosophila DD2R is involved in learning and memory, although dopaminergic (DA) neural circuits and D1 receptors are known to mediate Drosophila aversive learning. The present study, for the first time, demonstrated that DD2R is involved in olfactory associative learning in Drosophila larvae. Further, we showed that both presynaptic and postsynaptic DD2Rs mediate aversive and appetitive learning in the fly larvae as down-regulation of DD2R in DA and mushroom body (MB) neurons resulted in impaired olfactory learning (Qi, 2014).

Multiple studies have proved that dopamine signaling is necessary in Drosophila aversive learning. However, it is uncertain whether dopamine signaling is involved in appetitive learning. Several laboratories reported that DA signaling is not necessary for appetitive learning, which is mediated by another biogenic amine, octopamine In contrast, DA signaling has been shown to be necessary for appetitive learning; inhibition of DA release resulted in reduced appetitive learning. Furthermore, D1 receptor mutants (e.g., dDA1) showed impaired appetitive learning (Qi, 2014).

This study has demonstrated that dopamine mediates not only aversive learning, but also appetitive learning. Both learning behaviors are impaired when DD2R-RNAi is expressed in DA neurons or in MB neurons. Interestingly, aversive learning was completely impaired, while appetitive learning was only partially impaired when DD2R-RNAi was expressed in DA neurons. A possible explanation is that the effect of DD2R-RNAi is partial as RNAi down-regulates the target gene expression. Another possibility is that DA is not the only modulatory neurotransmitter mediating appetitive learning; another biogenic amine, octopamine, is involved in appetitive learning. Therefore, octopamine can mediate appetitive learning to a certain extent even if DA signaling is impaired. In contrast, no modulatory neurotransmitter other than DA is known to be involved in aversive learning (Qi, 2014).

Reduced locomotion due to expression of DD2R-RNAi has been reported. The current findings do not support that result since the larvae carrying DD2R-RNAi showed no changes in sensory and motor function, compared to WT and control fly strains. This discrepancy can be explained through the following reasons. first, it may be related to developmental-specific effects. This study used third-instar larvae while adult flies were used by Draper. Second, there are differences in locomotion assays. Draper quantified total activity counts, amount of time active, and number of activity-rest bouts. In the current study, crawling speed was measured. Third, DD2R-RNAi expression patterns are different. Draper used Act5C-Gal or elav-Gal4 to express DD2R-RNAi ubiquitously or pan-neuronally, respectively. In contrast, DD2R-RNAi was only expressed in DA or MB neurons in the current study. Therefore, neural circuits affected by DD2R-RNAi can be different, resulting in different behaviors. It is also possible that expression level of DD2R-RNAi is different due to different drivers (Qi, 2014).

Since the identification of Drosophila D2 receptor DD2R, two studies have revealed the autoreceptor function of DD2R. D2R agonists have been reported to reduce DA release in the Drosophila larval central nervous system. It was also shown that DD2R autoreceptors suppress excitability of DA neurons in Drosophila primary neuronal cultures. This study showed that DD2R is involved in mediating both appetitive and aversive olfactory learning. DD2R-RNAi in DA neurons down-regulates DD2R autoreceptor function. Thus excitability of DA neurons is increased, leading to an increase of DA release. Our results indicate that excessive DA release impairs olfactory learning. Indeed, it has been shown that olfactory learning is impaired in Drosophila DA transporter mutant fumin, likely due to increased synaptic DA levels. In contrast, a lack of DA release is known to cause impaired learning in Drosophila larvae. Therefore, it appears that homeostatic regulation of DA release by DD2R is important for both appetitive and aversive olfactory learning as either too much or too little synaptic DA causes impaired learning. Taking these facts into consideration, a model is proposed to explain the role of presynaptic DD2R autoreceptors. Presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. If presynaptic DD2R function is suppressed, then more DA is released into MB neurons. Increased DA tone in the MB impairs both aversive and appetitive learning behaviors (Qi, 2014).

