InteractiveFly: GeneBrief

Serotonin receptor 7: Biological Overview | References

BIOLOGICAL OVERVIEW

The 5-HT₇ receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT₇ receptor, as well as homologs for the mammalian 5-HT_1A, and 5-HT₂, receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and has been demonstrated to mediate similar behavioral responses. This study reports on the expression and function of the 5-HT₇ receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, 5-HT₇ activity was found to be essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly (Becnel, 2011).

Serotonin (5-HT) is a monoamine neurotransmitter that regulates a variety of behaviors and physiological processes including circadian rhythms, sleep, appetite, aggression, locomotion, perception and sexual behavior in mammals. In mammals, there are fourteen different receptors than can be organized into seven families. The many effects of serotonin are primarily mediated through G-protein coupled receptors, which initiate multiple effector pathways. Misregulation of serotonin signaling in humans has been implicated in neuropsychiatric disorders including depression, anxiety, anorexia nervosa, and schizophrenia (Becnel, 2011).

In mammals, 5-HT₇ mRNA has been observed in both the CNS and peripheral tissues including the suprachiasmatic nucleus of the hypothalamus, thalamus, hippocampus and cortex, as well as coronary artery, gastrointestinal tract, kidney, and spleen (Nichols, 2008). 5-HT₇ receptors are expressed postsynaptically in the cortex, hippocampal formation and other parts of the brain (Neumaier 2001). They are, however, found both pre- and postsynaptically in the SCN (Belenky, 2001). Studies using antagonists and a knock out mouse model show involvement of 5-HT₇ receptor activity in regulating mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles (Guscott, 2005; Guscott, 2003; Hedlund, 2003; Hedland, 2005; Janssen, 2004; Monti, 2006; Monti, 2008; Sprouse, 2005; Becnel, 2011 and references therein).

With regard to sleep, 5-HT₇ receptors modulate neuronal function in a number of areas of the brain that have been implicated in this behavior, including the SCN, DRN, thalamus and hippocampus (Thomas, 2004). The systemic administration of 5-HT₇ receptor antagonist to rats at the beginning of light periods reduces total amount of REM sleep, and direct administration of antagonist into the DRN reduces REM sleep and the number of REM sleep periods (Monti, 2006). Interestingly, 5-HT₇ receptors have also been implicated in the regulation of mammalian sexual behavior. Activation of this receptor in rats mediates an inhibitory effect of female sexual behavior (Siddiqui, 2006; Becnel, 2011 and references therein).

Drosophila has proven to be a very effective model system for investigating the function of mammalian systems and diseases. About 70% of human disease genes have functional orthologs in Drosophila (Bier, 2005), and the fly expresses functional orthologs of most mammalian neurotransmitter receptors, including receptors for dopamine, glutamate, acetylcholine, GABA, and serotonin, which mediate conserved behaviors. The fruit fly expresses orthologs of three of the seven mammalian receptor families: 5-HT_1A/BDro, 5-HT₂Dro and 5-HT₇Dro, and the molecular pathways linking serotonin receptor interactions with behaviors are likely to be conserved between the two systems. 5-HT_1A/BDro are expressed in adult mushroom bodies, as well as additional brain circuits (Yuan, 2005), and mediate aspects of sleep and aggression (Yuan, 2005; Johnson, 2009). The 5-HT₂Dro receptor has been previously characterized, and found to be expressed throughout the adult brain, including neurons within the protocerebrum and ellipsoid body, and mediates aspects of circadian behaviors and aggression (Johnson, 2009; Nichols, 2007; Becnel, 2011 and referencea therein)

This study reports on expression and function of the 5-HT₇Dro receptor in Drosophila. It is expressed within discreet circuits in the brain and ventral nerve cord, and is essential for normal courtship and mating (Becnel, 2011).

