InteractiveFly: GeneBrief

Odorant receptor 67d: Biological Overview | References

BIOLOGICAL OVERVIEW

Insects, like many other animals, use sex pheromones to coordinate their reproductive behaviours. Volatile pheromones are detected by odorant receptors expressed in olfactory receptor neurons (ORNs). Whereas fruit odours typically activate multiple ORN classes, pheromones are thought to act through single dedicated classes of ORN. This model predicts that activation of such an ORN class should be sufficient to trigger the appropriate behavioural response. This study shows that the Drosophila male-specific pheromone 11-cis-vaccenyl acetate (cVA) acts through the receptor Or67d to regulate both male and female mating behaviour. Mutant males that lack Or67d inappropriately court other males, whereas mutant females are less receptive to courting males. These data suggest that cVA has opposite effects in the two sexes: inhibiting mating behaviour in males but promoting mating behaviour in females. Replacing Or67d with moth pheromone receptors renders these ORNs sensitive to the corresponding moth pheromones. In such flies, moth pheromones elicit behavioural responses that mimic the normal response to cVA. Thus, activation of a single ORN class is both necessary and sufficient to mediate behavioural responses to the Drosophila sex pheromone cVA (Kurtovic, 2007).

Several lines of evidence initially suggested that Drosophila mating behaviours might be guided in part by pheromones detected by the class of ORNs that expresses the odorant receptor Or67d: (1) the Or67d ORNs innervate a sexually dimorphic glomerulus (DA1) in the antennal lobe (Kondoh, 2003; Stockinger, 2005); (2) these neurons constitute one of only three ORN classes that express the sex-specific transcripts of the behavioural sex determination gene fruitless (fru) (Couto, 2005; Fishilevich, 2005); (3) blocking the activity of all three classes of fru-positive ORNs impairs male courtship activity (Stockinger, 2005; Kurtovic, 2007 and references therein).

To assess the role of Or67d and the Or67d ORNs in Drosophila mating behaviour, mutant alleles were generated in which the open reading frame of Or67d was replaced with that of the yeast transcriptional activator GAL4. These mutant knock-in alleles allowed assessment of the function of Or67d itself, and also to use GAL4-responsive transgenes to study the function of the Or67d ORNs. Ends-in homologous recombination was used to produce a tandem duplication at the Or67d locus, consisting of one copy of the wild-type locus and one copy with the GAL4 replacement. By resolving this duplication, two independent mutant alleles were recovered that carried only the GAL4 replacement (Or67d^GAL4[1] and Or67d^GAL4[2]), and two independent control alleles in which the original intact locus was restored (Or67d^+[1] and Or67d^+[2]) (Kurtovic, 2007).

Using a UAS-mCD8-GFP reporter to label these cells with green fluorescent protein (GFP), it was confirmed that the Or67d^GAL4 knock-in drives transgene expression exclusively in the ORNs that also express Or67d. Previous studies using an Or67d promoter fragment to drive GAL4 expression did not fully clarify whether Or67d ORNs project to DA1 exclusively. Using the Or67d^GAL4 knock-in, it was confirmed that the DA1 glomerulus alone is targeted by Or67d ORNs, in both males and females. No reporter expression was detected anywhere else in adults, nor in embryos or larvae (Kurtovic, 2007).

The Or67d receptor is thought to mediate the detection of the male pheromone cVA ([Z]-11-octadecenyl acetate): cVA activates the T1 class of trichoid sensilla in which the Or67d neurons alone are housed, and ectopic expression of Or67d in other trichoid ORNs confers sensitivity to cVA (Ha, 2006). Indeed, using single-sensillum recordings, it was found that cVA elicits a rapid and robust firing response in the T1 sensilla of control Or67d⁺ males but not in those of Or67d^GAL4 mutants. Spontaneous activity was also greatly reduced in these mutants. Restoring Or67d function with a UAS-Or67d transgene fully rescued both the spontaneous and evoked responses. The responses to cVA were quantitatively indistinguishable in males and females across a 1,000-fold range of concentrations. To assess whether odorant receptors in other ORNs might also detect cVA, electroantennograms were used to simultaneously measure the responses of a large number of ORNs on the third antennal segment. Neither Or67d^GAL4 males nor females produced a detectable response to cVA, although both responded normally to ethanol. These genetic data confirm that Or67d mediates physiological responses to cVA, and show further that males and females respond equally to cVA and that Or67d is likely to be the only receptor for cVA (Kurtovic, 2007).

If cVA acts as a sex pheromone and Or67d is its sole receptor, then Or67d^GAL4 males or females should be impaired in their performance of one or more mating behaviours. To test this, male courtship behaviour was first monitored in single-pair courtship assays, using the courtship index (CI, the percentage of time for which the male courts during a 10-min assay) as a simple measure of overall courtship activity. Typically, wild-type males court with a CI of about 80% when paired with a virgin female, but only about 10% when paired with another male. When paired with virgin females, Or67d^GAL4 mutant males courted at levels comparable to those of the control Or67d⁺ males. In contrast, when paired with wild-type males, Or67d^GAL4 mutant males displayed a roughly threefold higher courtship activity than the Or67d⁺ controls. To confirm that this increased male-male courtship was indeed due to the loss of Or67d function, independent UAS-Or67d transgene insertions were introduced into each of the two Or67d^GAL4 lines. For both alleles, restoring Or67d function in this way suppressed male-male courtship back to its normal low levels (Kurtovic, 2007).

To assess whether Or67d also functions in female mating behaviour, individual mutant or control virgin females were paired with naive wild-type males in a series of small chambers and their latency to copulation was measured. About 50% of the control Or67d⁺ females copulated within 18 min, and about 60% within 30 min. In contrast, only about 20% of the Or67d^GAL4 females copulated within 18 min, and about 30% within 30 min. When Or67d expression was restored with the UAS-Or67d transgenes, the mutant females copulated as rapidly as the control females. The reduced receptivity of Or67d^GAL4 females was evidently not due to a lower attractiveness of these females to males, because they were courted as vigorously as Or67d⁺ females (Kurtovic, 2007).

These behavioural data imply that cVA is a dual-purpose sex pheromone, acting in males to inhibit mating (Mane, 1983) and in females to promote mating. To test directly whether cVA inhibits male courtship behaviour, and if so whether this requires Or67d, cVA was applied to the abdomens of virgin females and these females were offered to mutant or control males in single-pair courtship assays. Indeed, application of cVA suppressed courtship by Or67d⁺ control males but not by Or67d^GAL4 mutant males. cVA also suppressed courtship by Or67d^GAL4 males carrying the UAS-Or67d transgene. Thus, cVA acts through Or67d to inhibit male courtship behaviour. However, it should be noted that cVA is not the sole mediator of sex discrimination in Drosophila, because Or67d mutant males still courted females much more avidly than they courted other males. This implies the existence of either additional inhibitory cues from the male, stimulatory cues from the female, or both (Kurtovic, 2007).

General odorants are thought to activate many different receptors, with odour identity encoded by the specific combination of receptors that are activated. In this model, no single ORN class encodes a specific odour, and odour perception is thought to arise through spatial integration of ORN signals in higher-order olfactory circuits. In contrast, odours of particular biological significance, such as pheromones, may activate only a single class of ORN, such that this ORN class alone communicates an unambiguous signal to the brain through a dedicated 'labelled line.' This study has shown that Or67d is required for physiological and behavioural responses to cVA, but this does not distinguish between the combinatorial and labelled-line models for signal processing. For this, a method of activating the Or67d ORNs artificially was required. In a combinatorial model, stimulation of Or67d ORNs alone is not predicted to induce a behavioural response, but in the labelled-line model it should (Kurtovic, 2007).

Attempts were made to stimulate Or67d ORNs artificially with a heterologous ligand-receptor pair. For this, the sex pheromones of moths were examined. The female silkmoth Bombyx mori emits the pheromone bombykol ([E,Z]-10,12-hexadecadien-1-ol) to attract and stimulate the male (Butenandt, 1959). To the same effect, the female tobacco budworm Heliothis virescens produces a sex pheromone blend, one component of which is (Z)-11-hexadecenal. The receptor BmOR1 has recently been identified as a receptor for bombykol, on the basis of its ability to confer cellular responses to bombykol in Xenopus oocytes and Drosophila ORNs (Sakurai, 2004; Nakagawa, 2005; Krieger, 2005; Syed, 2006). Similarly, using single-sensillum recordings it was found that replacing Or67d with BmOR1 conferred a new response to bombykol in the Or67d ORNs. The Heliothis receptors HR13-16 have been identified as candidate pheromone receptors (Krieger, 2004), and by the same strategy this study found that HR13 conferred sensitivity to (Z)-11-hexadecenal in the Or67d ORNs. Thus, despite more than 300 million years of evolutionary divergence, these moth pheromone receptors are fully functional when expressed in Drosophila ORNs (Kurtovic, 2007).

To test whether artificial activation of the Or67d ORNs suppressed courtship, bombykol or hexadecenal were applied to virgin females, and these females were offered to naive males expressing either BmOR1 or HR13, respectively, in the Or67d ORNs. Courtship activity of these males was suppressed to a similar degree to that observed when cVA-treated females were offered to wild-type males. In contrast, both Or67d⁺ and Or67d^GAL4 males vigorously courted the bombykol-treated or hexadecenal-treated females, and the receptor replacement males vigorously courted females treated with solvent alone. Thus, courtship suppression is strictly dependent on both the presence of the moth pheromone on the female and the corresponding pheromone receptor in the Or67d ORNs in the males. It is inferred that the activation of the Or67d ORNs alone is sufficient to inhibit male courtship behaviour, in accordance with the labelled-line hypothesis for pheromone detection (Kurtovic, 2007).

Tracing this labelled line into higher olfactory centres should help to reveal how the activation of Or67d ORNs suppresses male mating behaviour, and perhaps also how the same signal might have the opposite effect in females. These neurons connect in the DA1 glomerulus to two distinct classes of second-order projection neurons (PNs): GABAergic vPNs and cholinergic iPNs. Both the DA1 vPNs and iPNs project their axons to a putative sex-pheromone processing centre in the lateral horn of the protocerebrum, specifically targeting two sexually dimorphic regions, one enlarged in males and the other enlarged in females (Jefferis, 2007). It will be interesting to test whether the DA1 PNs make sexually dimorphic patterns of inhibitory or excitatory connections in these regions, and if so, to what extent such dimorphic circuitry might shape distinct male and female responses to the cVA signal (Kurtovic, 2007).

Or67d mediates 11-cis-vaccenyl acetate-induced responses in Drosophila

Insect pheromones elicit stereotypic behaviors that are critical for survival and reproduction. Defining the relevant molecular mechanisms mediating pheromone signaling is an important step to manipulate pheromone-induced behaviors in pathogenic or agriculturally important pests. The only volatile pheromone identified in Drosophila is 11-cis-vaccenyl acetate (VA), a male-specific lipid that mediates aggregation behavior. VA activates a few dozen olfactory neurons located in T1 sensilla on the antenna of both male and female flies. This study identified a neuronal receptor required for VA sensitivity. Two mutants were identified lacking functional T1 sensilla; the expression of the VA receptor is dramatically reduced or eliminated. Importantly, misexpression of this receptor in non-T1 neurons, normally insensitive to VA, confers pheromone sensitivity at physiologic concentrations. Sensitivity of T1 neurons to VA requires Lush, an extracellular odorant-binding protein (OBP76a) present in the sensillum lymph bathing trichoid olfactory neuron dendrites. This study shows that Lush is also required in non-T1 neurons misexpressing the receptor to respond to VA. These data provide new insight into the molecular components and neuronal basis of volatile pheromone perception (Ha, 2006).

With the goal of identifying all genetic loci required for VA pheromone detection, a screen was undertaken to recover mutants with abnormal electrophysiological responses to VA pheromone. Two mutants were identified out of 1200 lines that lack functional T1 sensilla. tod¹ and tot¹ have normal basiconic and non-T1 sensilla, but no T1 sensilla. tod¹ and tot¹ are both recessive and fail to complement one another, revealing these mutants lack T1 sensilla as a result of lesions in independent genes. In normal flies, there is a mixture of T1 and non-T1 subtypes in the trichoid zone, but the proximal part of this zone is enriched in T1 sensilla. Morphologically, all trichoid sensilla are indistinguishable. However, T1 and non-T1 sensilla are clearly distinguishable by electrophysiology. In a survey of over 2000 animals, recordings from random trichoid sensilla in the proximal zone identify VA-sensitive T1 sensilla 89% of the time and non-T1 11% of the time based on spontaneous activity rate and sensitivity to VA. VA could evoke activity in T1 neurons from 0.35 ± 0.14 spikes per second before stimulation to 36.89 ± 3.43 after stimulation. VA did not induce activity in non-T1 neurons. Recordings from either tod¹ mutants or tot¹ mutants from the T1 zone always identified non-T1 sensilla. These sensilla have the classic characteristics of the non-T1 type, including lack of response to VA, more than one neuron present in the sensillum, and a high rate of spontaneous activity. Trichoid and large basiconic sensilla from across the antenna were surveyed in tod¹ mutants and tot¹ mutants, and no VA-responsive neurons could be identified. Therefore, there does not appear to be a simple mislocalization of T1 sensilla to a different part of the antenna, but a complete loss of the T1 functional type (Ha, 2006).

tod¹ and tot¹ mutants lack functional T1 sensilla. Therefore, T1-neuron specific gene products should be absent in these mutants. It was reasoned that a neuronal receptor mediating VA responses might be a member of the odorant receptor family expressed specifically by T1 neurons. Indeed, Ors have been shown to specify odor specificity to olfactory neurons in a number of systems, including Drosophila. Therefore, candidate members of the Drosophila Or gene family were screened for reduced expression in tod¹ and tot¹ mutants. An Or67d spliced transcript was found to be clearly present in wild-type antennas, but is reduced or absent in tod¹ and tot¹ mutants, even after 40 cycles of amplification. Or83b, expressed in most olfactory receptor neurons was present in all three samples, as were all other Or genes tested. These results suggest Or67d expression is specifically reduced or eliminated in tod¹ and tot¹ mutants, and correlates with the loss of the T1 functional class in these genetically distinct mutants (Ha, 2006).

