InteractiveFly: GeneBrief

muscarinic Acetylcholine Receptor, A-type & muscarinic Acetylcholine Receptor, B-type: Biological Overview | References

BIOLOGICAL OVERVIEW

Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. This study shows that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. These results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons (Bielopolski, 2019).

Animals learn to modify their behavior based on past experience by changing connection strengths between neurons, and this synaptic plasticity is often regulated by metabotropic receptors. In particular, neurons commonly express both ionotropic and metabotropic receptors for the same neurotransmitter, where the two may mediate different functions (e.g., direct excitation/inhibition vs. synaptic plasticity). In mammals, where glutamate is the principal excitatory neurotransmitter, metabotropic glutamate receptors (mGluRs) have been widely implicated in synaptic plasticity and memory. Given the complexity of linking behavior to artificially induced plasticity in brain slices, it would be useful to study the role of metabotropic receptors in learning in a simpler genetic model system with a clearer behavioral readout of synaptic plasticity. One such system is Drosophila, where powerful genetic tools and well-defined anatomy have yielded a detailed understanding of the circuit and molecular mechanisms underlying associative memory. The principal excitatory neurotransmitter in Drosophila is acetylcholine, but, surprisingly, little is known about the function of metabotropic acetylcholine signaling in synaptic plasticity or neuromodulation in Drosophila. This study addresses this question using olfactory associative memory (Bielopolski, 2019).

Flies can learn to associate an odor (conditioned stimulus, CS) with a positive (sugar) or a negative (electric shock) unconditioned stimulus (US), so that they later approach 'rewarded' odors and avoid 'punished' odors. This association is thought to be formed in the presynaptic terminals of the ~2000 Kenyon cells (KCs) that make up the mushroom body (MB), the fly's olfactory memory center. These KCs are activated by odors via second-order olfactory neurons called projection neurons (PNs). Each odor elicits responses in a sparse subset of KCs so that odor identity is encoded in which KCs respond to each odor. When an odor (CS) is paired with reward/punishment (US), an odor-specific set of KCs is activated at the same time that dopaminergic neurons (DANs) release dopamine onto KC presynaptic terminals. The coincident activation causes long-term depression (LTD) of synapses from the odor-activated KCs onto mushroom body output neurons (MBONs) that lead to approach or avoidance behavior. In particular, training specifically depresses KC-MBON synapses of the 'wrong' valence (e.g. odor-punishment pairing depresses odor responses of MBONs that lead to approach behavior), because different pairs of 'matching' DANs/MBONs (e.g. punishment/approach, reward/avoidance) innervate distinct regions along KC axons (Bielopolski, 2019).

Both MB input (PNs) and output (KCs) are cholinergic, and KCs express both ionotropic (nicotinic) and metabotropic (muscarinic) acetylcholine receptors. The nicotinic receptors mediate fast excitatory synaptic currents, while the physiological function of the muscarinic receptors is unknown. Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors; out of the three mAChRs in Drosophila (mAChR-A, mAChR-B and mAChR-C), mAChR-A (also called Dm1, mAcR-60C or mAChR) is the most closely homologous to mammalian mAChRs. Mammalian mAChRs are typically divided between 'M1-type' (M1/M3/M5), which signal via Gq and are generally excitatory, and 'M2-type' (M2/M4), which signal via Gi/o and are generally inhibitory. Drosophila mAChR-A seems to use 'M1-type' signaling: when heterologously expressed in Chinese hamster ovary (CHO) cells, it signals via Gq protein (Collin, 2013; Ren, 2015) to activate phospholipase C, which produces inositol trisphosphate to release Ca2+ from internal stores (Bielopolski, 2019).

Recent work indicates that mAChR-A is required for aversive olfactory learning in Drosophila larvae, as knocking down mAChR-A expression in KCs impairs learning (Silva, 2015). However, it is unclear whether mAChR-A is involved in olfactory learning in adult Drosophila, given that mAChR-A is thought to signal through Gq, and in adult flies Gq signaling downstream of the dopamine receptor Damb promotes forgetting, not learning. Moreover, it is unknown how mAChR-A affects the activity or physiology of KCs, where it acts (at KC axons or dendrites or both), and how these effects contribute to olfactory learning (Bielopolski, 2019).

This study shows that mAChR-A is required in KCs for aversive olfactory learning in adult Drosophila. Surprisingly, genetic and pharmacological manipulations of mAChR-A suggest that mAChR-A is inhibitory and acts on KC dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in an MB output neuron, MB-MVP2, that is required for aversive memory retrieval. It is suggested that dendritically acting mAChR-A is required for synaptic depression between KCs and their outputs (Bielopolski, 2019).