To explore the role of the 5-HT₇Dro receptor in the fly, an enhancer GAL4 driver strain was created and used characterized the putative CNS expression and function of the receptor. Using the 5-HT₇Dro-GAL4 driver to drive expression of a UAS-mCD8::GFP transgene, GFP expression in third larval instar brain was found to be localized to discreet circuits within the brain hemispheres, as well as to specific neurons in the ventral ganglion. In the adult brain, there is a high level of 5-HT₇Dro-GAL4 expression in large-field R neurons that innervate the ellipsoid body, as well as in neurons in the brain that tightly cluster with the PDF-positive LNv clock neurons and innervate the optic lobes. There is moderate expression detected in the olfactory and gustatory regions of the brain, and weak expression in other central complex structures like the fan shaped body. 5-HT₇Dro-GAL4 expression appears to be post-synaptic both in larvae and adults. This is consistent with the observed post-synaptic expression for the 5-HT₇ receptor in vertebrate CNS. In the ventral nerve cord, expression is detected in several sets of postsynaptic neurons and in all neuromeres of the fused ganglion. Importantly, there is a close apposition between receptor-expressing processes and those containing serotonin. Although a majority of GAL4 driver strains present relevant/accurate expression data of the intended gene, it should be emphasized here that 5-HT₇Dro-GAL4 enhancer expression may not represent the entire expression pattern of the native 5-HT₇Dro receptor, or may be even expressing in cells that do not express the native receptor. It should also be emphasized that the mCD8:GFP construct used in this study to examine expression is a membrane-bound form of the GFP protein that highlights all cellular membranes. Whereas the GFP expression patterns observed in this study provide clues as to the cells and structures that express the 5-HT₇Dro-GAL4 element, it is unable to provide information regarding the subcellular localization of the GPCR on the cellular membrane. Unfortunately, attempts to generate anti-sera to 5-HT₇Dro for further validation of native protein expression were unsuccessful (Becnel, 2011).

Treatment with the 5-HT₇ receptor antagonist SB258719 interferes with courtship and mating behaviors. Inhibition of courtship behavior frequency decreases as the courtship and mating process progresses from hardly any disruptive effect with respect to early behaviors involving sensory cues (orient and wing vibration), to more pronounced effects for intermediate behaviors (licking, curling, attempts), to complete loss of successful copulation at higher levels of the drug. In corroboration with the pharmacological studies, knockdown of 5-HT₇Dro message within 5-HT₇Dro-GAL4 expressing neurons produces behavioral changes consistent with the pharmacological results. The early behaviors of orientation and wing song are not affected much, the intermediate behaviors of licking and curling are significantly disrupted, and successful copulation is eliminated. There are some subtle differences between methods, however, like for curling latencies, which could be due to the nature of receptor inactivation (more acute pharmacological methods vs. constitutive nature of the RNAi knockdown) (Becnel, 2011).

From these results, there appears to be little 5-HT₇Dro receptor involvement in the early stages, where antagonist treated males were observed to be receptive to females present in the mating chamber, and are able to initiate the first elements of the mating process. During the intermediate stages, involving licking and curling, there is likely more involvement of 5-HT₇Dro receptor signaling. A decrease in licking behavior may result in decreased curling, and decreased curling may result in decreased successful copulation attempts, with the effects compounding at each successive behavior leading to an overall failure at successful copulation. These effects could arise from alterations in physical performance and coordination, sensory perception, olfaction, or to decreases in receptivity or interest, or a lack of ‘motivation’ to perform the higher intensity physical behaviors. These effects are thought not to be due to deficits in overt locomotor activity because the drug treated flies demonstrate normal levels of measured activity, or in coordination because at drug levels where mating frequency is decreased the flies that do perform, perform well with latencies not significantly different than control pairs. General alteration of sensory perception is also not a likely reason because males recognize females and readily perform behaviors related to visual and acoustic cues (orienting and wing vibration) despite administration of antagonist or knockdown of message. Whereas olfaction is necessary for receptivity, it is unlikely impeded following antagonist administration because flies fed 3 mM SB as well as the knockdown flies exhibit normal aversion and sensitivity to odors in olfactory tests. Nevertheless, there still may be an olfactory or pheromone component if the limited 5-HT₇Dro-GAL4 expression detected in the olfactory lobes correlates with select neurons necessary for reception of specific courtship related pheromones, or the expression does not correlate appropriately with native 5-HT₇Dro expression and localization in the olfactory lobes (Becnel, 2011).