This study also showed that olfactory learning in Drosophila larvae is impaired by down-regulation of postsynaptic DD2R in MB neurons. As the role of DD2R is inhibitory, the effects of DD2R-RNAi in MB neurons are expected to increase neuronal excitability, and thus olfactory learning is impaired by hyperexcitability in MB neurons. This observation might not be consistent with the physiological findings that learning and memory are mediated by enhanced neuronal excitability and synaptic transmission. Such well-known examples are long-term facilitation (LTF) and long-term potentiation (LTP). In this study, DD2R-RNAi is expressed throughout the larval stage. Therefore, hyperexcitability is chronic and thus this increased baseline activity interferes with coding new information in the MB. Indeed, olfactory learning is impaired in Drosophila by the chronic increase of excitatory cholinergic synaptic transmission due to the phosphodiesterase gene dunce mutation, resulting in increased cAMP levels. Taken together, temporal increases in excitability are key physiological changes underlying associative learning and thus DD2R-RNAi interferes with this change by inducing chronic hyperexcitability in MB neurons (Qi, 2014).

In addition to DD2R, there are Drosophila D1-like receptors (dDA1 and DAMB) that are known to be highly expressed in MB neurons. In fact, dDA1 null mutants showed defects in olfactory learning. Since D1-like receptors increase neuronal excitability via the cAMP-PKA signaling pathway, dDA1 mutant MB neurons are less depolarized when DA is released at the synaptic terminal in the MB, and thus cannot mediate olfactory learning. Proper excitability of MB neurons should be maintained by balancing actions of D1- and D2-like receptors in MB neurons (Qi, 2014).

It has been proposed that the adenylyl cyclase gene rutabaga in MB is a coincidence detector for CS and US in Drosophila olfactory learning and memory. Therefore, on the basis of the current results, a model is proposed to explain postsynaptic mechanisms underlying aversive and appetitive learning. Postsynaptic DD2Rs in MBNs inhibit neuronal excitability while dDA1 stimulates neural circuits associating CS with US in MB. DA receptors dDA1 and DD2R regulate AC in MB neurons in the opposite direction to maintain homeostatic balance of MB neuronal excitability, which is an important physiological element for Drosophila larval olfactory learning (Qi, 2014).

In conclusion, this study examined the role of D2-like receptor DD2R in Drosophila olfactory associative learning. The results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning (Qi, 2014).

The data strongly support the hypothesis that presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. If presynaptic DD2R function is suppressed, then more DA is released. Increasing DA tone to MB neurons impairs both aversive and appetitive learning behaviors. Postsynaptically, DD2R-RNAi impaired olfactory associative learning most likely by inducing chronic hyperexcitability in MB neurons. Therefore, the role of postsynaptic DD2R is to maintain the proper excitability in MB neurons during learning. Taken together, this study, for the first time, demonstrated that DD2R plays an important role in Drosophila olfactory associative learning (Qi, 2014).

Suppression of dopamine neurons mediates reward

Massive activation of dopamine neurons is critical for natural reward and drug abuse. In contrast, the significance of their spontaneous activity remains elusive. In Drosophila melanogaster, depolarization of the protocerebral anterior medial (PAM) cluster dopamine neurons en masse signals reward to the mushroom body (MB) and drives appetitive memory. Focusing on the functional heterogeneity of PAM cluster neurons, this study identified that a single class of PAM neurons, PAM-γ3, mediates sugar reward by suppressing their own activity. PAM-γ3 is selectively required for appetitive olfactory learning, while activation of these neurons in turn induces aversive memory. Ongoing activity of PAM-γ3 gets suppressed upon sugar ingestion. Strikingly, transient inactivation of basal PAM-γ3 activity can substitute for reward and induces appetitive memory. Furthermore, the satiety-signaling neuropeptide Allatostatin A (AstA) is a key mediator that conveys inhibitory input onto PAM-γ3. These results suggest the significance of basal dopamine release in reward signaling and reveal a circuit mechanism for negative regulation (Yamagata, 2016)

Serotonergic modulation of aggression in Drosophila involves GABAergic and cholinergic opposing pathways

Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. This study has identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. These data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression (Alekseyenko, 2019).