This study shows that mAChR-A is required in γ KCs for aversive olfactory learning and short-term memory in adult Drosophila. Knocking down mAChR-A increases KC odor responses, while the mAChR-A agonist muscarine suppresses KC activity. Knocking down mAChR-A prevents aversive learning from reducing responses of the MB output neuron MB-MVP2 to the conditioned odor, suggesting that mAChR-A is required for the learning-related depression of KC-->MBON synapses (Bielopolski, 2019).

Why is mAChR-A only required for aversive learning in γ KCs, not αβ or α'β' KCs? Although the mAChR-A MiMIC gene trap agrees with single-cell transcriptome analysis that α'β' KCs express less mAChR-A than do γ and αβ KCs, transcriptome analysis indicates that α'β' KCs do express some mAChR-A. Moreover, γ and αβ KCs express similar levels of mAChR-A. It may be that the RNAi knockdown is less efficient at affecting the physiology of αβ and α'β' KCs than γ KCs, whether because the knockdown is less efficient at reducing protein levels, or because αβ and α'β' KCs have different intrinsic properties or a different function of mAChR-A such that 40% of normal mAChR-A levels is sufficient in αβ and α'β' KCs but not γ KCs. This interpretation is supported by the finding that mAChR-A RNAi knockdown significantly increases odor responses only in the γ lobe, not the αβ or α'β' lobes. Alternatively, γ, αβ and α'β' KCs are thought to be important mainly for short-term memory, long-term memory, and memory consolidation, respectively; as this study tested only short-term memory, mAChR-A may carry out the same function in all KCs, but only its role in γ KCs is required for short-term (as opposed to long-term) memory. Indeed, the key plasticity gene DopR1 is required in γ, not αβ or α'β'4 KCs, for short-term memory. It may be that mAChR-A is required in non-γ KC types for other forms of memory besides short-term aversive memory, such as appetitive conditioning or other phases of memory like long-term memory. The finding that mAChR-A is required in γ KCs for aversive short-term memory is consistent with the finding that mAChR-A knockdown in KCs disrupts training-induced depression of odor responses in MB-MVP2, an MBON postsynaptic to γ KCs required for aversive short-term memory. However, the latter finding does not rule out the possibility that other MBONs postsynaptic to non-γ KCs may also be affected by mAChR-A knockdown in KCs (Bielopolski, 2019).

mAChR-A seems to inhibit KC odor responses, because knocking down mAChR-A increases odor responses in the calyx and γ lobe, while activating mAChR-A with bath or local application of muscarine decreases KC odor responses. Some details differ between the genetic and pharmacological results. In particular, while mAChR-A knockdown mainly affects γ KCs, with other subtypes inconsistently affected, muscarine reduces responses in all KC subtypes. What explains these differences? mAChR-A might be weakly activated in physiological conditions, in which case gain of function would cause a stronger effect than loss of function. Similarly, pharmacological activation of mAChR-A is likely a more drastic manipulation than a 60% reduction of mAChR-A mRNA levels. Although network effects from muscarine application cannot be entirely ruled out, the effect of muscarine does not stem from PNs or APLand locally applied muscarine would have little effect on neurons outside the mushroom body (Bielopolski, 2019).

How does mAChR-A inhibit odor-evoked Ca2+ influx in KCs? Given that mAChR-A signals through Gq when expressed in CHO cells (Ren, 2015), that muscarinic Gq signaling normally increases excitability in mammal, and that pan-neuronal artificial activation of Gq signaling in Drosophila larvae increases overall excitability, it may be surprising that mAChR-A inhibits KCs. However, Gq signaling may exert different effects on different neurons in the fly brain, and some examples exist of inhibitory Gq signaling by mammalian mAChRs. M1/M3/M5 receptors acting via Gq can inhibit voltage-dependent Ca2+ channels, reduce voltage-gated Na +currents, or trigger surface transport of KCNQ channels, thus increasing inhibitory K+ currents. Drosophila mAChR-A may inhibit KCs through similar mechanisms (Bielopolski, 2019).

What is the source of ACh which activates mAChR-A and modulates odor responses? In the calyx, cholinergic PNs are certainly a major source of ACh. However, KCs themselves are cholinergic and release neurotransmitter in both the calyx and lobes. KCs form synapses on each other in the calyx, possibly allowing mAChR-A to mediate lateral inhibition, in conjunction with the lateral inhibition provided by the GABAergic APL neuron (Bielopolski, 2019).

What function does mAChR-A serve in learning and memory? The results indicate that mAChR-A knockdown prevents the learning-associated weakening of KC-MBON synapses, in particular for MBON-γ1pedc>α/β, aka MB-MVP2. One potential explanation is that the increased odor-evoked Ca2+ influx observed in knockdown flies increases synaptic release, which overrides the learning-associated synaptic depression. However, increased odor-evoked Ca2+ influx per se is unlikely on its own to straightforwardly explain a learning defect, because other genetic manipulations that increase odor-evoked Ca2+ influx in KCs either have no effect on, or even improve, olfactory learning. For example, knocking down GABA synthesis in the inhibitory APL neuron increases odor-evoked Ca2+ influx in KCs and improves olfactory learning (Bielopolski, 2019).