A key gene known to be significantly involved in courtship behaviors is fruitless (fru), where almost every stage of the mating process has been shown to be disrupted by certain alleles of the locus. With the exception of one fru allele, satori, where males exclusively courts males, fru mutants indiscriminately court both males and females. In Drosophila, there are also several other known mutants that rarely court females or males. These include he's not interested (hni), tapered (ta), pale, cuckold (cuc) and courtless (crl). Two of these genes have been characterized: courtless encodes a ubiquitin-conjugating enzyme that is also involved in spermatogenesis, and pale encodes a tyrosine hydroxylase that catalyzes the synthesis of dopamine. In experiments with both the 5-HT₇ receptor antagonist and the RNAi knockdown, male flies displayed a general disinterest in mating with either females or other males, and females appeared non-responsive to male courtship attempts (Becnel, 2011).

Ectopic expression of the white gene, which encodes for the transporter for the biosynthetic precursor of serotonin, tryptophan, can induce inter-male courtship (Zhang, 1995). These observations, along with others, suggested that serotonin may be involved in courtship in the fly (interestingly, alterations in 5-HT levels have been shown to elicit homosexual behavior in mammals. In early studies examining co-expression of the male-specific Fru^M protein and 5-HT, it was found that in wild-type males there were no 5-HT CNS neurons that co-expressed Fru^M, with the exception of a small cluster of serotonergic cells at the posterior tip of the ventral nerve cord. The possibility, however, still remained that perhaps Fru^M expression was mediating the effects of serotonin postsynaptically. The current results show there is no overlap between Fru^M and 5-HT₇Dro-GAL4 expression. Therefore, it would appear that 5-HT₇Dro and Fru^M do not directly interact in their modulation and control of courtship and mating behaviors (Becnel, 2011).

What then is the role that 5-HT₇ receptor signaling plays in reducing receptivity at each stage? One possibility is that pheromone release from the female may be disrupted, resulting in a decrease of courtship. This is thought to be unlikely, however, because control males paired with 5-HT₇ receptor antagonist SB258719 (SB) fed females continue to attempt copulation with SB fed females that are not receptive, and will continually chase these females in the mating chamber, remaining uninterested despite repeated attempts by the males. Furthermore, when males fed SB were paired with control females, females that seemed to seek out the male, which would then run away from the female. Therefore, it would appear that because the control fly of the pair seeks out and attempts to initiate courtship behaviors, SB treatment and manipulation of 5-HT₇ receptor function does not interfere with pheromone release, or potentially other sensory cues, related to receptivity. If the default behavior is to initiate courtship in the absence of pheromones, however, then it may still be possible that SB is interfering with pheromone reception at later stages if they are required for the maintenance and intensification of courtship. In the SB/control pairing experiments, the SB fed male + control female pairs were the least successful at courtship behavior. One interpretation of these results are that males are essential to initiating certain elements of mating that are regulated by 5-HT₇ receptor function, and when these do not occur it may contribute to further lack of receptivity by females. Alternatively, males and females may be only responding to the SB drug differently (Becnel, 2011).

Because of the high level of expression of the 5-HT₇Dro-GAL4 driver in the large-field R field neurons that innervate the ellipsoid body, it was hypothesized that receptor expression in these neurons is relevant for normal courtship and mating. Furthermore, that the 5-HT₇ receptors expressed by these neurons regulate receptivity/interest of one fly for its partner. Little is known regarding the exact function of the ellipsoid body, and the neurons that feed into it. The overall structure is believed to be involved in mediating higher order behaviors, including aspects of learning and memory, stress response, flight control, and gravitaxis. It is comprised of many distinct cell types and individual circuits including 10 types of small field neurons, and 4 known major types of large-field R neurons. Previous reports have indicated that there are about 40 large-field R neurons in each of two clusters in the central brain, and based upon the number of large-field R neurons in which 5-HT₇Dro-GAL4 GFP expression was detected, there may be nearly 50, suggesting that there are additional subtypes of this neuron beyond those already identified. Significantly, 5-HT₇Dro-GAL4 may be the first GAL4 driver line reported that predominantly and strongly drives expression in the putative full set of large-field R neurons within the central brain, and as such may be a valuable strain to use to define the functional role of these cells and the development of ellipsoid body circuits. A subset, the R4m neurons, are cholinergic and express NMDA receptors. It remains to be seen which of the major subsets of large-field R neurons contribute to courtship and mating behaviors. Although it is hypothesized that the ellipsoid body mediates courtship behaviors, it is entirely possible that other neurons that are weakly expressing 5-HT₇Dro-GAL4, or neurons perhaps not expressing the 5-HT₇Dro-GAL4 element in the brain, are contributing to or completely mediating these observed effect on courtship and mating (Becnel, 2011).

