Modulation of Drosophila male behavioral choice

Abstract

The reproductive and defensive behaviors that are initiated in response to specific sensory cues can provide insight into how choices are made between different social behaviors. We manipulated both the activity and sex of a subset of neurons and found significant changes in male social behavior. Results from aggression assays indicate that the neuromodulator octopamine (OCT) is necessary for Drosophila males to coordinate sensory cue information presented by a second male and respond with the appropriate behavior: aggression rather than courtship. In competitive male courtship assays, males with no OCT or with low OCT levels do not adapt to changing sensory cues and court both males and females. We identified a small subset of neurons in the suboesophageal ganglion region of the adult male brain that coexpress OCT and male forms of the neural sex determination factor, Fruitless (Fru^M). A single Fru^M-positive OCT neuron sends extensive bilateral arborizations to the suboesophageal ganglion, the lateral accessory lobe, and possibly the posterior antennal lobe, suggesting a mechanism for integrating multiple sensory modalities. Furthermore, eliminating the expression of Fru^M by transformer expression in OCT/tyramine neurons changes the aggression versus courtship response behavior. These results provide insight into how complex social behaviors are coordinated in the nervous system and suggest a role for neuromodulators in the functioning of male-specific circuitry relating to behavioral choice.

Modulation of classical neurotransmitter action on target neurons adds great flexibility to synaptic output between neurons and is suggested to be at the core of important behavioral processes like learning and memory (1–3). In vertebrates, amines like serotonin, dopamine, and norepinephrine; peptides like arginine vasopressin, and oxytocin; gonadal steroids; and various glucocorticoids serve as well known neuromodulatory substances (4–6). Through selective actions at individual synaptic sites, neuromodulators coordinate the output of neuronal ensembles to generate behavioral patterns of varying complexity (7, 8).

An elegant example of coordinating network output comes from studies with the stomatogastric ganglion of crustaceans (9, 10). In this small neuronal ensemble, neuromodulators function either singly or in various combinations at multiple sites in the ganglion to alter the patterned output of the ganglion and thereby the movement of food through the stomach. An example of changing network ensembles in vertebrates is seen in studies of vole social behavior. Here, the distribution of oxytocin, vasopressin, and dopamine receptors within different brain regions appears linked to the differences seen in social behavior between prairie voles and montane voles (11).

In this paper, we focus on the roles of octopamine (OCT), a phenolamine structurally related to the catecholamine norepinephrine, in modulating the choice between courtship and aggression in male flies. Norepinephrine has been shown to be important in many aspects of vertebrate behavior, including arousal, anxiety, learning and memory, opiate reward, and aggression (12–16). Among invertebrates, OCT influences foraging behavior in honey bees (17); resets aggressive motivation in crickets (18); and functions in appetitive associative learning (19, 20), ethanol tolerance development (21), ovarian muscle contraction (22), and possibly aggression levels in Drosophila (23). Like their vertebrate amine neuron counterparts (24–26), OCT neurons in Drosophila (i) are few in number but have enormous fields of innervation covering essentially all neuropil areas in the fly brain (27–29) and (ii) function by activating multiple G protein-coupled receptors (30, 31).

Aggression and courtship usually are mutually exclusive behaviors; however, see ref. 32. By examining the choices made between these behaviors by male flies, a powerful approach is offered with which to study the genetic and neural basis of complex behaviors. Multiple decision-making actions are required for each of these behaviors, including the processing of chemosensory and visual information and deciding whether another fly is a potential opponent or a potential mate. Using aggression and competitive courtship assays, we find that OCT is necessary for pairs of Drosophila males to respond to the sensory cues presented and to coordinate expression of the appropriate response: aggression. Feminizing OCT/tyramine (TYR) neurons in males also changes the aggression vs. courtship response behavior. Because the gene fruitless directs both courtship and aggression in flies (33–35), we analyzed the expression patterns of OCT and the male forms of Fruitless (Fru^M) and found them to be coexpressed in distinct suboesophageal ganglion (SOG) neurons in the male brain. This region receives the contact gustatory pheromone information thought to facilitate sex and species discrimination (36, 37). The arborizations of one of the Fru^M-octopaminergic neurons were found to project bilaterally and appear to ramify in the posterior antennal lobe, multiple SOG layers, as well as the lateral accessory lobe (ventral body). These results offer insight into how sensory cues are integrated and modulated in the nervous system to direct sex-specific complex behaviors and indicate a role for the neuromodulator OCT in the functioning of the male-specific circuitry relating to behavioral choice.