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both 'classical' and 'modulatory' signals that could produce diverse and/or complementary effects in associated circuits. This study establishes that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identifed the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. These findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network (Sherer, 2020).

Addressing the functional complexities of 'one neuron, multiple transmitters' is critical to understanding how neuron communication, circuit computation, and behavior can be regulated by a single neuron. Over many decades, significant progress has been made elucidating the functional properties of neurons co-expressing neuropeptides and small molecule neurotransmitters, where the neuropeptide acts as a co-transmitter and modulates the action of the neurotransmitter. Only recently have studies begun to examine the functional significance of co-transmission by a fast-acting neurotransmitter and a slow-acting monoamine (Sherer, 2020).

This study has demonstrated that OA neurons express dVGLUT and has utilized a new genetic tool to remove dVGLUT in OA-glutamate neurons. Quantifying changes in the complex social behaviors of aggression and courtship revealed that dVGLUT in brain OGNs is required to promote aggressive behavior and a specific behavioral pattern, the lunge. In contrast, males deficient for dVGLUT function do not exhibit an increase in inter-male courtship. These results establish a previously undetermined role for dVGLUT in OA neurons located in the adult brain and reveal glutamate uncouples aggression from inter-male courtship. It has been suggested that classical neurotransmitters and monoamines present in the same neuron modulate each other's packaging into synaptic vesicles or after release via autoreceptors. For example, a reduction of dVGLUT in DA-glutamate neurons resulted in decreased AMPH-stimulated hyperlocomotion in Drosophila and mice suggesting a key function of dVGLUT is the mediation of vesicular DA content. In this study, the independent behavioral changes suggests enhancing the packaging of OA into vesicles is not the sole function of dVGLUT co-expression and suggests differences in signaling by OA from OGNs on courtship-related circuitry (Sherer, 2020).

Co-transmission can generate distinct circuit-level effects via multiple mechanisms. One mechanism includes spatial segregation; the release of two neurotransmitters or a neurotransmitter and monoamine from a single neuron occurring at different axon terminals or presynaptic zones. Recent studies examining this possible mechanism have described (1) the release of GLU and DA from different synaptic vesicles in midbrain dopamine neurons and (2) the presence of VMAT and VGLUT microdomains in a subset of rodent mesoaccumbens DA neurons. This study expressed a new conditionally expressed epitope-tagged version of VMAT in OGNs and visualized endogenous dVGLUT via antibody labeling. Within OGNs, the colocalization of VMAT and dVGLUT puncta was not complete suggesting the observed behavioral phenotype differences may be due to spatial differences in OA signaling (Sherer, 2020).

A second mechanism by which co-transmission may generate unique functional properties relies on activating distinct postsynaptic receptors. In Drosophila, recent work has identified a small population of male-specific neurons that express the alpha-like adrenergic receptor, OAMB, as aggression-promoting circuit-level neuronal targets of OA modulation independent of any effect on arousal and separately knockdown of the Rdl GABAa receptor in a specific doublesex+ population stimulated male aggression (Watanabe, 2017). Future experiments identifying downstream targets that express both glutamate and octopamine receptors would be informative, as well as using additional split-Gal4 lines to determine if segregation of transporters is a hallmark of the majority of OGNs. Finally, a third possible mechanism is Glu may be co-released from OGNs and act on autoreceptors to regulate presynaptic OA release (Sherer, 2020).