Courtship and mating has been shown by others to involve additional neurotransmitter systems including dopamine, which has been shown to play a role in both female receptivity as well as male-male courtship, and GABA, which also plays a role in female receptivity. Various brain structures including the mushroom bodies, protocerebrum, and optic lobes have also been shown to be involved in courtship and mating in the fly. With regards to courtship and mating, the 5-HT₇Dro circuitry, the ellipsoid body, or both, may be essential to the integration and processing of information received from the various sensory stimuli to correctly initiate physical courtship behaviors. Significantly, proper function of Gαs-coupled 5-HT₇Dro receptor signaling within these neurons may also be essential to this process. Interestingly, 5-HT₇ receptors have also been implicated in the regulation of mammalian sexual behavior, although the response is reverse to that in flies. Blockade of this receptor with antagonist in female rats increases lordosis activity, and it was hypothesized that in mammals 5-HT₇ receptors exert a tonic inhibitory effect on female sexual behavior (Becnel, 2011).

In summary, this study has generated a 5-HT₇Dro-GAL4 reporter; it was used to characterize the putative expression of the 5-HT₇Dro receptor. The receptor is highly expressed in the brain in large-field R neurons in the adult, as well as in small groups of cells that cluster with PDF-positive LNvs neurons. Functional studies utilizing pharmacological and genetic methods indicate that this receptor is necessary for normal courtship and mating (Becnel, 2011).

Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila

Learning and memory in Drosophila is a complex behavior with many parallels to mammalian learning and memory. Although many neurotransmitters including acetylcholine, dopamine, glutamate, and GABA have been demonstrated to be involved in aversive olfactory learning and memory, the role of serotonin has not been well defined. This study presents evidence of the involvement of individual serotonin receptors in olfactory learning and memory in the fly. A pharmacological approach was followed, utilizing serotonin receptor agonists and antagonists, to demonstrate that all serotonin receptor families present in the fly are necessary for short-term learning and memory. Isobolographic analysis utilizing combinations of drugs revealed functional interactions are occurring between 5-HT_1A-like and 5-HT₂, and 5-HT₂ and 5-HT₇ receptor circuits in mediating short-term learning and memory. Examination of long-term memory suggests that 5-HT_1A-like receptors are necessary for consolidation and important for recall, 5-HT₂ receptors are important for consolidation and recall, and 5-HT₇ receptors are involved in all three phases. Importantly, the pharmacological results were validated with genetic experiments, and hypomorph strains for 5-HT₂Dro and 5-HT_1BDro receptors, as well as knockdown of 5-HT₇Dro mRNA, were shown to significantly impair performance in short-term memory. These data highlight the importance of the serotonin system and individual serotonin receptors to influence olfactory learning and memory in the fly, and position the fly as a model system to study the role of serotonin in cognitive processes relevant to mammalian CNS function (Johnson, 2011).

Serotonin 5-HT_1A/1BDro, 5-HT₂Dro, and 5-HT₇Dro receptors are involved in aspects of both short-term and long-term conditioned stimulus olfactory aversive learning and memory processes. One significant finding of this work is that structures extrinsic to the MBs may be involved at some level in olfactory learning and memory, as 5-HT₂Dro and 5-HT₇Dro receptors are not known to express in the MBs. For each of the receptor drugs tested, there are no disruptive effects on locomotor activity, and this study has shown that there are no confounding effects on olfaction or shock reactivity. Importantly, to validate the pharmacological data, genetic methods were used, and insertion alleles and knockdown of receptor mRNA also were found to produce significant deficits in performance. Together, these genetic data are consistent with the pharmacological data and demonstrate that each serotonin receptor is necessary for aspects of normal olfactory learning and memory in the fly (Johnson, 2011).