The most intuitive explanation would be that mAChR-A acts at KC synaptic terminals in KC axons to help depress KC-MBON synapses. Yet overexpressed mAChR-A localizes to KC dendrites, not axons, and functionally rescues mAChR-A hypomorphic mutants, showing that dendritic mAChR-A suffices for its function in learning and memory. Does this show that mAChR-A has no role in KC axons? The inability to detect GFP expressed from the mAChR-A MiMIC gene trap suggests that normally there may only be a small amount of mAChR-A in KCs. It may be that with mAChR-A-FLAG overexpression, the correct (undetectable) amount of mAChR-A is trafficked to and functions in axons, but due to a bottleneck in axonal transport, the excess tagged mAChR-A is trapped in KC dendrites. While the results do not rule out this possibility, a general bottleneck in axonal transport seems unlikely as many overexpressed proteins are localized to KC axons. It is more parsimonious to take the dendritic localization of mAChR-A-FLAG at face value and infer that mAChR-A functions in KC dendrites (Bielopolski, 2019).

How can mAChR-A in KC dendrites affect synaptic plasticity in KC axons? mAChR-A signaling might change the shape or duration of KC action potentials, an effect that could potentially propagate to KC axon terminals. Such changes in the action potential waveform may not be detected by calcium imaging, but could potentially affect a 'coincidence detector' in KC axons that detects when odor (i.e. KC activity) coincides with reward/punishment (i.e., dopamine). This coincidence detector is generally believed to be the Ca2+-dependent adenylyl cyclase Rutabaga. Changing the waveform of KC action potentials could potentially affect local dynamics of Ca2+ influx near Rutabaga molecules. In addition, rutabaga mutations do not abolish learning (mutants have ~40-50% of normal learning scores), so there may be additional coincidence detection mechanisms affected by action potential waveforms. Testing this idea would require a better understanding of biochemical events underlying learning at KC synaptic terminals (Bielopolski, 2019).

Alternatively, mAChR-A's effects on synaptic plasticity may not occur acutely. Although purely developmental effects of mAChR-A were ruled out through adult-only RNAi expression, knocking out mAChR-A for several days in adulthood might still affect KC physiology in a not-entirely-acute way. Perhaps, as with other G-protein-coupled receptors, muscarinic receptors can affect gene expression -- if so, this could have wide-ranging effects on KC physiology: for example, action potential waveform, expression of key genes required for synaptic plasticity, etc. Another intriguing possibility is suggested by an apparent paradox: both mAChR-A and the dopamine receptor Damb signal through Gq, but mAChR-A promotes learning while Damb promotes forgetting. How can Gq mediate apparently opposite effects? Perhaps Gq signaling aids both learning and forgetting by generally rendering synapses more labile. Indeed, although damb mutants retain memories for longer than wildtype, their initial learning is slightly impaired; damb mutant larvae are also impaired in aversive olfactory learning. Although one study reports that knocking down Gq in KCs did not impair initial memory, the Gq knockdown may not have been strong enough; also, that study shocked flies with 90 V shocks, which also gives normal learning in mAChR-A knockdown flies (Bielopolski, 2019).

Such hypotheses posit that mAChR-A regulates synaptic plasticity 'competence' rather than participating directly in the plasticity mechanism itself. Why should synaptic plasticity competence be controlled by an activity-dependent mechanism? It is tempting to speculate that mAChR-A may allow a kind of metaplasticity in which exposure to odors (hence activation of mAChR-A in KCs) makes flies' learning mechanisms more sensitive. Indeed, mAChR-A is required for learning with moderate (50 V) shocks, not severe (90 V) shocks. Future studies may further clarify how muscarinic signaling contributes to olfactory learning (Bielopolski, 2019).

Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit

ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in the larval visual system, focus was placed on investigating the physiological properties and behavioral relevance of larval visual interneurons. The ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Galphao signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, these studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system (Qin, 2019).