Deciphering the signaling complexity that allows neural networks to integrate external stimuli with internal states to generate context-appropriate social behavior is a challenging endeavor. Neuromodulators including monoamines are released to signal changes in an animal's environment and positively or negatively reinforce network output. In invertebrates, a role for OA in responding to external chemosensory cues as well as promoting aggression has been well-established. In terms of identifying specific aggression circuit-components that utilize OA, previous results determined OA neurons directly receive male-specific pheromone information and the aSP2 neurons serve as a hub through which OA can bias output from a multi-functional social behavior network towards aggression. The ability of OA to bias behavioral decisions based on positive and negative reinforcement was also recently described for food odors. In vertebrates, it has been proposed that DA-GLU cotransmission in the NAc medial shell might facilitate behavioral switching. The finding that the majority of OA neurons are glutamatergic, suggests that the complex social behavior of aggression may rely on small subsets of neurons that both signal the rapid temporal coding of critical external stimuli as well as the frequency coding of such stimuli resulting in the enhancement of this behavioral network. One implication of the finding regarding the separable OA-dependent inhibition of inter-male courtship is the possibility of identifying specific synapses or axon terminals that when activated gate two different behavioral outcomes. A second implication is that aggressive behavior in other systems may be modified by targeting GLU function in monoamine neurons (Sherer, 2020).

Finally, monoamine-expressing neurons play key roles in human behavior including aggression and illnesses that have an aggressive component such as depression, addiction, anxiety, and Alzheimer's. While progress is being made in addressing the functional complexities of dual transmission, the possible pathological implications of glutamate co-release by monoamine neurons remains virtually unknown. Analyzing the synaptic vesicle and release properties of monoamine-glutamate neurons could offer new possibilities for therapeutic interventions aimed at controlling out-of-context aggression (Sherer, 2020).

Sexual dysfunction precedes motor defects, dopaminergic neuronal degeneration, and impaired dopamine metabolism: Insights from Drosophila model of Parkinson's disease

Sexual dysfunction (SD) is one of the most common non-motor symptoms of Parkinson's disease (PD) and remains the most neglected, under-reported, and under-recognized aspect of PD. Studies have shown that Dopamine (DA) in the hypothalamus plays a role in regulating sexual behavior. But the detailed mechanism of SD in PD is not known. Drosophila melanogaster shares several genes and signaling pathways with humans which makes it an ideal model for the study of a neurodegenerative disorder such as PD. Courtship behavior of Drosophila is one such behavior that is closely related to human sexual behavior and so plays an important role in understanding sexual behavior in diseased conditions as well. In the present study, a sporadic SD model of PD using Drosophila was developed and SD phenotype was observed based on abnormalities in courtship behavior markers. The Drosophila SD model was developed in such a way that at the window of neurotoxin paraquat (PQ) treatment [PQ is considered a crucial risk factor for PD due to its structural similarity with 1-methyl-4-phenyl pyridinium (MPP+), the active form of PD-inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)], it does not exhibit mobility defects but shows SD. The whole brain tyrosine hydroxylase immunostaining showed no observable dopaminergic (DAergic) degeneration (number of DA neurons and fluorescence intensity of fluorescently labeled secondary antibodies that target anti-TH primary antibody) of the SD model. Similarly, there was no significant depletion of brain DA and its metabolite levels (HVA and DOPAC) as determined using HPLC-ECD (High-Performance Liquid Chromatography using Electrochemical Detector). The present study illustrates that the traits associated with courtship and sexual activity provide sensitive markers at the earlier stage of PD onset. This PQ-induced SD fly model throws an opportunity to decipher the molecular basis of SD under PD conditions and to screen nutraceuticals/potential therapeutic molecules to rescue SD phenotype and further to DAergic neuroprotection (Koza, 2023).

A circuit node that integrates convergent input from neuromodulatory and social behavior-promoting neurons to control aggression in Drosophila

Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. A systematic, unbiased screen identified OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. The results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM(+) neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals (Watanabe, 2017).

A rich behavioral literature has implicated OA in the control of invertebrate aggression, although the direction of its effects differs between species. Classic studies in lobsters have shown that injection of OA into the hemolymph promotes a subordinate-like posture, while injection of serotonin (5HT) produces a dominant-like posture. In contrast, hemolymph injections of OA in crickets restore aggressiveness to subordinated animals, mimicking the arousing effects of episodes of free flight. OA has also been suggested to play a role in aggressive motivation restored to defeated crickets by residency in a shelter. In Drosophila, null mutations of TβH strongly suppressed aggressiveness, suggesting a positive-acting role for OA in flies as in crickets. Interestingly, intra-hypothalamic infusion of NE in mammals can also enhance aggression. However, little is known about the neurons on which these amines act directly to influence aggression, in any organism (Watanabe, 2017).