Having identified a candidate receptor correlating with the presence of T1 neurons, attempts were made to establish whether the expression pattern of Or67d in the antenna was consistent with the known T1 neuron distribution. in situ hybridization was performed using fluorescently labeled antisense RNA probes to Or67d to characterize expression of this putative receptor. Antisense probes to Or67d specifically label cells on the ventral–lateral surface of the third antennal segment. Serial sections reveal the labeled cells are concentrated in the proximal T1 zone. These probes failed to identify similar positive cells in antenna tod¹ or tot¹ (3C) consistent with the functional loss of T1 sensilla. Therefore, Or67d expression correlates well with the known distribution of T1 sensilla in wild-type antenna and the absence of T1 sensilla in the mutants (Ha, 2006).

Expression of Or67d in the T1 zone and its absence in tod¹ and tot¹ mutants is consistent with Or67d being the T1 VA receptor, but does not prove this receptor is responsible for VA sensitivity. For example, Or67d may have some functional role specific to T1 neurons that is unrelated to VA sensitivity. Alternatively, a subset of non-T1 class olfactory neurons may also be absent in tod¹ and tot¹ that specifically express Or67d. Therefore, to definitively prove Or67d mediates VA sensitivity, Or67d was misexpressed in olfactory neurons that normally do not express this receptor and are VA insensitive. Previous work has shown that coexpression of an extra odorant receptor in a Drosophila olfactory neuron results in an odor sensitivity profile that is the combination of the sensitivity of the individual receptors. Therefore, Or67d was misexpressed in all neurons by driving Or67d expression with the pan-neuron promoter, ELAV. VA sensitivity of wild-type animals and those misexpressing Or67d in non-T1 sensilla was examined. Neurons in wild-type non-T1 sensilla are insensitive to VA. However, animals misexpressing Or67d in all neurons have non-T1 neurons that are highly responsive to VA. Indeed, dose–response analysis reveals these neurons are nearly as sensitive to VA as wild-type T1 neurons. By all other criteria, these neurons are non-T1 and not T1 neurons. They display high spontaneous activity and contain multiple neurons, and their distribution is typical of the non-T1 functional class. The only difference observed in these neurons compared with wild-type controls was VA sensitivity. Conferring VA sensitivity on non-T1 neurons by expressing Or67d receptors demonstrates that this receptor is both necessary and sufficient to confer VA sensitivity on non-T1 neurons (Ha, 2006).

Lush protein is required in the T1 sensillum lymph for T1 neurons to be sensitive to VA. Non-T1 sensilla also express Lush protein in the sensillum lymph. Is Lush also required for sensitivity of non-T1 neurons misexpressing Or67d? The lush¹ mutation was crossed into the stock misexpressing Or67d in all neurons. Lush protein is critical for non-T1 neurons to respond to VA as well, because when the lush¹ mutation is crossed into the misexpressing flies, VA sensitivity is lost in non-T1 neurons. This clearly demonstrates that Lush is required in the extracellular space in order for non-T1 neurons misexpressing Or67d to be responsive to VA. Therefore, both the receptor Or67d and a specific extracellular binding protein, Lush, are required for VA sensitivity (Ha, 2006).

Receptors for mate recognition in Drosophila; Or65a and Or67d detect male-specific pheromones

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila. The trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, were systematically examined by electrophysiological analysis. None respond strongly to food odors but all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone transferred from males to females during mating. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin-female extract as well. Thus, males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. All members of the Odor receptor (Or) gene family that are expressed in trichoid sensilla were then systematically tested by using an in vivo expression system. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. A model is proposed describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic (van der Goes van Naters, 2007).

The responses of ORNs in trichoid sensilla of the antenna were measured by single-unit electrophysiology. All three trichoid-sensilla subtypes, T1, T2, and T3, which contain one, two, and three ORNs, respectively, were tested. These three subtypes occupy distinct but overlapping regions of the antennal surface and together comprise more than 20% of the sensilla in the antennae. Initially, 86 compounds were tested, most of which are found in fruits or are fermentation products. These compounds were tested on 60 trichoid sensilla, 30 from males and 30 from females. The compounds were tested in mixtures, and no mixture elicited a response greater than 20 impulses/s, which represents less than 10% of the maximal response of these ORNs. Some mixtures inhibited the spontaneous activity of T2 and T3 sensilla and produced decreases of 10-20 impulses/s in the action-potential rate. The three most inhibitory odors were subsequently determined to be 1-hexanol, hexyl acetate, and butyl acetate. The paucity of strong excitatory responses to food odors is consistent with the results of an earlier screen with a limited number of chemicals; in this earlier screen, no strong responses were found, although modest responses were elicited by trans-2-hexenal and cis-vaccenyl acetate (cVA) (van der Goes van Naters, 2007).

The odor of live flies was tested. 50 flies were placed in a glass tube that was closed at both ends with a cotton mesh. Air was puffed through the tube toward the antenna of a fly mounted for electrophysiological recording. 75 individual trichoid sensilla, of all three subtypes, were tested for responses to the odors of both males and virgin females. Air passing over male flies elicited a strong response from ORNs in a large group of trichoid sensilla. These ORNs did not respond to the odor of virgin females. These sensilla correspond to the T1 subtype, each of which houses a single ORN. T1 sensilla are found on both male and female antennae; in both cases they respond to the odor of males but not of virgin females. The T2 and T3 sensilla did not produce responses to fly odors when they were tested in this paradigm (van der Goes van Naters, 2007).

These experiments showed that at least some trichoid sensilla respond to fly odors. However, whether other trichoid sensilla might show responses to fly odors was tested in a more sensitive assay. A new paradigm was developed. Because flies approach each other closely during courtship, it was reasoned that some pheromone-sensitive sensilla might be adapted for short-range information reception. Some of the chemical cues that influence courtship behavior in Drosophila are present in the cuticle, i.e., on the surface of the fly, and are long-chain unsaturated hydrocarbons of very limited volatility. Although some of these cues are believed to be detected via the taste system, it seemed possible that the olfactory system might also contribute to the reception of cuticular components at very close range during courtship (van der Goes van Naters, 2007).

Accordingly, rather than adding odor stimuli to an air stream directed at the fly from a distance, stimuli were presented by approaching the antenna with the tip of a glass capillary carrying the odor. This procedure was designed to simulate the proximity of two interacting flies. As an initial test of the feasibility of this paradigm, 500 pl of a solution of cVA was draw into the capillary. cVA has been shown to act as an anti-aphrodisiac pheromone in Drosophila; there is also evidence for its playing a role as an aggregation pheromone. As the capillary tip approached certain trichoid sensilla, the impulse rates of certain ORNs increased and reached a maximum of >200 impulses/s upon physical contact of the capillary tip with the sensillum shaft. Control stimuli prepared with the hexane solvent alone gave no response (van der Goes van Naters, 2007).

Having established a short-range delivery paradigm, the responses, initially to cVA, of trichoid sensilla were systematically examined across the entire antennal surface. Mature male flies contain approximately 1 μg of cVA, primarily in the ejaculatory bulb. A capillary tip was loaded with 5 ng of cVA (0.005 fly equivalent) and 189 trichoid sensilla were approached individually. Strong responses of >100 impulses/s in were observed 169 of the 189 sensilla. Previous reports had shown that the ORN in T1 sensilla responds to cVA, and this study confirmed this finding. Responses to 5 ng of cVA exceeded 200 impulses/s in T1 sensilla. Also in agreement with the previous reports, some sensilla immediately adjacent to the zone containing T1 did not respond to cVA. However, it was determined that, in addition to the T1 subtype, a large number of sensilla more distolateral on the antennal surface also contained ORNs that are sensitive to cVA in this paradigm. Neurons in the distolateral sensilla responded to the cVA stimulus with a rate increase of more than 100 impulses/s. Thus, there appear to be at least two populations of sensilla with ORNs that respond to this pheromone (van der Goes van Naters, 2007).

To expand the scope of this analysis from a single defined pheromone, cVA, to a broad representation of the cuticular pheromone profile, hexane extracts of males and virgin females were prepared. Approximately 500 pl of extract was drawn into the capillary tip; this amount is equal to 0.25% of the material extracted from a single fly (van der Goes van Naters, 2007).

When a male extract was used as the odor source, all 147 trichoid sensilla tested, from all regions of the antennal surface, yielded responses. Different ORNs began to respond to the approaching odor source at different distances. The T1 sensilla, which house a single ORN, appeared to be particularly sensitive; they showed responses greater than 20 impulses/s when the odor source came within a 1 cm radius. As the odor source became still closer, the impulse rates increased rapidly. ORNs in T2 and T3 sensilla appeared to be less sensitive and had impulse rates increasing only after the odor source approached a distance of 200 μm, as determined with an ocular micrometer. The responses were dose dependent; when the dose was increased from 0.25% fly equivalent to 5% fly equivalent, the response radius increased from 200 μm to 500 μm (van der Goes van Naters, 2007).

When an extract from virgin females was used as the stimulus, strong responses were observed in ORNs of all trichoid sensilla except T1. Thus, T1 sensilla appear to be tuned to male odor, whereas T2 and T3 sensilla yield strong responses to both males and virgin females. Sensitivity to male and virgin-female extracts was comparable in T2 and T3 sensilla. These in vivo recordings, taken together, demonstrate that trichoid sensilla respond to fly odors and that the odors of males and virgin females are registered differently across the ensemble of trichoid sensilla. A limitation of the analysis is that it is difficult to ascribe responses to individual ORNs within trichoid sensilla. With the exception of T1, trichoid sensilla contain multiple ORNs. In recordings, this is evident from summation and cancellation events between impulses in the traces. In most cases it was not possible to discriminate the activities of the individual ORNs because the action potentials, as recorded extracellularly, did not differ significantly in size or shape. Because of the inability to classify action potentials with confidence, it was not possible to determine whether there is a functional subdivision among the ORNs sharing a sensillum. To address this limitation, advantage was taken of another experimental system, the 'empty neuron' system, in an effort to analyze the responses of trichoid sensilla at a higher resolution (van der Goes van Naters, 2007).

Drosophila contains a family of 60 Or (Odor receptor) genes, and the following 12 family members have been reported to map to individual ORNs of trichoid sensilla: Or2a, Or19a, Or19b, Or23a, Or43a, Or47b, Or65a, Or65b, Or65c, Or67d, Or83c, and Or88a. Each of these 12 Or genes were expressed in the 'empty neuron' system, an in vivo expression system based on a mutant ORN, ab3A, that resides in a basiconic sensillum. The endogenous receptor genes of this ORN, Or22a and Or22b, are deleted, and the promoter of Or22a drives ectopic expression of another odor receptor in ab3A via the UAS-GAL4 system. The odor responses conferred upon ab3A by the ectopically expressed receptor are then measured by single-unit electrophysiology (van der Goes van Naters, 2007).

The 12 trichoid receptors were systematically tested in the empty-neuron system with a panel of fly-derived chemicals: hexane extracts of males and virgin females, material from the genital regions of flies (males, virgin females, and mated females), and cVA. The genital odors were obtained by drawing a glass capillary, with a tip pulled to a diameter of 3 μm, across the genital region of a fly such that material visibly coated the tip. Preliminary experiments showed that the responses could be quantified most reproducibly not during the approach of a stimulus to the antenna but after the capillary tip contacted the sensillum. Responses mediated by the trichoid receptors were were therefore quantified by determining impulse rates of the ORN after contact. The 12 receptors were expressed and tested in both male and female recipients with all six stimuli, and no differences between the responses of male and female flies were identified (van der Goes van Naters, 2007).

Of the 12 receptors, four mediated responses to fly odors in this system. All four, Or47b, Or65a, Or67d, and Or88a, responded to male extract, and their action-potential frequencies increased by 50-200 impulses/s. Two of these receptors, Or65a and Or67d, did not respond to extract from virgin females. The sex specificity of Or65a and Or67d is consistent with a role for these receptors in the detection of male-specific pheromones. The other two receptors, Or47b and Or88a, responded to extract from virgin females; these responses were comparable to those they gave to male extracts. It was noted that both Or47b and Or88a were previously tested in the empty-neuron system with a panel of 110 odors, most of which were present in fruits and were of widely varying chemical structures, and no excitatory responses were recorded. These results are consistent with the hypothesis that Or47b and Or88a detect a pheromone secreted by both males and females (van der Goes van Naters, 2007).