ON and OFF selectivity, the differential neuronal responses elicited by signal increments or decrements, is an essential component of visual computation and a fundamental property of visual systems across species. Extensive studies of adult Drosophila optic ganglia and vertebrate retinae suggest that the construction principles of ON and OFF selective pathways are shared among visual systems, albeit with circuit-specific implementations. Anatomically, dedicated neuronal pathways for ON vs. OFF responses are key features in visual circuit construction. Specific synaptic contacts are precisely built and maintained in laminar and columnar structures during development to ensure proper segregation of signals for parallel processing. Molecularly, light stimuli elicit opposite responses in ON and OFF pathways through signaling events mediated by differentially expressed neurotransmitter receptors in target neurons postsynaptic to the photoreceptor cells (PRs). This has been clearly demonstrated in the mammalian retina, where light-induced changes in glutamatergic transmission activate ON-bipolar cells via metabotropic glutamate receptor 6 (mGluR6) signaling and inhibit OFF-bipolar cells through the actions of ionotropic AMPA or kainate receptors. In the adult Drosophila visual system, functional imaging indicates that ON vs. OFF selectivity emerges from visual interneurons in the medulla. However, despite recent efforts in transcriptome profiling and genetic analyses, the molecular machinery mediating signal transformation within the ON and OFF pathways has not yet been clearly identified (Qin, 2019).

Unlike the ~6000 PRs in the adult visual system, larval Drosophila eyes consist of only 12 PRs on each side. Larval PRs make synaptic connections with a pair of visual local interneurons (VLNs) and approximately ten visual projection neurons (VPNs) in the larval optic neuropil (LON). VPNs relay signals to higher brain regions that process multiple sensory modalities. Despite this simple anatomy, larvae rely on vision for negative phototaxis, social clustering, and form associative memories based on visual cues. How the larval visual circuit effectively processes information and supports visually guided behaviors is not understood (Qin, 2019).

Recent connectome studies mapped synaptic interactions within the LON in the first instar larval brain, revealing two separate visual pathways using either blue-tuned Rhodopsin 5 (Rh5-PRs) or green-tuned Rhodopsin 6 (Rh6-PRs). Rh5-PRs project to the proximal layer of the LON (LONp) and form direct synaptic connections with all VPNs, whereas Rh6-PRs project to the distal layer of the LON (LONd) and predominantly target one cholinergic (cha-lOLP) and one glutamatergic (glu-lOLP) local interneurons. The two PR pathways then converge at the level of VPNs (Qin, 2019).

These connectome studies also revealed potential functions for cha- and glu-lOLP. The pair of lOLPs, together with one of the VPNs, the pOLP, are the earliest differentiated neurons in the larval optic lobe and are thus collectively known as optic lobe pioneer neurons (OLPs). Besides relaying visual information from Rh6-PRs to downstream VPNs, the lOLPs also form synaptic connections with each other and receive neuromodulatory inputs from serotonergic and octopaminergic neurons, suggesting that they may act as ON and OFF detectors. This proposal is further supported by recent studies on the role of the Rh6-PR/lOLP pathway in larval movement detection and social clustering behaviors. However, it remains unclear how the lOLPs support differential coding for ON and OFF signals without anatomical separation at either the input or output level (Qin, 2019).

This study investigated the lOLPs' physiological properties and determined the molecular machinery underlying their information processing abilities. Functional imaging studies revealed differential physiological responses towards light increments and decrements in cha-lOLP and glu-lOLP, indicating their functions in detecting ON and OFF signals. Furthermore, it was found that light-induced inhibition on glu-lOLP is mediated by mAchR-B/Gαo signaling, which generates the sign inversion required for the OFF response and encodes temporal information between the cholinergic and glutamatergic transmissions received by downstream VPNs. Lastly, genetic manipulations of glu-lOLP strongly modified the physiological responses of VPNs and eliminated dark-induced pausing behaviors. Together, these studies identify specific cellular and molecular pathways that mediate OFF detection in Drosophila larvae, reveal functional interactions among key components of the larval visual system, and establish a circuit mechanism for ON vs. OFF discrimination in this simple circuit (Qin, 2019).

The Drosophila larval visual circuit, with its small number of components and complete wiring diagram, provides a powerful model to study how specific synaptic interactions support visual computation. Built on knowledge obtained from connectome and behavioral analyses, the current physiological and genetic studies revealed unique computational strategies utilized by this simple circuit for processing complex outputs. Specifically, the results indicate that ON vs. OFF discrimination emerges at the level of the lOLPs, a pair of second-order visual interneurons. In addition, the essential role is demonstrated of glu-lOLP, a single glutamatergic interneuron, in meditating OFF detection at both the cellular and behavior levels and identify mAchR-B/Gαo signaling as the molecular machinery regulating its physiological properties (Qin, 2019).

Functional imaging studies using genetically encoded calcium and voltage indicators provide valuable information regarding the physiological properties of synaptic interactions among larval visual interneurons and projection neurons. However, optical recording approaches have certain technical limitations, including the kinetics and sensitivities of the voltage and calcium sensors, as well as the imaging and visual stimulation protocols. In addition, although glu-lOLP displays a biphasic response towards the light stimulation, calcium reductions and increases for only the initial set of physiological characterizations were quantified. Compared to the delayed calcium rise, the light-induced calcium reductions have low amplitudes and high variabilities, possibly due to the half-wave rectification of the intracellular calcium previously described in adult visual interneurons. For the genetic experiments, focus was placed on evaluating the activation of glu-lOLP, which is reflected by the increase of intracellular calcium signals that lead to neurotransmitter release (Qin, 2019).