For short-term memory (STM), it cannot yet be said which components require serotonin receptors. They may be only necessary for learning, or memory, or they may be involved in both. Future studies will address this issue in more depth. Although there are a number of genetic tools that can be used to examine neuronal and receptor function, pharmacological methods can often provide unique and complementing information. Therefore, this study followed a pharmacological approach that incorporated dose response strategies to examine relative contributions of each receptor to short-term learning and memory, the nature of functional interactions between individual receptor types and circuits, and to begin to dissect out individual roles in specific components of LTM. The results here support the notion of some degree of receptor selectivity for these drugs, as do previous studies examining serotonin receptors in the fly where it has been shown that these drugs can have very different behavioral effects from one another in multiple behaviors. Nevertheless, the exact affinities for and selectivity of these drugs at their intended target receptor remain to be fully validated in Drosophila, and some caution must be exercised when interpreting these data. Interestingly, the data show that both agonists and antagonists at the same receptors disrupt performance. One may predict that if an antagonist is disruptive, then an agonist may be enhancing and vice versa. This is not always the case with GPCR signaling, and there are reports in the literature of both types of ligands at a given receptor producing disruptive effects. Because serotonin primarily plays a modulatory role in the CNS, the receptors likely require a dynamic response to properly regulate learning and memory processes. The homologous mammalian receptors each exhibit constitutive activity, and the fly receptors are predicted to have similar constitutive activity associated with them. If a certain level of basal activity and dynamic response to input levels of serotonin is required for normal performance and homeostasis, then both agonists and antagonists (inverse agonists, as ketanserin, WAY100635, and SB258719) would lead to a reduction of the ability of the receptors to dynamically respond to serotonin, leading to a degradation of performance. For example, similar phenomena are seen with increased and decreased receptor activity in human behaviors. It was also observed that whereas drugs were able to completely disrupt STM performance, genetic methods only produced ~50%-75% reduction in performance. It is likely that with pharmacological methods, it was possible to completely disrupt receptor function as drug levels increase, but for both the hypomorph strains and the RNAi knockdown studies there still exists a population of normal receptors, albeit reduced in expression, that confer some degree of functionality to the circuitry. Another factor may be time of administration. For the STM experiments, drugs were administered for 48 h to reach steady state levels, which may have greater or even different effects than would be evident with acute administration (Johnson, 2011).

With respect to LTM processes, the effects of IC50 concentrations of drug in the food were used to assess the role of the serotonin receptors on acquisition, consolidation, and retrieval. The data suggest that each receptor/circuit has their own unique contribution to LTM, where 5-HT_1A-like receptors are critical for consolidation and important for retrieval, 5-HT₂ receptors are important for consolidation and retrieval, and 5-HT₇ receptors are important for all three components. The possibility remains, however, that the differential results observed on LTM may simply be due to the drugs having differential off-target effects at other GPCRs that are important for LTM in addition to the core response mediated by the individual 5-HT receptors (Johnson, 2011).

Having established the involvement of serotonin receptors in learning and memory, how might they function in this capacity? The 5-HT_1A/₁BDro receptors are expressed postsynaptically in the MBs. Localization of these receptors to the MBs strongly implies that they directly influence MB function. Significantly, the 5-HT_1A/₁BDro receptors are coupled to Gαi and inhibition of adenylate cyclase activity, and when stimulated lead to a reduction of cAMP levels. Levels of cAMP have been demonstrated to be extremely important for learning and memory (Johnson, 2011).

As in mammals, the 5-HT_1A-like receptors are also expressed presynaptically and are predicted to have autoreceptor function. The data indicate that administration of the 5-HT_1A receptor agonist U92016A in combination with the 5-HT_1A receptor antagonist WAY100635 work synergistically to attenuate STM function in the fly. Although these agents are both targeting 5-HT_1A receptors, binding affinity and/or functional selectivity at pre- vs. postsynaptic receptors could be promoting a superadditive rather than a subadditive relationship between these two agents. For example, the agonist U92016A may have greater affinity or efficacy at presynaptic receptors and reduce 5-HT release through autoreceptor activity, and the antagonist WAY100635 may have greater affinity or efficacy at postsynaptic receptors to block reception of the signal that together produce a superadditive decrease in 5-HT effects (Johnson, 2011).

A similar synergistic behavioral effect was observed in previous studies with combinations of agonists and antagonists for 5-HT_1A-like receptors with respect to aggressive behaviors. Different affinities and efficacies for drugs acting at the same G-protein coupled receptor located at different biological sites is a well-established phenomenon termed functional selectivity, and this pre/postsynaptic phenomenon plays a significant role in the action of drugs, such as aripiprazole, in humans (Johnson, 2011).