Results

To reduce or eliminate the function of OCT neurons, we used the previously described Tyramine β-hydroxylase (Tβh) mutant lines (38). The Tβh gene encodes the enzyme necessary to convert TYR to OCT, and null mutants (Tβh^nM18) produce no detectable OCT, whereas the hypomorphic Tβh^MF372 strain generates low levels of OCT (see Materials and Methods and ref. 38). The revertant Tβh^M6 allele was used as the control. The original alleles were generated by P-element manipulations on the same chromosome (38). We performed subsequent manipulations to replace the w¹¹¹⁸ allele and backcrossed to Canton-S (CS) to maintain comparable genetic backgrounds (see Materials and Methods). We verified OCT, dopamine, and serotonin levels in each Tβh allele by using HPLC [supporting information (SI) Table 1].

Aggression Assays with Classical Mutant Lines.

Under experimental conditions previously described (39), pairs of male or female flies fight each other by using defined sets of aggressive behavioral patterns. When two males of the same genotype were paired in this assay, we observed that both control and OCT-deficient males extended and vibrated their wings toward the other male fly a limited number of times per 30-min fight [3.4 wing extensions per Tβh^nM18 fight (n = 14 of 19 fights) and 2.6 wing extensions per Tβh^M6 fight (n = 15/22 fights)]. Bilateral and unilateral wing extensions were included (Fig. 1 and SI Movies 1 and 2). The unilateral wing extension pattern resembled the singing displayed toward females during courtship rituals. Whether this pattern is initiated in aggression assays via specific sensory cues is uncertain. However, control males invariably transitioned from the wing extension pattern to aggression: the appropriate behavioral response to the presence of a second male in the territory (Fig. 1 a and c–e). The most common aggressive behavioral pattern observed after a wing extension was a “lunge” (Fig. 1e) or “front fencing” as described in refs. 39 and 40 and demonstrated in SI Movie 1.

Fig. 1.

Manipulating OCT levels and the sex of OCT/TYR neurons changes the usage of aggressive and courtship behaviors. (a and b) Graph depicting the percentage of aggression (black) and courtship (gray) transitions after a wing extension. Control males (Tβh^M6 and UAS-tra/+) predominantly transition to aggression (aggression/total transitions, n = 31/37 and 20/23). Experimental males [Tβh^nM18, Tβh^MF372, dTdc2-Gal4;UAS-tra, and dTdc2-Gal4 (new insertion #1);UAS-tra] transition to aggression and courtship or predominantly to courtship (courtship/total transitions: n = 24/42, 11/14, 24/31, and 30/39). The number of courtship vs. aggressive transitional patterns was compared among control and experimental genotypes with the Fisher exact probability test. Significant frequency differences were observed between Tβh^M6 and Tβh^nM18 patterns (P = 0.0002), Tβh^M6 and Tβh^MF372 patterns (P = 0.0006), and UAS-tra/+ and dTdc2-Gal4#1;UAS-tra patterns (P = 0.000001). (c–e) A still frame series of a wing extension followed by an aggressive lunge behavior. Wing vibration is not visible at this resolution or in a single clip, but see SI Movies 1 and 2. The clips show a Tβh^nM18 male approaching and starting a wing extension (c), continuing the wing extension (d), and demonstrating an aggressive lunge (e, arrow). (f–h) A still frame series of courtship behavior after a wing extension. These individual clips also are of a Tβh^nM18 male pair. One male is performing a wing extension and tapping (f), followed by abdomen bending (arrow) (g), and the courting male is rejected by the courted male (wing flick), and the wing extension ends (h) (see SI Movies 1 and 2).