To process light and dark information in parallel, both mammalian and adult fly visual systems utilize anatomical segregation to reinforce split ON and OFF pathways. In the larval visual circuit, however, almost all VPNs receive direct inputs from both cha-lOLP and glu-lOLP as well as the Rh5-PRs. Therefore, the response signs of the VPNs cannot be predicted by their anatomical connectivity to ON and OFF detectors. Based on the cumulative evidence obtained through genetic, anatomical, and physiological studies, it is proposed that temporal control of inhibition potentially contributes to ON vs. OFF discrimination in larvae. While cha-lOLP displays clear ON selectivity, the OFF selectivity in glu-lOLP is strengthened by the extended suppression of its light response by mAchR-B/Gαo signaling. This temporal control may also produce a window of heightened responsiveness in cha-lOLP and ON-VPNs towards light signals, similar to the case in mammalian sensory systems where the temporal delay of input-evoked inhibition relative to excitation sharpens the tuning to preferred stimuli. Together, the temporal separation between cholinergic and glutamatergic transmission could reinforce the functional segregation in the VPNs and lead to distinct transmissions of ON and OFF signals. Although further functional validations are needed, temporal control of inhibition provides an elegant solution that may be of general use in similar contexts where parallel processing is achieved without anatomically split pathways (Qin, 2019).

The connectome study identified ten larval VPNs which receive both direct and filtered inputs from two types of PRs and transmit visual information to higher brain regions, including four LNvs (PDF-LaNs), five LaN, nc-LaN1, and two pVL09, VPLN, and pOLP17. Based on these studies on LNvs and pOLP, it is expected the functional diversity in VPNs generated by differential expression of neurotransmitter receptors or molecules involved in electric coupling will be observed. Besides basic ON vs. OFF discrimination, VPNs are also involved in a variety of visually guided behaviors. The temporal regulation of their glutamatergic and cholinergic inputs as well as the local computation within the LON are among potential cellular mechanisms that increase the VPNs' capability to process complex visual information. Further physiological and molecular studies of the VPNs and behavioral experiments targeting specific visual tasks are needed to elucidate their specific functions (Qin, 2019).

Besides the similarities observed between larval lOLPs and the visual interneurons in the adult fly visual ganglia, an analogy can be drawn between lOLPs and interneurons in mammalian retinae based on their roles in visual processing. Cha-lOLP and glu-lOLP carry sign-conserving or sign-inverting functions and activate ON- or OFF-VPNs, respectively, performing similar functions as bipolar cells in mammalian retinae. At the same time, lOLPs also provide inhibitory inputs to either ON- or OFF-VPNs and thus exhibit the characteristics of inhibitory amacrine cells. The dual role of lOLPs is the key feature of larval ON and OFF selectivity, which likely evolved to fulfill the need for parallel processing using limited cellular resources (Qin, 2019).

Lastly, these studies reveal signaling pathways shared between mammalian retinae and the larval visual circuit. Although the two systems are constructed using different neurochemicals, Gαo signaling is responsible for producing sign inversion in both glu-lOLP and the ON-bipolar cell. In mGluR6-expressing ON-bipolar cells, light increments trigger Gαo deactivation, the opening of TrpM1 channels, and depolarization. In larval glu-lOLP, how light induces voltage and calcium responses via mAchR-B signaling has yet to be determined. Gαo is known to have functional interactions with a diverse group of signaling molecules including potassium and calcium channels that could directly link the light-elicited physiological changes in glu-lOLP. Genetic and physiological studies in the larval visual circuit will facilitate the discovery of these target molecules and contribute to the mechanistic understanding of visual computation (Qin, 2019).

Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking

G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and this study focused on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in approximately 90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, the conserved tryptophan residue was mutagenized in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function (Rizzo, 2018).