Male genital material elicited strong responses from Or65a, Or67d, and Or88a. Genital material from virgin females did not elicit a strong response from any of the 12 receptors. However, material from the genital region of females that were mated 1-4 hr previously produced responses from these three receptors, which, yielded firing rates comparable to those observed with male genital material. These results suggest that during copulation the male transfers compounds that activate these receptors (van der Goes van Naters, 2007).

One compound that the male transfers to the female during copulation is cVA. The sensitivity of Or67d to cVA is consistent with previous observations; expression studies have shown that Or67d is expressed in T1 sensilla, which are sensitive to cVA, and ectopic expression of Or67d in other trichoid sensilla conferred sensitivity to cVA. However, the results indicate that there are multiple receptors for cVA. Both Or67d and Or65a responded most strongly to cVA among a panel of six related compounds. The two receptors differed in their specificities, however; Or67d gave a relatively stronger response than did Or65a to cis-vaccenyl alcohol, for example. It is noted that the detection of a second cVA receptor, which has not been reported previously, may reflect the sensitivity of the short-range delivery paradigm that was designed (van der Goes van Naters, 2007).

The response specificity of Or67d, as measured in the empty-neuron system, is nearly identical to that of the ORN in the T1 sensillum. However, it is noted that the magnitude of the response to cVA in the expression system is approximately half that in T1. Dose-response curves show that the response threshold is also lower in the native T1 sensillum; it appears as though the T1 neuron can detect a dose of approximately 10⁻⁴ ng, whereas the expressed Or67d receptor may require a dose of approximately 10⁻² ng for detection. Slower rise and decay rates were also found, along with higher levels of spontaneous firing in the expression system. These results suggest that the expression system may lack a component that is present in the endogenous context; for example, the odorant-binding protein Lush was found to be required for normal response to cVA in T1 sensilla (van der Goes van Naters, 2007).

Whereas Or67d mediates responses to cVA in T1 sensilla, Or65a is expressed in the ORNs of trichoid sensilla that are more distolateral on the antenna and that also respond to cVA. It is noted that the Or65a gene is in close proximity to Or65b and Or65c and that the three genes are coexpressed in a single ORN. Although neither Or65b nor Or65c mediated responses to any of the fly odors tested in the empty-neuron system, the possibility was considered that they might contribute to the response of the ORN if they were coexpressed with Or65a, perhaps via heterodimer formation. Accordingly, all pairwise combinations of the three receptor genes were co-expressed. It was found that coexpression of Or65b or Or65c with Or65a did not increase the response mediated by Or65a to any stimulus or change the level of spontaneous activity. Coexpression of Or65b and Or65c yielded little, if any, response to any stimulus (van der Goes van Naters, 2007).

Finally, it is noted with interest that although Or88a conferred responses to male genital material, it did not mediate responses to cVA, suggesting that it detects an additional pheromone that is also transferred from males to females upon mating (van der Goes van Naters, 2007).

This study has identified four receptors that mediate responses to fly odors. Or47b and Or88a mediate responses to the odors of both males and virgin females. Or65a and Or67d mediate responses to cVA, a male-specific lipid that is present in male genital material, is presumably extracted in hexane extracts, and is transferred to females upon mating. Or88a also responds to a compound in male genitalia, but this compound is distinct from cVA (van der Goes van Naters, 2007).

The responses of these receptors suggest a working model of the olfactory basis of mate recognition by males. In this model, neural activity mediated by Or47b and Or88a reports the proximity of a fly, either male or female. This olfactory recognition may contribute to the recognition mediated by other sensory modalities; recognition of conspecifics is a prerequisite to successful courtship. The activity of Or65a, Or67d, or both would indicate that the partner is a male or a recently mated female; thus, when the antenna of a male is in close proximity to another fly, the activation of Or65a and/or Or67d would report that the other fly is unsuitable as a mate. The lack of a signal from these receptors would permit continued courtship activity by the male (van der Goes van Naters, 2007).

A well-documented phenomenon can be interpreted in terms of this model. Mature males not only court virgin females but also vigorously court newly eclosed males. Young males, like virgin females, lack cVA and would not be expected to activate Or65a and Or67d, allowing courtship to proceed (van der Goes van Naters, 2007).

Why would Or65a and Or67d not be activated in the antenna of a male by material in its own genital region? Perhaps very little of the internal genital material is released to the air unless the region is manipulated by a capillary tip or washed in hexane, and perhaps what little is released under natural conditions can normally be detected only at very close range; if cVA were released in large amounts and inhibited mating over a long range, then mating might be inhibited at sites where flies congregate and often mate, such as rich food sources. It is also possible that the fly adapts to the ambient level of cVA, produced by its own genital region, and is sensitive to increases above that level (van der Goes van Naters, 2007).

Why are there two cVA receptors, expressed in two distinct ORNs, in different subtypes of trichoid sensilla? There is evidence that cVA serves two functions as a pheromone in Drosophila. (1) cVA has been shown to act as an anti-aphrodisiac, detering males from courting with a recently mated female. (2) cVA is deposited by females during egg laying, and there is evidence that it enhances the attractiveness of the oviposition substrate to other flies. Perhaps Or65a and Or67d activate two distinct behavioral circuits and thereby separately mediate two functions of cVA in conjunction with other cues (van der Goes van Naters, 2007).

Interestingly, no receptor for female-specific odors was identifed, although there is evidence that 7,11-heptacosadiene and 7,11-nonacosadiene, two female-specific hydrocarbons, act as aphrodisiacs. It is possible that some of the trichoid receptors respond to these compounds, which were not tested individually, or other female-specific compounds but do not function efficiently in the expression system. It is also possible that these compounds are detected by gustatory receptors, perhaps members of the Gr family. One class of gustatory neuron, which expresses Gr68a, has been shown to be required for normal courtship. Finally, the possibility is noted that some of the receptors that did not respond to the tested stimuli might detect pheromones of other Drosophila species (van der Goes van Naters, 2007).

It is striking that no differences were observed between males' and females' antennal responses to any of the fly odors tested. This similarity is in stark contrast to the extreme sexual dimorphism in antennal responses to pheromones in moths, such as Bombyx mori and Manduca sexta. The similarity in Drosophila peripheral olfactory responses suggests that in the fly, differences in male and female behavioral responses may be determined by differences in reception of other classes of sensory input, such as taste information, or by differences in the transmission or processing of olfactory information. It is possible that cVA, for example, is sensed through the same peripheral mechanisms in males and females but that only in males is the primary representation transformed in a way that accords it a negative valence (van der Goes van Naters, 2007).

In summary, a systematic analysis was carried out of the trichoid sensilla, one of the three major types of sensilla on the Drosophila antenna. These sensilla appear to be specialized for sensing fly odors, as opposed to food odors. The differential activity of ORNs in trichoid sensilla provides an olfactory basis for a male's ability to discriminate suitable from unsuitable mating partners. The molecular basis of these responses was further explored and four odor receptors were identified that mediate responses to fly odors. A model is proposed in which olfactory information flows through these receptors according to a simple logic. Although the full repertoire of pheromones and receptors has yet to be characterized, it is possible that the model may be richly elaborated without undergoing an alteration in its fundamental logic (van der Goes van Naters, 2007).

An essential role for a CD36-related receptor in pheromone detection in Drosophila; SNMP is required for responses of OR67d to cVA when ectopically expressed in OSNs

The CD36 family of transmembrane receptors is present across metazoans and has been implicated biochemically in lipid binding and transport. Several CD36 proteins function in the immune system as scavenger receptors for bacterial pathogens and seem to act as cofactors for Toll-like receptors by facilitating recognition of bacterially derived lipids (Hoebe, 2005; Philips, 2005; Stuart, 2005). A Drosophila CD36 homologue, Sensory neuron membrane protein (SNMP), is expressed in a population of olfactory sensory neurons (OSNs) implicated in pheromone detection. SNMP is essential for the electrophysiological responses of OSNs expressing the receptor OR67d to (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA), a volatile male-specific fatty-acid-derived pheromone that regulates sexual and social aggregation behaviours. SNMP is also required for the activation of the moth pheromone receptor HR13 by its lipid-derived pheromone ligand (Z)-11-hexadecenal9, but is dispensable for the responses of the conventional odorant receptor OR22a to its short hydrocarbon fruit ester ligands. SNMP is required for responses of OR67d to cVA when ectopically expressed in OSNs not normally activated by pheromones. Because mammalian CD36 binds fatty acids, it is suggested that SNMP acts in concert with odorant receptors to capture pheromone molecules on the surface of olfactory dendrites. This work identifies an unanticipated cofactor for odorant receptors that is likely to have a widespread role in insect pheromone detection. Moreover, these results define a unifying model for CD36 function, coupling recognition of lipid-based extracellular ligands to signalling receptors in both pheromonal communication and pathogen recognition through the innate immune system (Benton, 2007).

Insect odorant receptors represent a novel class of polytopic membrane proteins unrelated to vertebrate G-protein-coupled chemosensory receptors. The functional insect odorant receptor is a heteromer of a ligand-binding subunit and the highly conserved OR83b co-receptor, which mediates transport to sensory cilia. Little is known about how this complex recognizes odours and evokes neuronal depolarization. To isolate novel components involved in insect olfactory detection, a bioinformatic approach was used to identify molecules that exhibit the same insect-specific orthology and olfactory-specific tissue expression as these receptors. Two-thousand one-hundred and thirty-five Drosophila genes with insect-specific orthologs were identified by comparing the fruit fly (Drosophila melanogaster), mosquito (Anopheles gambiae) and eight non-insect genomes using the OrthoMCL algorithm. Broadly expressed genes were excluded by selecting only the 616 genes with fewer than two expressed sequence tags. All classes of known insect chemosensory genes were recovered, including odorant receptors, gustatory receptors, odorant and other chemosensory binding proteins, and putative odour-degrading enzymes. The remaining genes were classified on the basis of predicted protein domains and included many implicated in immunity and defence (Benton, 2007).

Three-hundred and thirty-nine uncharacterized genes were screened for selective expression in the antenna (the major olfactory organ of Drosophila) by reverse transcriptase-polymerase chain reaction (RT-PCR). Of these, focus was placed on Snmp, an antennal-enriched gene related to the CD36 receptor family. The Anopheles homologue of Snmp was also antennal-specific, consistent with the previously described olfactory-specific expression pattern of the silk moth (Antheraea polyphemus) homologue Snmp-1. SNMPs form an insect-specific sub-group of the CD36 family, explaining how Drosophila Snmp emerged from the bioinformatic screen (Benton, 2007).

In the antenna, Snmp was found prominently expressed in a lateral-distal population of OSNs that co-express Or83b, in non-neuronal support cells that surround these OSNs, and in support cells elsewhere in the antenna and chemosensory organs on the proboscis. Genetic labelling of SNMP-expressing OSNs with mouse CD8 fused to green fluorescent protein (CD8-GFP) revealed that these neurons target nine glomeruli in the antennal lobe -- DA1, VA1d, VA1l/m, DL3, DA4m, DA4l, DA2, DC3 and DC1 -- corresponding to those innervated by OSNs of the trichoid sensilla, which are involved in pheromone detection (Benton, 2007).

Using a peptide antibody, SNMP was found concentrated in trichoid sensory cilia, where it co-localized with OR83b, but only at very low levels in the cell bodies and axons, similar to moth SNMP-1. No SNMP was observed in non-trichoid OSNs, but it was expressed in support cells throughout the antenna. All anti-SNMP immunoreactivity was abolished in an snmp-null mutant, confirming antibody specificity. Although the localization of SNMP in OSN cilia was similar to that of odorant receptors, it did not depend on OR83b when a functional SNMP-GFP fusion protein was expressed in OSNs innervating basiconic sensilla. Therefore, SNMP ciliary trafficking is independent of both specific ligand-binding odorant receptors and OR83b. Whether SNMP might still contact odorant receptors in trichoid cilia was examined by using the fluorescent protein fragment complementation assay. SNMP and OR83b bearing complementary fragments of a yellow fluorescent protein (YFP) reporter were generated and functionally verified. Reconstitution of the fluorescent YFP signal in sensory cilia was observed only when both fusion proteins were expressed. As the YFP fragments do not self-associate, this reconstitution could only result if SNMP and OR83b were brought into close proximity (<80 Å), providing evidence that SNMP is closely apposed to, although not necessarily directly interacting with, odorant receptors in the sensory compartment (Benton, 2007).

Snmp null mutants were generated by gene targeting. snmp mutants are viable and fertile with no gross morphological or locomotor defects. The function of SNMP was examined in the sub-population of trichoid sensilla innervated by neurons expressing OR67d -- the best-characterized Drosophila pheromone receptor that recognizes cVA. In snmp mutants, neither the expression of Or67d nor the ciliary localization of GFP-OR67d or OR83b was affected and axonal projections of snmp mutant OR67d-expressing neurons to the antennal lobe were wild type. The expression of Lush, an odorant-binding protein secreted by trichoid sensilla support cells into the lymph was normal. Thus Snmp is dispensable for the development of trichoid OSNs and support cells (Benton, 2007).