In contrast, males with no OCT or low OCT levels transitioned from a wing extension to both aggressive and courtship behavioral patterns. Of the wing extensions performed by Tβh^nM18 (null) males, 42% were followed by aggressive behaviors and 46% were followed by tapping, licking, or abdomen bending as seen during courtship rituals. The courtship transitions are significantly more frequent in pairs of Tβh^nM18 males than pairs of control Tβh^M6 males (Fisher's exact probability test, two-tailed, P = 0.0002). Hypomorphic Tβh^MF372 males also transition to both courtship and aggression behavioral patterns, with courtship being the predominant behavior (Fig. 1 a and f–h). The transition pattern frequency in Tβh^MF372 males also is significantly different from that in control males (Fisher's exact probability test, two-tailed, P = 0.0006). These results suggest that, without OCT, the same sensory information can elicit two different behavioral responses: courtship or aggression.

Feminization of Male OCT/TYR Neurons.

Wing extensions during courtship are exclusively male behaviors (41). Therefore, having to make a transitional choice between the two behaviors only relates to male flies. To determine whether these neurons are important for appropriate male–male behavioral output, we feminized the small set of OCT/TYR neurons in an otherwise masculine CNS and asked whether a behavioral change occurs. Male neurons can be feminized by eliminating expression of Fru^M through RNAi (42) or by turning on the transformer (tra) gene. In these studies, we used the dTdc2(tyrosine decarboxylase)-Gal4 (27) line, which directs GAL4 expression in both OCT- and TYR-containing neurons, and crossed these to UAS-tra flies (43). We use the term OCT/TYR neurons to describe these cells because the dTdc2-Gal4 driver expresses in both, although only a subset of the OCT neurons may be of functional importance in the present studies (see below).

These partially feminized males (dTdc2-Gal4;UAS-tra) also direct wing extensions to other transgenic males but follow the wing extensions predominantly with courtship behavior (Fig. 1b). To rule out nonspecific Gal4 insertion effects, we mobilized the original dTdc2-Gal4 line, identified a new insertion line (dTdc2-Gal4#1) (see Materials and Methods), and repeated the experiment. The pattern frequencies from the experimental groups are significantly different from the control (UAS-tra/+, heterozygous progeny of the parent line) [Fisher's exact probability test, two-tailed, P = 0.0001 (dTdc2-Gal4#1;UAS-tra)], and these results confirm that at least a subset of the OCT/TYR neurons may be important in modulating sex-specific behavioral choice.

Cuticular Hydrocarbon Profiles.

Because the smell and sight of another male should present as threatening stimuli and suppress reproductive behavior, we tested the possibility that the OCT-deficient flies may not display normal male sensory cues. The system of pheromonal communication in Drosophila depends, at least in part, on fatty-acid-derived hydrocarbons that are present on the fly cuticle (cuticular hydrocarbons) (44). We specifically examined the levels of 7-tricosene (a male courtship inhibitor) (45) in Tβh^nM18, Tβh^MF372, and Tβh^M6 males and did not observe significant differences (SI Table 2).

OCT Modulates Male–Female and Male–Male Courtship Behavior.