Pharmacological identification of cholinergic receptor subtypes: modulation of locomotion and neural circuit excitability in Drosophila larvae

Acetylcholine (ACh) is an abundant neurotransmitter and neuromodulator in many species. In Drosophila melanogaster ACh is the neurotransmitter used in peripheral sensory neurons and is a primary excitatory neurotransmitter and neuromodulator within the central nervous system (CNS). The receptors that facilitate cholinergic transmission are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs). This receptor classification is shared in both mammals and insects; however, both the pharmacological and functional characterization of these receptors within the Drosophila nervous system has lagged behind its mammalian model counterparts. In order to identify the impact of ACh receptor subtypes in regulating the performance of neural circuits within the larval CNS, this study used a behavioral and electrophysiological approach to assess cholinergic modulation of locomotion and sensory-CNS-motor circuit excitability. Intact and semi-intact 3^rd instar larvae were exposed to ACh receptor agonists and antagonists to observe their roles in behavior and regulation of neural circuit excitability and to investigate AChR pharmacological properties in vivo. This was combined with targeted AChR RNAi-mediated knockdown to identify specific receptor subtypes facilitating ACh modulation of circuit efficacy. A contribution by both mAChRs and nAChRs was identified in regulation of locomotor behavior, and it was revealed that they play a role in modulation of the excitability of a sensory-CNS-motor circuit. A conspicuous role for mAChR-A and mAChR-C in motor neurons was identified in modulation of their input-output efficacy (Malloy, 2019).

Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca2+ signalling in glutamatergic interneurons

Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. This study reports a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. Nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, this study shows that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development (Jayakumar, 2016).

Muscarinic ACh receptors contribute to aversive olfactory learning in Drosophila

The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, understanding has advanced with regard to the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. This study evaluates the proposition that, as in mammals, muscarinic ACh receptors (mAChRs; see mAChR-A) contribute to memory formation in Drosophila. The results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila (Silva, 2015).

Different training protocols used in Drosophila have helped advance understanding of the cellular and genetic basis for learning and memory. One of the most studied and best understood is the associative learning of odors, where an odorant that has or does not has an intrinsic value for the animal (the CS) is paired with the US. Thus, the odorant acquires a new value for this animal. The type of memory generated depends on the quality of the US: while in some training protocols electric shock or aversive chemicals such as quinine or salt have been used as US to generate aversive memories, odors can also be paired with sugar to generate appetitive memories. Behavioral and genetic studies have demonstrated that this associative learning depends on the integrity of the major neuropil in the fly brain, the MB, and their principal neurons, the Kenyon Cells (KCs). Therefore, it has been accepted that the timely, coincident arrival of the information of the CS and the US to MB KCs is essential to generate new olfactory memories. This is valid not only for adult flies but also in animals at the larval stage (Silva, 2015).

The literature supports the idea that neurons containing and releasing BAs transmit the US information to the MB, both in adult flies and also in larvae. Remarkably, recent reports have advanced knowledge on the neural aminergic pathways innervating the MB, the specific receptors activated, and some of the cellular events gated by amines in KCs that could underlie the generation of new memories both in larva and the adult fly (Silva, 2015).

However, it is possible the CS is relayed to KCs through cholinergic inputs arising from the antennal lobe (AL) via the inner antennal cerebral tract. This is consistent with the idea that ACh is the main excitatory neurotransmitter in the insect brain. In mammals it is well known that ACh exerts its diverse actions by activation of the fast-acting ionotropic nicotinic receptors (nAChRs) and also metabotropic muscarinic ACh receptors (mAChRs). Ten different genes encode the different subunits for Drosophila nAChRs and although the exact subunit composition of native fly neuronal nAChRs is not known, cell physiology experiments have led to some insights on the functional properties of these channels. For instance, electrophysiological studies have shown that ACh activates α-bungarotoxin-sensitive nAChRs underlying fast excitatory synaptic currents in Drosophila brain neurons. Moreover, it has been recently shown in an in vitro preparation that the enhancement of the AL Projection Neuron-MB synapse depends on the activity of nAChRs. Consistent with all these data, imaging studies have shown that activation of nAChRs induces an increase in intracellular calcium that mediates cellular plasticity (Silva, 2015).

To date, one mAChR has been identified and cloned in Drosophila. The Drosophila mAChR shows high sequence homology to the vertebrate M1-type mAChR and accordingly it was shown to increase the metabolism of membrane phospholipids when expressed in heterologous systems. Interestingly, no information is available on the possibility that mAChRs are involved in olfactory processing in Drosophila, even though it has been shown that this receptor is highly expressed in MB. This lab has generated a new protocol to induce olfactory aversive memories in Drosophila larvae. By using this protocol, this study shows that mAChRs expressed in Drosophila MB contribute to larval olfactory aversive learning and memory (Silva, 2015).

The reciprocal training protocol regularly used in these experiments is aimed at getting a robust, reproducible memory of odors that is thought to be independent of the odorant dilutions used for training and/or memory testing, as in different sets of experiments the US-paired odorant is switched. This is different from training protocols where only one odorant is associated with an US. However, the current data show that even when using the reciprocal training protocol it is necessary to establish the adequate experimental conditions leading to an equilibrated distribution of animals exposed to the odorants in the test chamber before any training. In fact, two different experiments carried out with EA/AA dilution ratios of 10 lead to a different naïve preference: when this ratio is obtained starting from a 1:10 AA dilution, preference observed is above 60%; when a 1:100 AA dilution is used to prepare this dilution ratio, the preference observed is about 50%. This data suggests that it is important to control for naïve preference of animals for the odorants to be used in olfactory learning and memory experiments, as this complex behavior depends on the ability of animals to adequately sense and respond to odorant stimuli. Other factors that could also affect the performance of animals in this associative behavior include the presence of drugs (in the current case atropine) or the US (i.e., salt in these experiments). All these factors have been controlled in the experiments to make sure the results are indeed explained by the ability of animals to generate new memories (Silva, 2015).