Whether the responses of OR67d neurons to cVA stimulation were altered in snmp mutants was tested. The relatively low spontaneous activity of the OR67d neuron was observable as a sparse distribution of action potentials of uniform amplitude. On stimulation with cVA, wild-type neurons responded with a robust train of action potentials in a dose-dependent manner. snmp mutant neurons displayed no cVA-evoked electrophysiological responses at any concentration tested, but showed an increase in spontaneous activity. Both spontaneous and stimulus-evoked responses were fully restored by expression of the Snmp rescuing transgene in OR67d-expressing neurons, but not by expression in support cells surrounding these neurons. Expression of a distinct Drosophila CD36-related protein, NINAD, in OR67d-expressing neurons did not rescue electrophysiological defects of snmp mutants. Thus, SNMP has an essential, cell-autonomous and specific function in OR67d-expressing neurons in mediating responses to cVA (Benton, 2007).

cVA detection is also dependent on Lush and the OR67d/OR83b heteromeric receptor complex, suggesting that SNMP acts with these proteins in a signalling pathway. In contrast to snmp mutants, however, loss of lush, Or67d or Or83b severely decreased spontaneous activity of these neurons. Double-mutant analysis of this spontaneous activity phenotype revealed that Snmp is epistatic to lush, because OR67d-expressing neurons retained high levels of spontaneous activity in animals lacking both SNMP and Lush. In contrast, snmp Or83b double mutants were, like Or83b, electrically silent. Although the mechanism by which spontaneous activity is regulated in Drosophila OSNs is unknown, genetic analysis indicates that SNMP may act downstream of Lush and upstream of, or in parallel with, odorant receptors in the generation of action potentials (Benton, 2007).

To investigate the specificity of SNMP function, a second receptor, OR22a, which is responsive to fruit esters, such as ethyl butyrate and pentyl acetate, was ectopically expressed in OR67d neurons. Although chemically related to cVA, OR22a ligands lack the long hydrophobic tail of this fatty-acid-derived pheromone. Ectopic expression of OR22a in wild-type OR67d-expressing neurons conferred responses to a panel of known OR22a ligands in addition to the endogenous cVA response, but not to a control odour, geranyl acetate, which activates neither OR67d nor OR22a. In snmp mutants, ectopic OR22a-dependent responses were unaffected, but all cVA responses were lost. The broad expression of SNMP in trichoid OSNs indicates that it might have a general function in pheromone detection. Because no other volatile pheromones have been identified in Drosophila, whether SNMP is required for the activation of the moth (Heliothis virescens) pheromone receptor HR13 by (Z)-11-hexadecenal, a component of the sex pheromone blend of this species, was tested. As previously observed, expression of HR13 in OR67d-expressing neurons conferred responsiveness to this pheromone (Kurtovic, 2007). This response was almost completely abolished in snmp mutants and restored by transgenic rescue of Snmp. Together, these experiments reveal a specific and conserved function for SNMP in mediating pheromone-evoked neuronal activity. OR67d and HR13 share <15% amino acid identity and their ligands have chemically distinct head groups, suggesting that it is the fatty-acid-derived hydrocarbon tail common to these pheromones that necessitates SNMP (Benton, 2007).

Finally, it was asked whether SNMP is required for the activation of OR67d by cVA in neurons not normally responsive to pheromones. OR67d was ectopically expressed in basiconic OSNs that lack the endogenous OR22a ligand-binding odorant receptor, but retain OR83b. All action potentials in these neurons can therefore be ascribed to OR67d/OR83b activity. Or22a mutant neurons expressing OR67d without SNMP exhibited spontaneous firing, but did not respond to cVA. In contrast, when OR67d was co-expressed with SNMP, significant responses to this pheromone were observed; compared to the responses of native OR67d neurons, the frequency of action potentials was lower and exhibited slower rise and decay rates. Such differences may be due to the absence in basiconic sensilla of Lush or odour-degrading enzymes specialized to inactivate pheromone molecules (Benton, 2007).

In summary, through a bioinformatic screen for insect olfactory transduction molecules, Drosophila SNMP was identified as a CD36-related receptor broadly expressed in pheromone-sensing neurons: SNMP is an essential co-factor for detection of the fatty-acid-derived pheromone cVA. Since mammalian CD36 has an important biochemical function in binding and membrane translocation of fatty acids it is suggested that SNMP directly captures pheromone molecules on the surface of OSN cilia -- possibly retrieving them from odorant-binding proteins in the extracellular milieu -- and facilitates their transfer to the odorant-receptor-OR83b complex. OR67d ectopically expressed without SNMP can be activated by cVA when the pheromone was directly applied to the sensillar cuticle overlying the OSN, indicating that pheromone receptors can be directly stimulated by ligand. When pheromones are presented in an air stream to the receptor in its native environment, however, SNMP (and odorant-binding proteins) are essential. It is suggested that the combination of molecular specializations of pheromone-sensing trichoid neurons together contribute to the sensitivity of these cells and that SNMP-related proteins function in the detection of many insect pheromones (Benton, 2007).

The mechanistic basis of CD36 ligand interactions and signalling is still poorly understood in any biological system. These results have three important general implications: (1) SNMP has a specific role in the detection of fatty-acid-derived odour ligands. Because other CD36-related receptors are involved in binding and transport of lipid-based molecules, for example in the mammalian intestine (Ge, 2005), this protein family may represent specialized receptors for extracellular fatty ligands of diverse biological origin and function. (2) It was shown that SNMP acts in concert with other transmembrane odorant receptors in OSN cilia in mediating pheromone-evoked activity. Because CD36 was previously shown to act as a co-receptor for Toll-like receptors, it is suggested that CD36-related proteins have obligate transmembrane partners in all their cellular roles (Benton, 2007).

(3) These results reveal a molecular parallel in the mechanisms of intraspecific recognition through pheromone detection and pathogen recognition through the innate immune system. CD36 proteins in both invertebrates and vertebrates have been implicated in the recognition of specific lipid-derived products from bacterial cell walls, and coupling of this recognition through Toll-like receptors to initiate the innate immune response. Notably, mammalian CD36 has been proposed as a candidate fat taste receptor (Laugerette, 2005). Common molecular recognition mechanisms in immune and chemosensory systems may therefore be widespread (Benton, 2007).

Or67d is not required for the courtship inhibitory role of cis-vaccenyl acetate (cVA), transferred from males to females during mating. This function appears to be served by Or65a: Or67d is an 'appetitive' cVA receptor while Or65a is an 'aversive' receptor

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. This study shows that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males (Ejima, 2007).

In Drosophila, unsuccessful courtship decreases subsequent courtship. When the initial courtship object (trainer) is a virgin female, suppression has been shown to be the result of formation of an associative memory linking the failure to copulate with volatile stimulatory courtship cues specific to the age of the female trainer. Exposure to a mated female, on the other hand, results in a suppression of courtship toward all types of females and is believed to require an aversive pheromone. The cuticular hydrocarbon profiles of mature and immature females differ significantly, but these types of females also differ behaviorally. Mature virgins are receptive to courtship, while immature virgins and mated females show characteristic rejection behaviors. Immature females kick, fend, and run away, while mated females extrude their ovipositors. To determine whether female behavior or appearance had any role in the development of age-specific or general courtship suppression, males were trained and tested with decapitated females in dim red light. Memory index was expressed as a ratio of the courtship index (CI) during the 10 min test period to the mean CI of a sham-trained males tested with the same type of female. The use of a ratio allows direct comparison of the strength of memory between conditions, with a value of CI_test/mCI_sham = 1 indicating no memory (Ejima, 2007).

Consistent with results with mobile trainer females, decapitated virgins provoke an age-specific suppression, while decapitated mated female trainers cause general suppression of courtship. These data indicate that the specificity of learning with different trainer types does not stem from behavioral differences in the trainer female's response to courtship or from visual cues specific to the trainer type. Generalization of learning with a mated female trainer is therefore the result of chemosensory cues. In all subsequent experiments, decapitated trainers and testers were used, except where noted (Ejima, 2007).

In the previous experiment, males were placed in the same chamber as the trainer female and therefore could obtain both olfactory and gustatory information about that female. To investigate the nature of the generalization cue, attempts were made to reconstitute generalized learning with virgin trainers and mated female extracts. Placing a filter containing a hexane extract of mated female in the chamber with either a mature or immature trainer female caused a generalization of learning, as demonstrated by the ability of mature trainers to generate memory against immature testers and vice versa. To determine whether the active component of the mated female bouquet was volatile, a two-compartment courtship chamber was used, and a pheromone source (fly corpse or filter with extract) was placed across a mesh from the side of the chamber containing the male and the trainer female. For both mature and immature trainers, the presence of volatile compounds from either a mated female or a male was sufficient to cause generalization of courtship suppression, although the effects of these pheromones appeared more potent with mature trainers. In the absence of a courtship object, the presence of a filter with extract or a corpse did not generate suppression of courtship toward tester females (Ejima, 2007).

Next the identity of the generalization cue was addressed. The mated female and mature virgin trainers that were used were of the same age (4-5 days old) and might be expected to have similar cuticular hydrocarbon profiles, so any compound that differed between these two classes of females might have a role in generalization. Hexane washes of 4- to 5-day-old virgins and 4- to 5-day-old mated females that had been mated 24 hr before extraction were compared by using gas chromatography-flame ionization detection and mass spectrometry. Qualitatively, the two types of females appear identical with the exception of one peak, cis-vaccenyl acetate (cVA), which is undetectable in virgins but present at significant levels in mated females. cVA is a major component of mature male cuticular hydrocarbon and is not synthesized by females. Its presence in both males and mated females makes it a good candidate for being the generalization cue for courtship learning (Ejima, 2007).

Quantitation of mature virgin and mated female hydrocarbon levels shows a significant difference in cVA levels. There is also a small, but statistically insignificant, increase in 7-tricosene (7-C23:1). 7-tricosene is a major component of the male cuticular hydrocarbon and is believed to have inhibitory effects on male-male courtship. Transfer of 7-tricosene to females has been shown to occur via cuticular contact during copulation, but it is largely gone by 8 hr after mating. Consistent with this, larger amounts of 7-tricosene are seen on virgins that have been courted, but not copulated, when they are extracted immediately after the courtship. Mature virgins and mated females that have been aged 24 hr after copulation have lower and statistically indistinguishable levels of 7-tricosene. The loss over time (presumably through passive transfer and grooming) of 7-tricosene and the nonvolatile nature of this compound make it an unlikely candidate for the generalizing cue. It is also significant to note that no decreases in mated females are seen of hydrocarbons such as 7,11-heptacosadiene (7,11-nC27:2), 7,11-nonacosadiene (7,11-nC29:2), and 9-pentacosene (9-C25:1) that are believed to be stimulatory pheromones for courtship conditioning. Thus, the only consistent mated female-specific difference in hydrocarbon content that was found was in cVA (Ejima, 2007).

How does cVA, a male lipid, become part of the mated female pheromonal profile? Like 7-tricosene, cVA could be transferred directly by contact during courtship and/or copulation. Alternatively, the presence of cVA in the male ejaculatory bulb suggests that it can be transferred with sperm during copulation. To determine the major mode of cVA transmission, cVA levels were measured on virgin females, virgin females that were courted in a small chamber and extracted immediately, and females that were extracted 24 hr after complete copulation or disrupted copulation. Only females that copulated long enough to receive ejaculate have significant levels of cVA. Females that did not copulate and were merely courted by the male had virtually no cVA, even though they had significant amounts of passively acquired 7-tricosene. This suggests that transfer of cVA occurs via ejaculate and that mated females store cVA (Ejima, 2007).

These data support a role for cVA as a generalizing cue, but the presence of other volatile compounds in mated female and male extracts might still be required. To test the sufficiency of cVA, varying amounts of purified cVA were applied to filters across the mesh in a two compartment courtship chamber, males were trained with either mature or immature virgins, and then tested with a virgin of the other age. In both cases, cVA was sufficient to generalize memory. With mature virgin testers, 0.2 ng of cVA was enough to generalize memory. The average amount of cVA present on a mated female 24 hr after mating is 9.3 ± 3.5 ng, so the effects of synthetic cVA are occurring in the biologically relevant dose range. In contrast to results with mature virgins, pairing cVA with immature trainers is less effective. Only large amounts of cVA (200 μg) produce generalized learning. cVA alone (with no trainer female) is ineffective, as is cis-vaccenol (cVOH), a putative metabolite of cVA with either trainer type (Ejima, 2007).

The circuitry underlying generalization is of great interest for understanding this behavior. As a first step, attempts were made to identify the olfactory receptor neurons that carry the aversive cVA signal. cVA has been shown to be sensed by a subset of trichoid sensilla in the Drosophila antenna, which includes the T1 type sensillum that expresses Or67d. By using the 'empty neuron' preparation, which allows the decoding of odor specificity for Drosophila olfactory receptors (ORs), it was found that there is an additional cVA-responsive receptor, Or65a, and that Or65a and Or67d differ in their response to cVOH, with Or67d responding strongly and Or65a not responding. Or65a is one of the several ORs expressed in neurons of the T3 sensilla (Ejima, 2007).