This study applied a novel, unbiased approach to identify OARNs relevant to aggression in Drosophila. Importantly this screen was based not on mutations in OAR genes, but rather upon genetic silencing of neurons that express GAL4 under the control of different OAR gene cis-regulatory modules (CRMs). This screen was agnostic with respect to which OAR gene is involved, or in which neurons that OAR is expressed. It yielded a small population of male-specific, FruM+ OA-sensitive neurons, called aSP2, the activity of which is required for normal levels of aggressiveness. No significant change in UWEs (male-male courtship) was observed when these neurons were activated or silenced. Nevertheless, neuronal silencing in the parental R47A04-GAL4 line increased male-male courtship, perhaps reflecting an inhibitory role for non-aSP2 neurons in that line. Therefore, while it is not possible to completely exclude a role for aSP2 neurons to suppress male-male courtship, the evidence does not strongly support it (Watanabe, 2017).

Multiple lines of evidence suggest that R47A04^aSP2 neurons are indeed OA responsive, likely via OAMB. First, these neurons are labeled by a CRM from the Oamb gene. Second, RNAi-mediated knockdown of Oamb in R47A04 neurons reduced aggression, phenocopying the effects of an Oamb null allele. (However, knockdown using the split-GAL4 R47A04^aSP2driver only yielded a trend to reduced aggression that did not reach significance, perhaps reflecting a floor effect in this assay.) Third, overexpression of Oamb cDNAs in these neurons using R47A04-GAL4 rescued the Oamb null mutant and enhanced the effect of OA feeding to promote aggression. Fourth,R47A04^aSP2 neurons were activated by bath-applied OA in brain explants, and this effect was also blocked by RNAi-mediated knockdown of Oamb. Taken together, these data strongly suggest that aSP2 neurons respond directly to OA to mediate its effects on aggression, although they do not exclude a role for other OA-responsive non-aSP2 neurons in line R47A04. While it was not possible to definitively establish which of the 27 different classes of OANs in Drosophila provide functional OA input to aSP2 cells, some candidate OA neurons labeled in retrograde PA-GFP experiments (VUM and VPM) have previously been implicated in aggression (Watanabe, 2017).

In Drosophila OA, like NE in vertebrates, is thought to promote arousal. Consistent with such a function, OAergic fibers are broadly distributed across the entire Drosophila CNS, as are NE fibers in vertebrates. Thus OARNs could enhance aggression by increasing arousal, and there is evidence for such a function in crickets. However, manipulations of R47A04^aSP2neurons that increased or decreased aggression did not affect locomotion, circadian activity, or sleep. This suggests that these neurons influence aggression directly and specifically, rather than by increasing generalized arousal. Other classes of OARNs not investigated in this study have been implicated in sleep-wake arousal (Watanabe, 2017).

Does OA promote aggression in a permissive or instructive manner? While it is clear that OA synthesis and release are required for aggression in Drosophila, whether increasing OA suffices to promote aggression is less clear. It was reported that NaCh Bac-mediated activation of Tdc2-GAL4 neurons enhanced aggression, but the current study neither this manipulation, nor activation of Tdc2 neurons using dTrpA1 or Chrimson, yielded consistent effects. Thus, while OA is essential for normal levels of aggression, it is not clear whether it plays an instructive role to promote this behavior (Watanabe, 2017).