The existence of one G-protein coupled metabotropic muscarinic ACh receptor (mAChR, aka mAChR-A) has been shown in Drosophila. This mAChR shows high sequence homology to vertebrate M1-type mAChRs and as expected induces the activation of PLC to modulate membrane phospholipids in heterologous systems. Recently a second mAChR was identified (a.k.a. mAChR-B). This second putative mAChR shows several differences in its amino acid sequence and pharmacological and physiological properties with all previously described vertebrate and invertebrate mAChRs, including the fact that it is not activated by muscarine or blocked by atropine or scopolamine, two well-known mAChR antagonists. Thus, it is not clear whether this is actually a mAChR. For all these reasons this study focused only on the mAChR-A (Silva, 2015).

Expression studies have shown that the mAChR is highly expressed in the adult MB and AL. Information obtained from high-throughput expression studies indicates that this receptor is also expressed in the larval CNS, although up to now there was no information on the expression of this receptor in specific larval brain regions. The current data show that the mAChR is expressed in somas and processes in the ventral nerve cord and the larval MB region, specifically in the calyx and larval MB lobes, positioning this receptor in the right place to modulate olfactory learning (Silva, 2015).

Two different but complementary approaches were used to assess the contribution of mAChRs to olfactory aversive learning in larvae. In one hand, animals were trained and tested in presence of atropine, a well-known antagonist for mAChRs. Data obtained show that these animals are unable to form an aversive olfactory memory, which suggests that mAChRs are required for memory formation. This approach does not speak of the site where mAChRs are acting to modulate memory formation, and therefore several situations could explain this result. Since cholinergic neurons convey the information of the CS to the MB, it is possible that mAChRs are presynaptically located in the AL Projection Neuron-MB synapse to modulate ACh release in the MB region, similar to what has already been suggested for nAChRs in an in vitro AL-MB synapse preparation. mAChRs could also be located in the aminergic terminals responsible for sending the information of the US to the MB, modulating this synapse. The modulation of the release of amines by cholinergic ligands is a possibility that has recently been shown in an in vitro fly brain preparation. It is also possible that mAChRs expressed in the MB neurons directly modulate the activity of these cells to induce memory formation. Since the current expression studies support this proposition, this study used a genetic approach to assess this last possibility. Remarkably, the specific expression of an RNAi^mAChR in MB fully inhibited the formation of new aversive olfactory memory in larvae. Altogether, these data demonstrate for the first time that mAChRs expressed in MB are required for the generation of aversive memory in Drosophila larvae (Silva, 2015).

The contribution of mAChRs in olfactory memory is something already established in other systems. For instance, it has been previously shown that mAChRs contribute to olfactory memories in honeybees. Interestingly, these data support the idea that the muscarinic receptors are only required for olfactory memory retrieval, not acquisition. Moreover, the effect of mAChRs on olfactory memory in honeybees depends specifically on the MB ?-lobe. It is not known whether mAChRs are required for specific memory phases or processes in Drosophila or if as in bees mAChRs are required in specific larval MB regions, but these are issues are currently being evaluated (Silva, 2015).

For example, it has been shown that the administration of scopolamine, a nonselective antagonist for M1-M5 vertebrate mAChRs, decreases different types of memory in mammals. Moreover, data obtained in mice expressing a mutation for the M1-type mAChR show defects on memory acquisition and consolidation. Remarkably, rats treated with scopolamine in the prelimbic cortex show deficient olfactory memory. These data show that mAChRs are important contributors in the generation of memories, particularly olfactory memory, in mammals as it is in insects. The data contribute to the understanding of the molecular underpinnings of memory formation in Drosophila but further support the proposition that regardless of obvious anatomical differences, the key contributors to complex phenomenon including olfactory learning and memory are conserved from arthropods to mammals (Silva, 2015).