With this information, the role was investigated of the olfactory receptor neurons that express these two receptors in sensing the aversiveness of cVA. Initial courtship levels provide a simple assay for this property of cVA. Naive males show lower levels of courtship toward mated females than toward virgins of the same age. This effect can be reproduced by addition of a cVA-laced filter across the mesh in the two-compartment courtship chamber with a mature virgin courtship object in the upper chamber with the male. Expression of tetanus toxin (TNT), which blocks synaptic release, under control of Or65a-GAL4, but not Or67d-GAL4, abolished the ability of cVA to inhibit initial courtship. Males heterozygous for Or65a-GAL4 or the UAS-TNT transgene showed cVA-dependent courtship suppression as did males expressing inactive toxin (TNT-VA) under control of Or65a-GAL4. These results indicated that ORNs expressing Or65a-GAL4, but not Or67d-GAL4, are required for sensing cVA as an aversive cue (Ejima, 2007).

Or65a has been reported to be expressed solely in ORNs that innervated the DL3 glomerulus of the antennal lobe with anti-GFP immunohistochemistry in animals expressing GFP under control of Or65a-GAL4 promoter fusions (Couto, 2005; Fishilevich; 2005). Or65a-GAL4, while it has strong expression in DL3, shows a somewhat broader pattern, with significant expression in VA1v, DC1, and DA4m. By using confocal microscopy to directly visualize GFP from a UAS-mCD8-GFP transgene in unfixed brains, Or65a-GAL4 line was compared to other published Or65a-GAL4 lines. The Or65a-GAL4 line used in this study was many times stronger than that published by Fishilevich (2005), which has predominant expression in DL3. GFP fluorescence in the Couto (2005) GAL4 line was barely detectable. To determine whether the weak, but more DL3-specific, Fishilevich driver would also block cVA effects, it was used to express active and inactive tetanus toxin. Consistent with the results with the Or65a-GAL4 used in this study, active tetanus toxin significantly abrogated the ability of cVA to suppress initial courtship, although the effect appeared weaker than with the line used in this study. Inactive tetanus toxin had no effect on cVA-mediated suppression. It is concluded that the aversive effects of cVA on initial courtship are most likely mediated by ORNs expressing Or65a (Ejima, 2007).

Since its identification as a male-specific lipid in the Drosophila ejaculatory bulb, there has been interest in cVA as a potential modifier of reproductive behavior. In this study, it has been shown pairing of cVA and a virgin female trainer is sufficient to reproduce the unique effects of exposure to a mated female: generalized suppression of male-female courtship. This study has identified neurons expressing Or65a-GAL4 as responsible for the aversive effects of cVA. These results provided the first molecular/genetic insights into the pheromones responsible for courtship learning (Ejima, 2007).

The results also provide some insight into the controversial nature of cVA's role in Drosophila behavior. The literature on cVA has posited roles for this lipid as both as an attractant and as an antiaphrodisiac, although this last function has been disputed. The social attractant role of cVA makes sense because it is deposited on eggs at feeding sites by females, and congregation at such sites is advantageous in terms of finding food and mates. The aversive role is equally plausible in light of cVA's transfer to females during mating, which would make it a marker of previous copulation. Understanding the molecular basis of cVA function and the circuitry subserving its behavioral effects will be necessary to completely unravel its multiple roles, but several important findings have emerged (Ejima, 2007).

(1) There are multiple cVA receptors, and they appear to have different behavioral roles. Or67d, which is expressed in T1, singly innervated sensilla, has a role in sensing the attractive properties of cVA (Xu, 2005; Ha, 2006). This receptor is not required for the courtship inhibitory role of cVA; this function appears to be served by Or65a, which is expressed in one of the three neurons of the T3 trichoid sensilla. These data suggest that Or67d is an 'appetitive' cVA receptor while Or65a is an 'aversive' receptor. Segregating the hedonic effects of this lipid by activating two independent receptors is an interesting way of establishing, at an early step, independent behavioral circuits for attraction and repulsion. The lack of behavioral redundancy between Or65a and Or67d neurons is also interesting in light of findings with the nonpheromonal olfactory receptor Or43b, where elimination of the receptor does not change the behavioral response to its preferred odorant. The interpretation of this result was that other olfactory receptors that recognized the odorant, but projected to different antennal glomeruli, could signal the same behavioral response. These results suggest that for some pheromone odorants the antennal lobe circuitry they connect to is critical to the behavioral output they engender (Ejima, 2007 and references therein).

(2) Responses to cVA appear to be context dependent. Having multiple sensory channels for cVA does not itself help the animal decide how to respond to this chemical; there must be some mechanism by which the environment or other cues can tell the animals which sensory channel is relevant for a particular situation. One way to achieve this would be to have the cVA channels be linked to other, situation-relevant, odor cues. In the case of both attraction and aversion, this appears to be the case. The first report of cVA as an attractant found that cVA was not attractive unless presented with food or food-associated odors. This studies assay set-up was designed to measure fast (in minutes) attractive responses in an open arena, as opposed to the long-term (days) maze/trap assays used by another, which did not uncover a role for food odor. The two paradigms may differ in sensitivity and relevance to particular behaviors, but the issue remains to be fully explored (Ejima, 2007 and references therein).

(3) Context also appears to be important for the aversive effects of cVA. Synthetic cVA is a very effective, and completely sufficient, generalizing cue when applied in small doses to mature virgin trainers, but is not very effective, requiring 10⁴ times more, when used with immature virgin trainers. The potency of mated female extracts with immature trainers is also less than with mature trainers, but the difference is not as exaggerated. This strongly suggests that some component of the mature female hydrocarbon profile that is not shared with immature virgins acts in concert with cVA to generalize learning. With the immature trainer, the mated female extract is supplying a low dose of cVA, but it also may supply a mature female compound that enhances the cVA effect. The identity of the compound(s) is unknown, but given that mature male extract is also able to allow generalization with immature trainers, it may be a hydrocarbon that is shared between mature males and females (Ejima, 2007).

The requirement for concurrent mature fly chemical signals for cVA to be an effective aversive cue and generalizer of learning is not unreasonable from an evolutionary point of view. Under normal circumstances, cVA is found only on males or mated females. The meaning of cVA in the presence of male hydrocarbons is clear: males should suppress courtship of other males because it is wasted reproductive energy. If a male in the wild sees cVA in the context of an immature female pheromone profile, however, it is likely that he has encountered a virgin at a feeding site where cVA-laced eggs have been deposited, and he should not suppress courtship (Ejima, 2007).

The underlying logic of suppressing courtship when presented with cVA in the context of a mature (and theoretically receptive) female is less obvious from a male's point of view. Copulation with a previously mated female is not ideal because she is already storing sperm from her previous mate, but there is still marginal gain; the second male's sperm can displace the first male's sperm. From the female's reproductive point of view, remating might also be advantageous because she will have more genetically diverse offspring, but it comes at a cost: it is correlated with reduced life span. The only player for whom remating does not have some advantage is the first male. It has been well documented that components of seminal fluid in Drosophila alter female behavior and reproduction to decrease remating. Transfer of cVA may be another facet of the successful male's strategy to decrease reproduction by competitor males. The effect of cVA on initial courtship decreases a second male's chances of success with that particular mated female, but the aftereffect, generalized suppression of his courtship drive, eliminates him as a competitor for other virgin females. The ability of cVA to engage the intrinsic plasticity machinery that allows animals to adapt to and learn from change to bring about a long-lasting change in another male's behavior could provide selective advantage to successfully copulating males (Ejima, 2007).

The Drosophila pheromone cVA activates a sexually dimorphic neural circuit

Courtship is an innate sexually dimorphic behaviour that can be observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate this behaviour are developmentally programmed. In Drosophila, courtship involves a complex yet stereotyped array of dimorphic behaviours that are regulated by Fru^M, a male-specific isoform of the fruitless gene. Fru^M is expressed in about 2,000 neurons in the fly brain, including three subpopulations of olfactory sensory neurons and projection neurons (PNs). One set of Fru⁺ olfactory neurons expresses the odorant receptor Or67d and responds to the male-specific pheromone cis-vaccenyl acetate (cVA). These neurons converge on the DA1 glomerulus in the antennal lobe. In males, activation of Or67d⁺ neurons by cVA inhibits courtship of other males, whereas in females their activation promotes receptivity to other males. These observations pose the question of how a single pheromone acting through the same set of sensory neurons can elicit different behaviours in male and female flies. Anatomical or functional dimorphisms in this neural circuit might be responsible for the dimorphic behaviour. This study reports a neural tracing procedure that employs two-photon laser scanning microscopy to activate the photoactivatable green fluorescent protein. Using this technique it was found that the projections from the DA1 glomerulus to the protocerebrum are sexually dimorphic. A male-specific axonal arbor was observed in the lateral horn whose elaboration requires the expression of the transcription factor Fru^M in DA1 projection neurons and other Fru⁺ cells. The observation that cVA activates a sexually dimorphic circuit in the protocerebrum suggests a mechanism by which a single pheromone can elicit different behaviours in males and in females (Datta, 2008).

In initial experiments, photoactivatable green fluorescent protein (PA-GFP) was expressed in flies in which the GAL4 enhancer-trap GH146 drives the expression of UAS-PA-GFP in 60% of the PNs that innervate most glomeruli in the antennal lobe. PA-GFP exhibits low-level fluorescence, sufficient to identify individual glomeruli, that is enhanced 100-fold after photoconversion with high-energy light. The PA-GFP was photoactivated with a two-photon laser scanning microscope to localize 710-nm light with submicrometre three-dimensional precision. Photoactivation of the antennal lobe neuropil, encompassing all glomeruli, results in intense labelling of the dendritic arbors of GH146 PNs. Diffusion of PA-GFP from the illuminated dendritic arbors allowed revealation of the cell bodies and axonal projections of the multiple GH146 PNs. Photoactivation of individual glomeruli (VM3 and DA1) reveals the dendritic arbors, cell bodies and projections of the subpopulation of GH146 PNs that innervate a single glomerulus (Datta, 2008).

An approach was devised to allow the tracing of individual PNs that innervate identified glomeruli. The DA1 glomerulus was exposed to low levels of photoconverting light and then the antennal lobe was rapidly imaged to identify the PN cell bodies that show modest increases in fluorescence intensity. Under these limiting conditions of photoactivation, diffusion of PA-GFP into axonal projections was not observed. Next a single weakly labeled PN cell body was strongly photoactivated at higher light intensity to reveal the axonal projections of an individual PN that innervates the DA1 glomerulus. Thus, two-photon laser scanning microscope-mediated activation of PA-GFP provides sufficient spatial resolution and photoconversion energy to reveal the neuronal processes of defined neuronal populations as well as individual neurons in the fly brain (Datta, 2008).

The development of a combined genetic and optical neural tracing method permits comparison of the topography of projections from Fru⁺ PNs that innervate the cVA-responsive DA1 glomerulus in male and female flies. Flies in which GAL4 is expressed under the control of the P1 fruitless promoter responsible for generating Fru^M (fru^GAL4) were crossed with flies harbouring the UAS-PA-GFP transgene. P1 transcripts from the modified fru^GAL4 allele do not undergo the sexually dimorphic splicing observed for the wild-type fru allele, and they therefore allow marking of Fru⁺ cells in both sexes. Unilateral photoactivation of the fly brain reveals many Fru⁺ cells, including neurons in the antennal lobe. Specific photoactivation of the DA1 glomerulus reveals six Fru⁺ PNs in both male and female flies that innervate this glomerulus. The cell bodies of these neurons reside in the lateral PN cluster, not the dorsal cluster as previously suggested (Datta, 2008).

It is possible that the sex-specific behavioural responses to cVA result from different functional responses of the DA1 glomerulus in the two sexes despite there being no apparent difference in the number or location of Fru⁺ DA1 PNs. Therefore the Ca²⁺-sensitive fluorescent protein GCaMP was expressed in Fru⁺ neurons, and two-photon imaging was used to examine increases in Ca²⁺ in the DA1 glomerulus in response to cVA. Large increases in Ca²⁺ within the DA1 glomerulus were detected by two-photon imaging after exposure of an intact, behaving fly to cVA. However, no differences were observed between male and female responses over a broad range of cVA concentrations (Datta, 2008).

These imaging experiments report local changes in the concentration of Ca²⁺ in both the presynaptic and postsynaptic compartments, because both Or67d-expressing neurons and DA1 PNs are Fru⁺. Therefore whether the electrophysiological properties of Fru⁺ DA1 PNs were sexually dimorphic was examined. The DA1 glomerulus was photoactivated to identify Fru⁺ DA1 PNs and the enhanced fluorescence was used to guide a patch electrode to the cell bodies. Recordings were made from Fru⁺ DA1 PNs in the loose patch configuration in an intact fly preparation and no significant difference was noted in the spike frequency or response kinetics between males and females when tested at several concentrations of cVA. These responses are comparable to those previously observed in whole-cell recordings of female DA1 PNs. This result demonstrates that male and female DA1 PNs show similar electrophysiological responses to cVA despite the previously noted dimorphism in the size of the DA1 glomerulus (Datta, 2008).