5-HT₂Dro receptors are located postsynaptically on neurons of the protocerebrum that are in close proximity to Kenyon cells of the MBs. These cells may normally serve to influence the function of Kenyon cells, and 5-HT₂Dro receptor activity may therefore conceivably be indirectly modulating function of the MBs. These receptors are coupled to Gαq, and are generally stimulatory in nature and in mammalian CNS are involved in cognitive processing and integrating sensory information. Their role in the fly may be similar and facilitating the integration of sensory information into the MBs. Interaction data show that simultaneous administration of 5-HT₂ receptor agonist and antagonist are interfering, which would be predicted for a receptor with only postsynaptic localization. Significantly, 5-HT_1A antagonists in combination with 5-HT₂ antagonists are interfering, indicating a functional interaction between the two receptor circuitries. In mammals, 5-HT_1A and 5-HT₂ receptors often functionally antagonize one another, and it may be that blockade of 5-HT₂Dro receptors counteracts the effects of blockade of 5-HT_1ADro receptors in the fly. In addition, there is expression of 5-HT₂Dro in a subset of cells of the EB that may be contributing to its role in learning and memory. This notion is supported by data indicating functional interactions occurring between 5-HT₂Dro and 5-HT₇Dro receptor circuitry, which has high expression in the EB (Johnson, 2011).

The results examining the 5-HT₇Dro receptor are very intriguing. Although the receptor is expressed weakly in other circuits and areas of the brain (Becnel, 2011), its strong expression in all large field R-neurons of the EB is suggestive of involvement of the EB at some level in olfactory learning and memory processes. Previous attempts to study the role of this structure in olfactory learning and memory have largely been unsuccessful. Walking and flying are mediated by the EB, and the use temperature sensitive off/on shibireTS or TRPM channels to inactivate the entire structure, or mutants that structurally disrupt the EB, has been shown to produce profound coordination and locomotor difficulties, precluding accurate testing of the EB’s role in behaviors. An attempt at a more precise analysis examined NMDA receptor function in a subset of EB neurons, and a role has been proposed for consolidation in LTM. Significantly, a subset of large field R-neurons has recently been demonstrated to be necessary for visual pattern memory. Because there are no direct connections between the central complex and the MB, it remains to be elucidated how structures of the central complex are involved in modulating both olfactory and visual memory. Furthermore, the precise 5-HT₇Dro expressing neurons extrinsic to the MBs, either within the central complex or elsewhere, modulating learning and memory remain to be determined in future studies (Johnson, 2011).

In summary, this study has provided the first evidence that serotonin receptors are necessary for normal olfactory learning and memory in the fly. STM is disrupted by both pharmacological agents and by genetic manipulations of serotonin receptor function. The use of pharmacological tools has allowed examination of receptor-receptor and receptor-circuitry interactions through isobolographic analysis, where it was determined that there are functional interactions between 5-HT_1A and 5-HT₂ circuitries, as well as 5-HT₂ and 5-HT₇ receptor circuitries. These interactions may be interpreted in a model such that 5-HT_1A-like receptors expressed within the MBs directly influence MB function for STM, and particularly consolidation in LTM, by virtue of their location in MB neurons. The 5-HT₂Dro expressing multipolar neurons in close proximity to the Kenyon cells of the protocerebrum, and neurons within the EB, may then be modulating the activity of the MBs in STM, and in consolidation and retrieval for LTM. The 5-HT₇Dro circuitry may be indirectly influencing MB function through modulation of 5-HT₂Dro circuits, or potentially other yet to be identified circuits. In this model, components of the central complex like the EB may be playing a master regulatory role for complex behaviors like STM and all three aspects of LTM, rather than a more specific and direct role in olfactory learning and memory per se. This is consistent with observations that 5-HT₇Dro receptor activity is required for other complex behaviors like normal courtship and mating (Becnel, 2011). This work is intended to be presented as an initial characterization of serotonin receptor involvement in olfactory learning and memory; however, additional work remains to fully elucidate the role of serotonin and its receptors in these processes (Johnson, 2011).

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).

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 (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. 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).