In principle, OA RNs could act directly in command-like neurons that mediate aggression, or rather in cells that play a modulatory role. It was found that aggression was increased by tonically enhancing the excitability of R47A04^aSP2 neurons using NaChBac, but not by phasically activating them optogenetically, arguing against a command-like function. Furthermore, the influence of TK FruM neurons, which do promote aggression when phasically activated, was not dependent on the activity of R47A04^aSP2 neurons, indicating that the latter are not functionally downstream of the former. Together, these data argue against a role for R47A04^aSP2 cells as command-like neurons, or as direct outputs of command neurons, for aggression. Rather, these cells exert a modulatory influence on agonistic behavior (Watanabe, 2017).

In searching for neurons that may interact with R47A04^aSP2 cells in their modulatory capacity, P1 neurons, a FruM+ population of 20 neurons/hemibrain was identified that controls male courtship, but which can also promote aggression when activated. It has been argued that this aggression-promoting effect is due to a subset of FruM neurons in the GAL4 line used in these studies, R71G01-GAL4. However, this study shows that conditional expression of FLP-ON Chrimson in a subset of neurons within the R71G01-GAL4 population using Fru-FLP. Nevertheless, these data do not exclude that the aggression-promoting neurons in the P1 cluster expressed Fru-FLP only transiently during development, nor do they exclude the possibility that different subpopulations of neurons within line R71G01 control courtship versus aggression; further studies will be required to resolve these issues (Watanabe, 2017).

The P1 cluster is known to project to downstream cells that are specific for courtship . The present study provides the first evidence that cells in this cluster also functionally activate (and physically contact) aggression-specific neurons. However, due to limitations of the genetic reagents employed, it is not certain that the behavioral, physiological, and anatomical interactions with aSP2 cells demonstrated in this study are all mediated by the same subset of neurons in the P1 cluster. With this caveat in mind, these data suggest that aSP2 neurons are functionally downstream of both a subset(s) of P1 neurons, as well as of OA neurons (Watanabe, 2017).

Feeding flies OA potentiated the activation of R47A04^aSP2 neurons by P1 neuron stimulation, in brain explants. Furthermore, activation of aggression by P1 stimulation was enhanced and suppressed by pharmacologically increasing or decreasing OA signaling, respectively. While some off-target effects of the drugs, or an action on non-aSP2 neurons expressing OARs, cannot be excluded these pharmacologic effects were overridden by opposite-direction genetic manipulations of R47A04^aSP2neuronal activity. Whether P1 neurons and OANs normally activate aSP2 neurons in vivo, simultaneously or sequentially, is not yet clear. Nevertheless it is striking that P1 and Tdc2 putative inputs occupy adjacent regions of aSP2 dendrites. Taken together, these findings suggest that aSP2 cells may serve as a node through which OA can bias output from a multifunctional social behavior network involving P1 neurons, in a manner that favors aggression. However, aSP2 neurons themselves do not appearto control directly the choice between mating and fighting (Watanabe, 2017).

Male-specific cuticular hydrocarbons such as 7-tricosene (7-T) are known to be required for aggression. Interestingly, it has recently been shown that gustatory neurons expressing Gr32a, which encodes a putative 7-T receptor, innervate OANs in the SEZ; these OANs are activated by 7-T in a Gr32a-dependent manner. SEZ-innervating OANs include the VPM/VUM subsets seen in PA-GFP retrograde labeling experiments. These data raise the possibility that R47A04^aSP2 neurons might be targets of VPM/VUM OANs activated by 7-T. If so, then they could provide a potential link between the influence of male-specific pheromones, OA, and central aggression circuitry. Studies of NE neurons in vertebrates have led to a prevailing view that this neuromodulator is released in a diffuse, 'sprinkler system'-like manner to control brain-wide states like arousal. Recent studies in Drosophila indicate that the broad, brain-wide distribution of OAergic fibers reflects the superposition of close to 30 anatomically distinct subclasses of OANs). The data presented in this study reveal a high level of circuit specificity for OARNs that mediate the effects of OA on aggression, mirroring the anatomical and functional specificity of OANs reported to control this behavior. If this anatomical logic is conserved, then such circuit specificity may underlie the actions of NE in mammals to a greater extent than is generally assumed (Watanabe, 2017).

Zygotically transcribed genes

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