The A- and B-type muscarinic acetylcholine receptors from Drosophila melanogaster couple to different second messenger pathways

Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the agonists acetylcholine and muscarine and blocked by several antagonists, among them atropine. In mammals five mAChRs (m1-m5) exist of which m1, m3, and m5 are coupled to members of the Gq/11 family and m2 and m4 to members of the Gi/0 family. Drosophila melanogaster and other arthropods have two mAChRs, named mAChR-A and mAChR-B, where the A-type has the same pharmacology as the mammalian mAChRs, while the B-type has a very low affinity to muscarine and no affinity to classical antagonists such as atropine. This study found that the D. melanogaster A-type mAChR is coupled to Gq/11 and D. melanogaster B-type mAChR to Gi/0. Furthermore, by comparing the second and third intracellular loops of all animal mAChRs for which the G protein coupling has been established, it was possible to identify several amino acid residues likely to be specific for either Gq/11 or Gi/0 coupling. Using these hallmarks for specific mAChR G protein interaction it was found that all protostomes with a sequenced genome have one mAChR coupled to Gq/11 and one to four mAChRs coupled to Gi/0. Furthermore, in protostomes, probably all A-type mAChRs are coupled to Gq/11 and all B-type mAChRs to G0/i (Ren, 2015).

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study two structurally related GPCRs were cloned from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine and muscarine and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Haga published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. Animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data, a model is proposed for the evolution of mAChRs (Collin, 2013).

Extrasynaptic muscarinic acetylcholine receptors on neuronal cell bodies regulate presynaptic function in Caenorhabditis elegans

Acetylcholine (ACh) is a potent neuromodulator in the brain, and its effects on cognition and memory formation are largely performed through muscarinic acetylcholine receptors (mAChRs). mAChRs are often preferentially distributed on specialized membrane regions in neurons, but the significance of mAChR localization in modulating neuronal function is not known. This study shows that the C. elegans homolog of the M1/M3/M5 family of mAChRs, gar-3, is expressed in cholinergic motor neurons, and GAR-3-GFP fusion proteins localize to cell bodies where they are enriched at extrasynaptic regions that are in contact with the basal lamina. GAR-3 can be activated by endogenously produced ACh released from neurons that do not directly contact cholinergic motor neurons. Together, these results suggest that humoral activation of asymmetrically localized mAChRs by ACh is an evolutionarily conserved mechanism by which ACh modulates neuronal function (Chan, 2013).

Search PubMed for articles about Drosophila mACHr-1 and mACHr-2

Bielopolski, N., Amin, H., Apostolopoulou, A. A., Rozenfeld, E., Lerner, H., Huetteroth, W., Lin, A. C. and Parnas, M. (2019). Inhibitory muscarinic acetylcholine receptors enhance aversive olfactory learning in adult Drosophila. Elife 8. PubMed ID: 31215865

Chan, J. P., Staab, T. A., Wang, H., Mazzasette, C., Butte, Z. and Sieburth, D. (2013). Extrasynaptic muscarinic acetylcholine receptors on neuronal cell bodies regulate presynaptic function in Caenorhabditis elegans. J Neurosci 33: 14146-14159. PubMed ID: 23986249

Collin, C., Hauser, F., Gonzalez de Valdivia, E., Li, S., Reisenberger, J., Carlsen, E. M., Khan, Z., Hansen, N. O., Puhm, F., Sondergaard, L., Niemiec, J., Heninger, M., Ren, G. R. and Grimmelikhuijzen, C. J. (2013). Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods. Cell Mol Life Sci 70(17): 3231-3242. PubMed ID: 23604020

Jayakumar, S., Richhariya, S., Reddy, O. V., Texada, M. J. and Hasan, G. (2016). Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca2+ signalling in glutamatergic interneurons. Elife 5 [Epub ahead of print] PubMed ID: 27494275

Malloy, C. A., Somasundaram, E., Omar, A., Bhutto, U., Medley, M., Dzubuk, N. and Cooper, R. L. (2019). Pharmacological identification of cholinergic receptor subtypes: modulation of locomotion and neural circuit excitability in Drosophila larvae. Neuroscience 411: 47-64. PubMed ID: 31102763

Qin, B., Humberg, T. H., Kim, A., Kim, H. S., Short, J., Diao, F., White, B. H., Sprecher, S. G. and Yuan, Q. (2019). Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit. Nat Commun 10(1): 4093. PubMed ID: 31501438

Ren, G. R., Folke, J., Hauser, F., Li, S. and Grimmelikhuijzen, C. J. (2015). The A- and B-type muscarinic acetylcholine receptors from Drosophila melanogaster couple to different second messenger pathways. Biochem Biophys Res Commun 462(4):358-64. PubMed ID: 25964087

Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. and Johnson, E. C. (2018). Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking. Biochem Biophys Res Commun 503(3): 1919-1926. PubMed ID: 30064912

Silva, B., Molina-Fernandez, C., Ugalde, M. B., Tognarelli, E. I., Angel, C. and Campusano, J. M. (2015). Muscarinic ACh receptors contribute to aversive olfactory learning in Drosophila. Neural Plast 2015: 658918. PubMed ID: 26380118

date revised: 26 September 2019

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