Search PubMed for articles about Drosophila 5-HT7 receptor

Becnel, J., et al. (2011). The serotonin 5-HT₇Dro receptor is expressed in the brain of Drosophila, and is essential for normal courtship and mating. PLoS ONE 6: e20800. PubMed ID: 21674056

Belenky. M. A. and Pickard, G. E. (2001). Subcellular distribution of 5-HT(1B) and 5-HT(7) receptors in the mouse suprachiasmatic nucleus. J. Comp. Neurol. 432: 371-388. PubMed ID: 11246214

Bier, E. (2005). Drosophila, the golden bug, emerges as a tool for human genetics. Nat. Rev. Genet. 6: 9-23. PubMed ID: 15630418

Guscott, M., et al. (2005). Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression. Neuropharmacology 48: 492-502. PubMed ID: 15755477

Guscott, M. R., et al. (2003). The hypothermic effect of 5-CT in mice is mediated through the 5-HT7 receptor. Neuropharmacology 44: 1031-1037. PubMed ID: 12763096

Hedlund, P. B., et al. (2003). No hypothermic response to serotonin in 5-HT7 receptor knockout mice. Proc. Natl. Acad. Sci. 100: 1375-1380. PubMed ID: 12529502

Hedlund, P. B., et al. (2005). 5-HT7 receptor inhibition and inactivation induce antidepressantlike behavior and sleep pattern. Biol. Psychiatry 58: 831-837. PubMed ID: 16018977

Janssen, P., et al. (2004). 5-HT7 receptor efficacy distribution throughout the canine stomach. Br. J. Pharmacol. 143: 331-342. PubMed ID: 15339857

Johnson, O., Becnel, J. and Nichols, C. D. (2009). Serotonin 5-HT(2) and 5-HT(1A)-like receptors differentially modulate aggressive behaviors in Drosophila melanogaster. Neuroscience 158: 1292-1300. PubMed ID: 19041376

Johnson, O., Becnel, J. and Nichols, C. D. (2011). Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila melanogaster. Neuroscience 192: 372-81. PubMed ID: 21749913

Monti, J. M. and Jantos, H. (2006). Effects of the 5-HT(7) receptor antagonist SB-269970 microinjected into the dorsal raphe nucleus on REM sleep in the rat. Behav. Brain Res. 167: 245-250. PubMed ID: 16290281

Monti, J. M., Leopoldo, M. and Jantos, H. (2008). The serotonin 5-HT(7) receptor agonist LP-44 microinjected into the dorsal raphe nucleus suppresses REM sleep in the rat. Behav. Brain Res. 191: 184-189. PubMed ID: 18466985

Neumaier, J. F., et al. (2001). Localization of 5-HT(7) receptors in rat brain by immunocytochemistry, in situ hybridization, and agonist stimulated cFos expression. J. Chem. Neuroanat. 21: 63-73. PubMed ID: 11173221

Nichols, C. D. (2007). 5-HT2 receptors in Drosophila are expressed in the brain and modulate aspects of circadian behaviors. Dev. Neurobiol. 67: 752-763. PubMed ID: 17443822

Nichols, D. E. and Nichols, C. D. (2008). Serotonin receptors. Chem. Rev. 108: 1614-1641. PubMed ID: 18476671

Siddiqui, A., Niazi, A., Shaharyar, S. and Wilson, C. A. (2007). The 5HT(7) receptor subtype is involved in the regulation of female sexual behaviour in the rat. Pharmacol. Biochem. Behav. 87: 386-392. PubMed ID: 17561239

Sizemore, T. R. and Dacks, A. M. (2016). Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster. Sci Rep 6: 37119. PubMed ID: 27845422

Sprouse, J., Li, X., Stock, J., McNeish, J. and Reynolds, L. (2005). Circadian rhythm phenotype of 5-HT7 receptor knockout mice: 5-HT and 8-OH-DPAT-induced phase advances of SCN neuronal firing. J. Biol. Rhythms 20: 122-131. PubMed ID: 15834109

Suzuki, Y., Schenk, J. E., Tan, H. and Gaudry, Q. (2020). A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe. Curr Biol. 30(6):1110-1118 PubMed ID: 32142699

Thomas, D. R. and Hagan, J. J. (2004). 5-HT7 receptors. Curr. Drug Targets CNS Neurol. Disord. 3: 81-90. PubMed ID: 14965246

Yuan, Q., Lin, F., Zheng, X. and Sehgal, A. (2005). Serotonin modulates circadian entrainment in Drosophila. Neuron 47: 115-127. PubMed ID: 15996552

Zhang, S. D. and Odenwald, W. F. (1995). Misexpression of the white (w) gene triggers male-male courtship in Drosophila. Proc. Natl. Acad. Sci. 92: 5525-5529. PubMed ID: 7777542

date revised: 1 March 2024

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