Next the projection patterns of Fru⁺ DA1 PNs were examined in the two sexes. Photoconversion of the DA1 glomerulus allowed the projection patterns of the population of DA1 PNs to be revealed in the lateral horn in living brains. Despite significant similarity in the axonal arbors of DA1 PNs in males and females, an increase was observed in the density of ventral axonal branches in the male. Quantification of differences in branch patterns in multiple individual male and female flies was hampered by variations in the orientation of the live brain during microscopy. Therefore the approach was altered to employ fixed brains stained with the antibody nc82 to label the synaptic neuropil of the lateral horn. An image registration algorithm was used to first 'warp' the nc82 channel of individual brains onto a reference brain and then map the PA-GFP fluorescence onto this reference brain. The registration error averaged less than 2 μm in any dimension when measured at the neuropil edge. It was observed that the projections from the DA1 glomerulus target the anterior ventromedial region of the LH. The projection pattern is triskelion-shaped, with ventral, lateral and dorsal branches. Fru⁺ DA1 projections from males have additional axonal branches that extend ventromedially. Superposition of the DA1 projections taken from ten male and ten female flies confirms this observation, indicating that information carried by Fru⁺ DA1 PNs is differentially segregated in the lateral horn of the two sexes. As a control a similar analysis of the PN projections from the Fru^- glomerulus VM3, which responds to alcohols and acetates, was performed. Superposition of the projections from VM3 reveals no consistent differences in the pattern of axonal projections in the lateral horn between the two sexes. These observations show that the image alignment procedure does not introduce sex-specific biases in projection patterns and that the dimorphic projection patterns that were observe for the Fru⁺ glomerulus DA1 are not a general feature of projections from all glomeruli (Datta, 2008).

The anatomical dimorphism observed at the level of the population of axons is also shown by the axons of single identified neurons. Tracing individual Fru⁺ DA1 neurons after warping revealed that the ventral axonal branches of male PNs define a male-specific region of protocerebral space (about 600 μm³). Each individual male in the data set sends at least one axon branch into this area. This area seems to partly overlap a region of neuropil in the lateral horn that was recently shown to be larger in male flies than in female flies. In addition, the total density of ventrally oriented axonal branches is significantly greater in males than in females. In contrast, the total innervation of the dorsal axonal arbor showed no statistically significant differences between sexes. No similar female-specific area was identified, although there are several smaller areas (particularly laterally) that appear to have an increased density of female axons. The data from single-axon tracing, along with observations from populations of DA1 neurons, indicate that DA1 PN projections are sexually dimorphic (Datta, 2008).

Fru mutant males court other males with high frequency. If the male-specific arbor contributes to the dimorphic behavioural response, it is expected that the DA1 PN projection patterns will be regulated by the fruitless gene. Therefore the axonal projections of single DA1 PNs were made visible in fru mutant males, and it was observed that DA1 PNs lack the characteristic male-specific axonal branches and exhibit a branching pattern more characteristic of wild-type females. However, the feminization is not complete in that the male-specific ventral axonal branches are significantly reduced but not completely eliminated in fru mutant males. Thus, the male pattern of projections of Fru⁺ DA1 PNs requires the male-specific isoform of fru, Fru^M (Datta, 2008).

It was also shown that the ectopic expression of Fru^M in females masculinizes the axonal arbor of their DA1 PNs. Projections of single Fru⁺ DA1 PNs in female flies that express Fru^M (fru^GAL4/fru^UAS-FruM) exhibit a striking increase in axonal projections to the ventral male-enhanced area. Quantitative analysis of these branches reveals that expression of Fru^M in females renders their ventral axon branch pattern statistically indistinguishable from that of males. The innervation patterns of individual neurons are sufficient for a computational discrimination algorithm to effectively distinguish individual females from Fru^M-expressing females with 100% accuracy, and individual males from fru mutant males with more than 91% accuracy. Thus, analysis of the PN projections of both single defined neurons and populations of neurons reveal that Fru⁺ DA1 PNs project to different regions of the protocerebrum in male and female flies. Moreover, this anatomical dimorphism in the neural circuit is controlled by the dimorphic transcription factor, Fru^M (Datta, 2008).

Next, whether the formation of the male-specific arbor requires the action of Fru^M in DA1 projection neurons was examined. The enhancer-trap MZ19 drives the expression of GAL4 in six DA1 PNs, about ten additional PNs that innervate two Fru^- glomeruli, and 25 extrinsic neurons of the mushroom body. Flies harbouring fru^GAL4, MZ19 or MZ19;fru^GAL4 all reveal expression of PA-GFP in six DA1 PNs. This suggests that the six lateral DA1 neurons labelled by the MZ19 and fru^GAL4 lines are identical. In accord with this observation, male and female DA1 neurons in MZ19 flies have a sexually dimorphic pattern of projections that closely resembles the dimorphic branching observed for Fru⁺ DA1 PNs. Therefore Fru^M expression was eliminated in male MZ19 neurons by expression of Tra, which directs the female-specific splicing of fruitless transcripts. Genetic feminization of male DA1 PNs in MZ19/UAS-tra flies results in two anatomical classes of DA1 projection neurons. Half of the genetically feminized DA1 PNs show a reduction in the male-specific arbor and closely resemble male DA1 projection neurons defective for Fru^M. The remaining genetically feminized neurons exhibit the wild-type male-specific branching patterns. Within a single male MZ19/UAS-tra fly, neurons of both anatomical classes were observed. These data suggest that Fru^M is required in DA1 PNs to generate a male-specific projection pattern, but its action in this genetic context is partly penetrant (Datta, 2008).

Also, whether the expression of Fru^M in female DA1 PNs masculinizes the DA1 axon arbor was examined. DA1 PNs in female MZ19; fru^UAS-FruM flies do not significantly innervate the male-specific area, although most send minor branches into the ventral region of the lateral horn. This is in contrast with observations with fru^GAL4/fru^UAS-FruM strains that exhibit a transformation of the female DA1 PN branching pattern into a complete male-specific arbor. Taken together, these results suggest that Fru^M is required in both DA1 PNs and in other Fru⁺ neurons to generate the male-specific pattern of ventral axon arborization in the lateral horn (Datta, 2008).

In Drosophila, courtship behaviour is governed by pheromonal excitation of peripheral olfactory pathways that ultimately activate behavioural circuits in higher brain centres. One pheromone elaborated by the male, cVA, suppresses male-male courtship but in females enhances receptivity to courting males. cVA activates the DA1 glomerulus, which is innervated by PNs that have sexually dimorphic projections in the lateral horn. This dimorphic circuit is under control of the transcription factor Fru^M, a male-specific isoform of fruitless. Moreover, the dimorphism in this circuit correlates with behaviour. In males mutant for Fru^M, cVA no longer suppresses male-male courtship and males exhibit a feminized pattern of DA1 projections. In females that express Fru^M, DA1 PNs exhibit a male pattern of axonal arbors in the lateral horn, and these females show reduced sexual receptivity. These observations are in accord with a mechanism in which the anatomical differences observed in Fru⁺ DA1 projection neurons contribute to the distinct behaviours elicited by cVA in the two sexes. In Drosophila, dimorphism in the Fru⁺ SP2 and mAL neurons has been observed, but the behavioural function of these circuits is unknown (Datta, 2008).

The anatomical dimorphism observed may be translated into a behavioural dimorphism if the connections between DA1 PNs and third-order neurons differ between the sexes. Third-order neurons whose dendrites innervate the ventral lateral horn may either receive greater input from male PNs or may restrict their synapses to the male-specific region of the DA1 axon arbor. The relatively small size of the male-specific arbor, about the volume of a glomerulus, implies a precision of connectivity in higher processing centres in the fly brain. The stereotyped and local precision of synaptic connections is an organizing principle in the antennal lobe and may be a common feature of invertebrate nervous systems (Datta, 2008).

Characterization of specific neural circuits that may mediate behaviour, as described in this study for the pheromone-responsive DA1 pathway, requires the development of tracing approaches that label defined populations of neurons. The distinction between genetic approaches -- including MARCM, Flp-Out and PA-GFP-based tracing -- and the histological approaches of Golgi and Cajal 100 years ago is the ability to use genetic markers to identify partners in the neural circuit more precisely. The targeted illumination of PA-GFP permits non-random, optically guided labelling of individual neurons from either anatomically or genetically defined subsets of neurons. Moreover, PA-GFP can be photoactivated in neurons in the living brain and allows electrophysiological recordings of labelled cells. This approach to neural tracing and recording in a defined circuit can be readily adapted to other brain regions in both the fly and mouse (Datta, 2008).

A dimorphic pheromone circuit in Drosophila from sensory input to descending output

Drosophila show innate olfactory-driven behaviours that are observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically programmed. Despite the numerical simplicity of the fly nervous system, features of the anatomical organization of the fly brain often confound the delineation of these circuits. This study identified a neural circuit responsive to cVA, a pheromone that elicits sexually dimorphic behaviours. Neural tracing using an improved photoactivatable green fluorescent protein (PA-GFP) was combined with electrophysiology, optical imaging and laser-mediated microlesioning to map this circuit from the activation of sensory neurons in the antennae to the excitation of descending neurons in the ventral nerve cord. This circuit is concise and minimally comprises four neurons, connected by three synapses. Three of these neurons are overtly dimorphic and identify a male-specific neuropil that integrates inputs from multiple sensory systems and sends outputs to the ventral nerve cord. This neural pathway suggests a means by which a single pheromone can elicit different behaviours in the two sexes (Ruta, 2010).

The male pheromone 11-cis-vaccenyl acetate (cVA) elicits male-male aggression and suppresses male courtship towards females as well as males. A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. In females, cVA activates the same sensory neurons to promote receptivity to males. cVA-induced aggregation behaviour is shown by both sexes. What neural circuits permit a single pheromone acting through the same set of sensory neurons to elicit several distinct and sexually dimorphic behavioural responses? (Ruta, 2010).

The sensory neurons that express the odorant receptor Or67d respond to cVA, and these neurons converge on the DA1 glomerulus in the antennal lobe. Projection neurons (PNs) that innervate the DA1 glomerulus terminate in the lateral horn of the protocerebrum. Comprehensive maps of Drosophila higher olfactory centers: spatially segregated fruit and pheromone representation. Previous experiments showed that the DA1 axons are sexually dimorphic and reveal a male-specific ventral axonal arborization in the lateral horn (Datta, 2008). This dimorphism by itself might explain the sexually dimorphic behaviours or, alternatively, it might presage iterative anatomical dimorphisms at each stage in the circuit to descending output. Therefore, a neural circuit was characterized that transmits information from the DA1 PNs to the ventral nerve cord (see Photoactivation identifies dimorphic lateral horn neurons). The analysis was restricted to neurons that express the sexually dimorphic transcription factor fruitless (Fru^M). Fru^M is expressed in both Or67d-expressing sensory neurons and DA1 PNs and governs the development of dimorphic neural circuitry including the male-specific axonal arborization of DA1 PNs. In addition Fru^M specifies many male-specific behaviours, including those that are mediated by cVA (Ruta, 2010).

In initial experiments PA-GFP, a photoactivatable GFP, was used to identify Fru+ third-order neurons whose dendritic processes are closely apposed to DA1 axon termini. A strategy was developed in which two-photon photoactivation is restricted to a small, circumscribed region of a neuron's axonal arborization with the expectation that this would label the postsynaptic cells by photoconversion of PA-GFP in their dendrites. To ensure that this limited activation could produce sufficient signal from the photoconverted fluorophore to illuminate third-order neurons and their most distal processes, two new enhanced PA-GFPs were generated, namely C3PA-GFP and SPA-GFP (Ruta, 2010).

Photoconversion of the DA1 glomerulus in flies expressing C3PA-GFP or SPA-GFP under the control of fru^GAL4 readily identified the axonal arborizations of the DA1 PNs. Then the volume of neuropil circumscribing the DA1 axon termini was photoactivated and four clusters of presumptive third-order neurons were reproducibly labelled in the lateral horn of male flies. Labelling of the two dorsal clusters, DC1 and DC2, was observed only in males; the clusters were either absent in the female or lacked projections into the ventral lateral horn. The lateral cluster LC1 was present in the two sexes but was dimorphic in both number and projection pattern. LC2 did not show an apparent numeric or anatomical dimorphism. Photoactivation of DA1 axon terminals in male flies that express C3PA-GFP pan-neuronally labelled few additional neurons and suggests that these four Fru+ clusters constitute the major potential recipients of DA1 input (Ruta, 2010).

These photoactivation experiments identify clusters of third-order neurons in the lateral horn that are anatomically poised to propagate dimorphic responses to cVA. However, anatomical proximity does not ensure functional connectivity. Therefore a method was developed to specifically activate individual glomeruli and simultaneously record from presumptive downstream neurons to determine whether the lateral horn clusters that were identified receive excitatory input from DA1 PNs. DA1 PNs were selectively stimulated by positioning a fine glass electrode in the centre of the DA1 glomerulus and iontophoresing acetylcholine, the neurotransmitter that excites PNs, into the glomerular neuropil. Varying the iontophoretic voltage allowed variation of the frequency of elicited action potentials systematically in DA1 PNs up to 250, a value close to the upper limit of cVA-elicited responses measured in these PNs (Datta, 2008; Schlief, 2007). Activation of the DA1 glomerulus over this voltage range excited DA1 PNs specifically and elicited no response in PNs innervating other glomeruli in the antennal lobe. Stimulation of the neighbouring glomeruli, VA1d and VA1lm, similarly elicited the specific excitation of their cognate PNs but did not activate DA1 PNs (Ruta, 2010).

Next, whether stimulation of the DA1 glomerulus would result in the excitation of neurons within the four clusters in the lateral horn that were identified, a result indicative of functional synaptic connections with DA1 PNs, was examined. The genetically encoded calcium indicator GCaMP3 was examressed in Fru+ neurons in male flies and two-photon imaging was used to monitor increases in Ca²⁺ concentration in the lateral horn clusters in response to DA1 excitation. Stimulation of the DA1 glomerulus elicited large increases in Ca²⁺ in neurons within the DC1 and LC1 clusters, with a far weaker response being observed in LC2. The small DC2 cluster is difficult to identify reliably because of the low basal fluorescence of GCaMP3; it was therefore not examined by optical imaging. The Ca²⁺ response in DC1 was specific for DA1 activation and was not observed when the stimulating electrode was repositioned in two neighbouring glomeruli, VA1d and VA1lm. These optical imaging experiments demonstrate that neurons within the DC1 and LC1 clusters extend processes in anatomical proximity to the DA1 axons and receive excitatory input from DA1 PNs. Immunostaining indicated that neurons within the LC1 cluster produce the inhibitory neurotransmitter GABA (γ-aminobutyric acid). Electrophysiological experiments suggested that DC1 neurons are excitatory but the neurotransmitter remains unknown (Ruta, 2010).

Focused was placed on the male-specific DC1 neurons to define a cVA-responsive circuit. The DC1 cluster consists of ~19.7 cell bodies (n = 10) in a spatially stereotyped location in the dorsal aspect of the anterior protocerebrum. Double labelling experiments revealed that the DC1 processes interdigitate richly with DA1 axons in the lateral horn. Photoactivation of single DC1 cell bodies indicated that the cluster is composed of several anatomical classes of neurons characterized by distinct branch patterns within the protocerebrum that are likely to receive and integrate inputs from both olfactory and non-olfactory brain centres (Ruta, 2010).

Electrophysiological recordings were performed to examine the response of DC1 neurons to both DA1 stimulation and cVA exposure. Selective stimulation of the DA1 glomerulus evoked action potentials in 66% of male-specific DC1 neurons recorded in the loose patch configuration. Among responsive DC1 neurons, it was observed that the sensitivity to DA1 stimulation differed. This functional heterogeneity within the DC1 cluster observed by both electrical and optical recording was consistent with the anatomical heterogeneity of dendritic fields in the lateral horn observed for single DC1 neurons (Ruta, 2010).

In accord with the imaging experiments, the electrophysiological response of DC1 neurons is selectively tuned to DA1 input. After recording the response of a DC1 neuron to DA1 stimulation, the stimulating electrode was repositioned into 6-11 other superficial glomeruli located throughout the antennal lobe. DC1 neurons activated by minimal DA1 stimulation were either weakly excited or unresponsive to strong stimulation of other glomeruli. Stimulation of the Fru+ VA1lm glomerulus failed to excite DC1 neurons despite the close proximity of DA1 and VA1lm axons (Jefferis, 2007). These observations demonstrate the specificity of glomerular excitation and reveal that olfactory input to DC1 is mediated largely by the DA1 glomerulus and not by the activation of at least 11 other glomeruli, suggesting that DC1 neurons receive olfactory stimulation only from cVA. Next cVA-evoked responses from DC1 neurons were recorded in an intact fly preparation. It was observed that 62% of DC1 neurons were responsive to cVA over a range of concentrations. The input-output relationship of DC1 neurons was similar whether action potentials were evoked in DA1 PNs through direct glomerular stimulation or by pheromonal excitation of the antenna, suggesting that DC1 neurons are excited primarily by means of DA1 input. Both Or67d-expressing sensory neurons and DA1 PNs have been shown to be selectively tuned to cVA. DC1 neurons showed similar odorant selectivity and fired only weakly in response to stimulation of the antenna with a cocktail of ten fruit-derived odorants that excite a majority of glomeruli. Thus, DC1 neurons are likely to receive direct excitatory feedforward input from DA1 PNs and respond selectively to cVA (Ruta, 2010).

Photoactivation of PA-GFP in presynaptic DA1 axonal arborizations, in concert with electrophysiology, has identified postsynaptic third-order neurons in the lateral horn that are responsive to cVA. The iterative use of this strategy could allow definition of the complete cVA circuit from sensory input to descending output. Tracing of photoactivated DC1 axons revealed that they terminate proximally within a triangular neuropil in the lateral protocerebrum (the lateral triangle) and extend distal processes to a previously uncharacterized tract within the superior medial protocerebrum (the SMP tract). The lateral triangle and SMP tract are sexually dimorphic neuropils that are absent in females (Ruta, 2010).

Photoactivation of the terminal arborizations of DC1 axons was performed to identify neurons innervating the lateral triangle and SMP tract. Dense labelling was observed in these structures arising from the rich male-specific projections of multiple classes of Fru+ neurons. Dimorphic LC1 neurons that receive direct innervation from DA1 PNs send inhibitory projections to the lateral triangle and SMP tract. Dimorphic mAL neurons were also observed extending from the subesophageal ganglion (SOG) and terminating within these neuropils. In addition, these neuropils are innervated by male-specific P1 interneurons implicated in the initiation of male courtship behaviour. Thus, the lateral triangle and SMP tract receive dimorphic projections from several brain regions including other sensory processing areas, suggesting that these neuropils may integrate sex-specific information from multiple sensory systems (Ruta, 2010).

Several neurons that innervate the lateral triangle and SMP tract also extend processes that descend into the ventral nerve cord, suggesting that these potential fourth-order descending neurons may transmit information from cVA-responsive sensory neurons to the ganglia of the ventral nerve cord. Descending neurons that innervate the lateral triangle and SMP tract were characterized by photoactivation of the cervical connectives conveying neural signals from the brain to the ventral nerve cord. In the brain, the processes of these descending neurons showed a marked dimorphism that was apparent in their extensive innervation of the male-specific SMP tract and lateral triangle. A descending neuron, DN1, absent in females, was observed in the ventral posterior aspect of the male brain, at the midline. Labelling of this male-specific cell body revealed short processes terminating within the lateral triangle and SMP tract, and a long descending process entering the ventral nerve cord and terminating within the thoracic and abdominal ganglia. Electroporation of DN1 with Texas Red dextran, followed by photoactivation of the DC1 cluster, revealed extensive intermingling of the green DC1 axons with the red dendrites of the descending neuron. This suggests that this descending neuron is anatomically poised to make direct synaptic contacts with third-order, cVA-responsive DC1 neurons (Ruta, 2010).

Whole-cell patch clamp recordings were performed on DN1 to discern whether it transmits pheromonal information to the ventral nerve cord. In response to either exposure of the antenna to cVA or direct stimulation of the DA1 glomerulus, DN1 received a barrage of excitatory postsynaptic potentials (EPSPs) bringing its membrane potential close to or past threshold. To determine whether this response was mediated by DC1 neurons, a microlesion technique was devised exploiting the spatial precision of a two-photon laser to effectively sever DC1 inputs into the lateral triangle and SMP tract. Optical recordings revealed that microlesioning of DC1 dendrites resulted in the immediate and selective loss of DC1 responses to DA1 stimulation without affecting the excitation of neighbouring LC1 dendrites and cell bodies. Severing the connections between DA1 and DC1 resulted in an almost complete loss of the response of DN1 to stimulation of DA1. The response of this descending neuron was far weaker than the response of early neural participants in this circuit. However, the observation that two-photon-mediated microlesions in DC1 resulted in a decrease of more than 70% in the DN1 response to stimulation of DA1 suggests that, despite its weak excitation, DN1 is a component of this circuit. A more potent response may require a more natural setting that integrates pheromonal input with other sensory signals. Taken together, these experiments suggest that male-specific DC1 neurons excite the male-specific DN1 through synaptic connections within the dimorphic lateral triangle and SMP tract. Thus, olfactory information may be processed by as few as three synapses within the brain before descending to initiate motor programs within the ganglia of the ventral nerve cord. Although a behaviour elicited by this circuit cannot yet be defined, it is presumed that it mediates a component of the innate behavioural repertoire initiated by cVA (Ruta, 2010).

This cVA-responsive circuit provides insights into the mechanism by which sensory information received by the antenna may be translated into motor output. First, the circuit is concise: as few as four neuronal clusters and three synapses bring pheromonal signals from the periphery to the ganglia of the nerve cord. This minimal circuit assumes monosynaptic connections between the neurons that were identified. This circuit is shallow but seems to include adequate synaptic connections to permit the integration of olfactory and non-olfactory information. Third-order lateral horn neurons reveal a capacity for multisensory integration with inputs to the DC1 cluster from the SOG and from the optic lobe. The lateral triangle and SMP tract also integrate sensory inputs from DC1 and LC1 as well as inhibitory projections from the SOG. This integration provides the opportunity for other sensory signals emanating from a cVA-scented fly to modulate the response to the pheromone (Ruta, 2010).

Second, multiple neural components within the circuit are anatomically dimorphic, and this could explain the different behaviours elicited by cVA in males and females. The initial neural components of the circuit, Or67d-expressing sensory neurons and DA1 PNs, are dedicated to the receipt of a singular olfactory stimulus, cVA, and are equally responsive to the pheromone in the two sexes. However, dimorphisms are observed in the synaptic connections between the PNs and the third-order lateral horn neurons and define a node from which sex-specific neural pathways emanate. The DA1 PNs reveal dimorphic axon arborizations, but this dimorphism is only one component of a highly dimorphic circuit. These dimorphic arborizations synapse with male-specific DC1 neurons that send axons to a male-specific neuropil (the lateral triangle and SMP tract). One output of this neuropil is a male-specific descending neuron, DN1. This circuit is likely to participate in the generation of cVA-elicited behaviours observed only in males. The identification of a sex-specific circuit including extensive neuropils present only in males suggests pathways for dimorphic behaviours that differ from earlier proposals that invoke the differential activation of circuits that are common to the two sexes. DA1 PNs also synapse onto the cluster of LC1 neurons that are present in both sexes but are numerically and anatomically dimorphic. The multiple dimorphic targets of a singular olfactory input could explain how a pheromone acting through the same sensory inputs may elicit different behaviours in the two sexes (Ruta, 2010).

Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

Nervous systems exhibit myriad cell types, but understanding how this diversity arises is hampered by the difficulty to visualize and genetically-probe specific lineages, especially at early developmental stages prior to expression of unique molecular markers. This study used a genetic immortalization method to analyze the development of sensory neuron lineages in the Drosophila olfactory system, from their origin to terminal differentiation. This approach was applied to define a fate map of nearly all olfactory lineages and refine the model of temporal patterns of lineage divisions. Taking advantage of a selective marker for the lineage that gives rise to Or67d pheromone-sensing neurons and a genome-wide transcription factor RNAi screen, the spatial and temporal requirements for Pointed, an ETS family member, was identified in this developmental pathway. Transcriptomic analysis of wild-type and Pointed-depleted olfactory tissue reveals a universal requirement for this factor as a switch-like determinant of fates in these sensory lineages (Chai, 2019).

By combining genetic drivers labeling small subsets of precursor cells with methods for immortalization of expression patterns within defined temporal windows, this study generated a fate map of the complete peripheral Drosophila olfactory system. This resource adds a novel developmental perspective on the Drosophila olfactory circuitry, complementing the maps of glomerular innervations and PN projections to higher brain centers. While the concentric spatial organization of SOPs is partially maintained in the distribution of olfactory sensilla, there is only a limited relationship with the organization of the axon projections of OSNs in the antennal lobe. Future immortalization of additional enhancer-GAL4 drivers with restricted antennal disc expression should help improve the resolution of the fate map, and identify unique sensilla lineage markers in addition to the at1 driver. This information will be important to investigate the developmental mechanisms that act within a particular arc to specify up to 5-6 different SOP types (Chai, 2019).

Previous screening for transcriptional determinants of OSN fates identified a small set of factors that act in a combinatorial manner to activate or repress olfactory receptor expression in specific OSN classes. Such factors are likely to act only at the end of more elaborate gene regulatory networks that ensure the specification of SOP type, and determination and coordination of OSN receptor expression and axon targeting. Genome-wide, constitutive RNAi screen of transcriptional regulators has identified a large number of new molecules that are likely to function in several of these processes. This study focused on the role of the ETS homolog, Pnt, because of its unique mutant phenotype, which reveals a role in limiting, rather than determining, Or67d neuron specification. With ab at1 lineage marker (GMR82D08-GAL4, a driver that labels SOPs that exclusively produce at1 sensilla), protein expression and gain- and loss-of-function analyses, evidence is provided that this transcription factor has a switch-like function in distinguishing the terminal Svp-expressing Naa cell from its Svp-negative sibling Nab. Interestingly, this role of Pnt appears to be distinct from other functions of this transcription factor where it serves as a nuclear read-out of various MAPK signaling pathways. Antennal transcriptomic analysis indicates that this role of Pointed is likely to be universal in olfactory sensilla. Moreover, the switch-like function is not the only role of Pnt in the antenna, as it also contributes to the specification of the correct global number of SOPs, and may more directly regulate the expression of specific olfactory receptor genes (e.g., Ir84a). Pnt's broad expression in the non-neuronal sublineage suggests it could also participate in support cell development (Chai, 2019).

With the OSN lineage driver, it is now possible to exploit single-cell RNA-seq and chromatin profiling technologies to examine the gene expression and epigenetic states of the at1 lineage from birth to maturity, and how these may be influenced by internal state and environmental conditions. While cellular-resolution level transcriptomic/epigenomic data are undeniably important to understand neural development, the combination of these with methods for visualizing specific lineages in vivo is essential for a complete view of how structural and functional diversity develops in the nervous system (Chai, 2019).

Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein

Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. This study shows that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, Lush. Lush undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in Lush that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, Lush^D118A, produces a dominant-active Lush protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of Lush^D118A reveals that it closely resembles cVA-bound Lush. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants (Laughlin, 2008).

This study has shown that cVA binds to the pheromone-binding protein Lush and induces conformational changes. Mutations predicted to reduce or enhance the conformational changes also reduce or enhance cVA sensitivity in vivo. One Lush mutant, Lush^D118A, is dominantly active, triggering robust action potentials in T1 neurons in the absence of pheromone. This effect is specific to T1 neurons, as basiconic and other trichoid olfactory neurons are unaffected by this protein. Lush^D118A activates T1 neurons through the putative cVA-activated neuronal receptor components, Or67d and SNMP, accounting for the specificity of the dominant Lush. The data reveal that pheromone molecules are not required for activation of T1 neurons and define a novel olfactory signaling paradigm in which the pheromone-induced conformational change in Lush mediates activation of T1 neurons (Laughlin, 2008).

cVA can trigger weak responses in T1 neurons in the absence of Lush when applied at high concentrations. Direct effects of cVA on Or67d/SNMP receptor complexes may mediate these Lush-independent responses, as these two components confer marginal cVA sensitivity to the empty neuron preparation (Benton, 2007). Alternatively, activated Lush may normally dimerize with an unknown cofactor that alone can weakly activate T1 receptors in the presence of cVA. However, the sensitivity for cVA in the absence of Lush is so poor that lush¹ mutants are blind to the pheromone in aggregation assays. In proximity experiments, cVA levels emanating from single male flies are below detection limits in the absence of Lush. Therefore, the Lush-independent activation of T1 neurons is unlikely to play a role in cVA responses in vivo (Laughlin, 2008).

Olfactory neurons are thought to be tuned to odorants exclusively by the odorant receptors they express. Indeed, in Drosophila melanogaster, activation of many odorant receptors results from direct binding of food odorants. Why does cVA reception require a binding protein intermediate? It is suggested that the binding protein may enhance sensitivity and specificity in the pheromone detection process. If a pheromone induces a stable, ligand-specific conformational change in a binding protein, single pheromone molecules could be detectable if the neuronal receptor complex is specifically tuned to that conformation. Further, if the conformation of the binding protein that activates the receptors is specific to the pheromone-bound state, other environmental stimuli are less likely to activate the neurons, even if they interact with the binding protein. Consistent with this idea, Lush increases the sensitivity of T1 neurons to cVA over 500-fold, but, remarkably, does not sensitize the neurons to structurally similar chemicals, such as vaccenyl alcohol or vaccenic acid. Indeed, Lush can bind a large array of chemicals, but only cVA activates T1 neurons. Other OBPs have been shown to bind to a wide range of unnatural compounds, including plasticizers and dyes, and the electrophysiological or behavioral responses to a specific ligand do not correlate with the binding affinity of the OBP for that ligand. Therefore, binding is clearly not sufficient for sensitization. However, by utilizing a ligand-specific conformational shift in a binding protein, detection of rare pheromone molecules is possible with high fidelity and sensitivity by creating an active binding protein species that diffuses within the sensillum lymph until it contacts and activates a receptor on the dendrites (Laughlin, 2008).

Attempted were made to reconstitute the cVA detection pathway in basiconic neurons lacking endogenous receptors. The CD36 homolog SNMP is expressed in most or all trichoid neurons and is required for sensitivity to cVA (Benton, 2007; Jin, 2008). SNMP colocalizes with the odorant receptor complex in T1 neuron dendrites (Benton, 2007), and antiserum to SNMP infused into the lymph of T1 sensilla phenocopies SNMP loss-of-function mutants, suggesting that SNMP directly mediates pheromone sensitivity (Jin, 2008). Expression of SNMP, Or67d, and Lush together in the empty neuron system failed to recapitulate T1 cVA sensitivity. Or67d alone was unresponsive, but adding Lush through the recording pipette did sensitize Or67d receptors slightly to cVA in the absence of SNMP, suggesting that Lush interacts directly with Or67d. Coexpressing SNMP and Or67d enhanced cVA sensitivity, but, surprisingly, adding Lush failed to further enhance sensitivity. These differences between the empty neuron responses and T1 neurons may reflect reduced levels of one or more components when expressed in basiconic sensilla or, more likely, indicate that additional components are missing. Indeed, in a screen for cVA-insensitive mutants, mutations were recovered in the known sensitivity factors as well as three additional unknown genes encoding factors that are essential for cVA sensitivity. It is expected that, when all of these components are identified and expressed in the basiconic neurons, full cVA sensitivity will be conferred (Laughlin, 2008).

OBPs, like Lush, are a large family of soluble proteins secreted into the lymph fluid surrounding the olfactory neurons. Proposed functions for OBPs include transporting ligands to the ORs, protecting the odor from degradation or deactivation by odorant-degrading enzymes (ODEs), and forming a complex with an odor that either directly activates ORs or binds to other accessory proteins, which ultimately results in OR activation. In vitro studies of the pheromone-binding protein (PBP) from Bombyx mori show that the OBP undergoes a conformational change at low pH that prevents ligand binding, suggesting that OBPs may function primarily as passive carriers and changes in the local pH stimulate pheromone release in the vicinity of the neuronal membrane. Furthermore, previous studies reported that high concentrations of moth pheromones can directly activate cognate pheromone receptors expressed in tissue culture and that DMSO is as effective as the pheromone-binding proteins at sensitizing the neurons to pheromone, leading to the conclusion that the binding proteins are pheromone solubilizers/carriers. However, similar studies implicate the binding proteins as factors in receptor specificity. The current data support the latter view. It is noted that Lush homologs in other insects and the 12 Drosophila species have conserved the amino acids predicted to form the salt bridge. Only Drosophila ananassae (D. ana) is predicted to lack the phenylalanine corresponding to F121 in melanogaster (replaced by leucine). A similar activation mechanism, therefore, is likely to occur in these species. Recent work in rodents reveals that vertebrate pheromones can be peptides or protein. It will be interesting to determine whether the conformational activation mechanism identified for Lush is conserved in analogous extracellular binding proteins in other species (Laughlin, 2008).

Love makes smell blind: mating suppresses pheromone attraction in Drosophila females via Or65a olfactory neurons

In Drosophila, the male sex pheromone cis-vaccenyl acetate (cVA) elicits aggregation and courtship, through the odorant receptor Or67d. Long-lasting exposure to cVA suppresses male courtship, via a second channel, Or65a. In females, the role of Or65a has not been studied. This study shows that, shortly after mating, Drosophila females are no longer attracted to cVA and that activation of olfactory sensory neurons (OSNs) expressing Or65a generates this behavioral switch: when silencing Or65a, mated females remain responsive to cVA. Neurons expressing Or67d converge into the DA1 glomerulus in the antennal lobe, where they synapse onto projection neurons (PNs), that connect to higher neural circuits generating the attraction response to cVA. Functional imaging of these PNs shows that the DA1 glomerulus is inhibited by simultaneous activation of Or65a OSNs, which leads to a suppression of the attraction response to cVA. The behavioral role of postmating cVA exposure is substantiated by the observation that matings with starved males, which produce less cVA, do not alter the female response. Moreover, exposure to synthetic cVA abolishes attraction and decreases sexual receptivity in unmated females. Taken together, Or65a mediates an aversive effect of cVA and may accordingly regulate remating, through concurrent behavioral modulation in males and females (Lebreton, 2014 PubMed).

Search PubMed for articles about Drosophila Or67d

Benton, R., Vannice, K. S. and Vosshall, L. B. (2007). An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature [Epub ahead of print]. PubMed ID: 17943085

Butenandt, A., Beckmann, R., Stamm, D. and Hecker, E. (1959). Über den Sexuallockstoff des Seidenspinners Bombyx mori. Reindarstellung und Konstitution. Z. Naturforsch. 14b: 283-284

Chai, P. C., Cruchet, S., Wigger, L. and Benton, R. (2019). Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system. Nat Commun 10(1): 643. PubMed ID: 30733440

Couto, A., Alenius, M. and Dickson, B. J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15: 1535-1547. PubMed ID: 16139208

Datta, S. R., et al (2007). The Drosophila pheromone cVA activates a sexually dimorphic neural circuit. Nature 452: 473-477. PubMed ID: 18305480

Ejima, A., et al. (2007). Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Curr. Biol. 17(7): 599-605. PubMed ID: 17363250

Fishilevich, E. and Vosshall, L. B. (2005). Genetic and functional subdivision of the Drosophila antennal lobe. Curr. Biol. 15: 1548-1553. PubMed ID: 16139209

Ge, Y. and Elghetany, M. T. (2005). CD36: a multiligand molecule. Lab. Hematol. 11: 31-37. PubMed ID: 15790550

Ha, T. S. and Smith, D. P. (2006). A pheromone receptor mediates 11-cis-vaccenyl acetate-induced responses in Drosophila. J. Neurosci. 26(34): 8727-33. PubMed ID: 16928861

Hoebe, K. et al. (2005). CD36 is a sensor of diacylglycerides. Nature 433: 523-527. PubMed ID: 15690042

Jefferis, G. S., et al. (2007). Comprehensive maps of Drosophila higher olfactory centers: spatially segregated fruit and pheromone representation. Cell 128(6): 1187-203. PubMed ID: 17382886

Jin, X., Ha, T. S. and Smith, D. P. (2008). SNMP is a signaling component required for pheromone sensitivity in Drosophila. Proc Natl Acad Sci U S A 105: 10996-11001. PubMed ID: 18653762

Kondoh, Y., Kaneshiro, K. Y., Kimura, K. and Yamamoto, D. (2003) Evolution of sexual dimorphism in the olfactory brain of Hawaiian Drosophila. Proc. R. Soc. Lond. B 270: 1005-1013. PubMed ID: 12803889

Krieger, J., et al. (2004). Genes encoding candidate pheromone receptors in a moth (Heliothis virescens). Proc. Natl Acad. Sci. 101: 11845-11850. PubMed ID: 15289611

Krieger, J., Grosse-Wilde, E., Gohl, T. and Breer, H. (2005). Candidate pheromone receptors of the silkmoth Bombyx mori. Eur. J. Neurosci. 21: 2167-2176. PubMed ID: 15869513

Kurtovic, A., Widmer, A. and Dickson, B. J. (2007). A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446(7135): 542-6. PubMed ID: 17392786

Laugerette, F. et al. (2005). CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J. Clin. Invest. 115, 3177-3184. PubMed ID: 16276419

Laughlin, J. D., Ha, T. S., Jones, D. N. and Smith, D. P. (2008). Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell 133: 1255-1265. PubMed ID: 18585358

Lebreton, S., Grabe, V., Omondi, A. B., Ignell, R., Becher, P. G., Hansson, B. S., Sachse, S. and Witzgall, P. (2014). Love makes smell blind: mating suppresses pheromone attraction in Drosophila females via Or65a olfactory neurons. Sci Rep 4: 7119. PubMed ID: 25406576

Mane, S. D., Tompkins, L. and Richmond, R. C. (1983). Male esterase 6 catalyzes the synthesis of a sex pheromone in Drosophila melanogaster females. Science 222: 419-421. PubMed ID: 17789533

Nakagawa, T., Sakurai, T., Nishioka, T. and Touhara, K. (2005). Insect sex-pheromone signals mediated by specific combinations of olfactory receptors. Science 307: 1638-1642. PubMed ID: 15692016

Philips, J. A., Rubin, E. J. and Perrimon, N. (2005). Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309: 1251-1253. PubMed ID: 16020694

Ruta, V., et al. (2010). A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468: 686-690. PubMed ID: 21124455

Sakurai, T., et al. (2004). Identification and functional characterization of a sex pheromone receptor in the silkmoth Bombyx mori. Proc. Natl Acad. Sci. 101: 16653-16658. PubMed ID: 15545611

Schlief, M. L. and Wilson, R. I. (2007). Olfactory processing and behavior downstream from highly selective receptor neurons. Nature Neurosci. 10: 623-630. PubMed ID: 17417635

Stockinger, P., Kvitsiani, D., Rotkopf, S., Tirian, L. and Dickson, B. J. (2005). Neural circuitry that governs Drosophila male courtship behavior. Cell 121: 795-807. PubMed ID: 15935765

Stuart, L. M. et al., (2005). Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170: 477-485. PubMed ID: 16061696

Syed, Z., Ishida, Y., Taylor, K., Kimbrell, D. A. and Leal, W. S. (2006). Pheromone reception in fruit flies expressing a moth's odorant receptor. Proc. Natl Acad. Sci. 103: 16538-16543. PubMed ID: 17060610

van der Goes van Naters, W. and Carlson, J. R. (2007). Receptors and neurons for fly odors in Drosophila. Curr. Biol. 17(7): 606-12. PubMed ID: 17363256

Xu, P., Atkinson, R., Jones, D. N. and Smith, D. P. (2005). Drosophila OBP Lush is required for activity of pheromone-sensitive neurons. Neuron 45: 193-200. PubMed ID: 15664171

date revised: 22 September 2022

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