If OCT modulates choice points between aggression and courtship in response to male sensory cues, then an additional possibility is that OCT mutant males may have difficulty distinguishing male and female sensory information. We tested this hypothesis in a series of courtship studies. First, we used a multiple male courtship assay in which two males of the same genotype were placed together with one CS virgin female. We asked whether control males (Tβh^M6), males that lack OCT (Tβh^nM18), or males with low OCT levels (Tβh^MF372) exhibited a courtship preference for a male or female partner by measuring the time a male spends courting the female or the second male during 10 min of interaction (see Materials and Methods and ref. 46). We specifically quantified time spent performing wing extensions/singing during this interval for two reasons. First, this behavior is easily scored, and, second, behaviors such as tapping (courtship) and fencing (aggression) when directed toward another male can be mistaken for the other. In addition, we used a 25-mm round chamber similar to that used by others (47) to clearly distinguish which fly was receiving the wing extension/song. The overall percentage of time spent singing in the 10-min assay to females by control males and males without OCT was quantitatively indistinguishable [Tβh^M6, 18.02 ± 1.25% (±SEM) versus Tβh^nM18, 15 ± 2.6%; P < 0.35, t test for independent samples), and these results compare with the courtship indices observed when total courtship behaviors were included (33, 47). In general, Tβh^MF372 males perform wing extensions significantly less (5.5 ± 2.3%; P < 0.001) than control males. The preference for females, however, was preserved.

Like the control Tβh^M6 males, the null Tβh^nM18 and hypomorph Tβh^MF372 males spend more time singing to females than to another male (Fig. 2a). However, Tβh^nM18 and Tβh^MF372 males also directed wing extensions to the second male for a significantly higher percentage of time than did control males (Fig. 2a). Control males directed wing extensions to another male 3% of the total time spent courting, whereas males with no or low OCT levels courted males 15.74% and 29.8% of the total time, respectively. Furthermore, males with feminized OCT/TYR neurons (dTdc2-Gal4; UAS-tra) also courted males for a greater percentage of time (17.2%) than controls (2.45%) (Fig. 2b). These results indicate that the abnormalities in modulating a sex-specific behavioral response observed in the aggression assays also are seen in the courtship assay setting.

Fig. 2.

OCT-deficient males court males significantly more than do controls. (a and b) We quantified the total time that a male performed wing extensions to a female or a second male during a 10-min interval in the multiple male assay. The box plot depicts the percentage of total time spent performing wing extensions first to females (red) and then males (blue). The upper and lower edges of the boxes correspond to the 25% and 75% quantiles. The median (50% quantile) is shown as a horizontal line in the box. The lines depict the 5% and 95% quantiles. Asterisk shows medians that are statistically different according to Wilcoxon rank sum for nonparametric data: Tβh^M6-Tβh^nM18 (P < 0.006), Tβh^M6-Tβh^MF372 (P < 0.001), and UAS-tra/+- dTdc2-Gal4;UAS-tra (P < 0.0001). Six assays were performed with dTdc2-Gal4;UAS-tra males and six with dTdc2-Gal4 #1;UAS-tra (new insertion) males. Results were not statistically different, and the data were pooled (n = 12 multiple male assays per line tested). (c and d) Box plot of the wing extension duration (in seconds) displayed by one male to a second fly (females are indicated in red and males are indicated in blue). The data were calculated by dividing the total wing extension time by the number of bouts for each male. The duration of male–male wing extensions by Tβh^M6 control males is significantly shorter than male–female wing extensions performed by the same males (Student's t test, P < 0.001) and male–male wing extensions performed by Tβh^nM18 and Tβh^MF372 males (asterisks; ANOVA for independent groups, P < 0.001). The duration of wing extensions to other males by UAS-tra/+ transgenic control males was significantly shorter (asterisk) than dTdc2-Gal4;UAS-tra males (Student's t test, P < 0.0001). The wing extension duration data were collected from the multiple male assays described above.

OCT Modulates Additional Courtship-Related Parameters.

Using the multiple male courtship assay, we examined several other parameters relating to male–male courtship. The average duration of individual bouts of the male–male wing extension was significantly longer in null and hypomorphic males (1.61 and 1.38 s) than in controls (0.70 s) (Fig. 2c). Likewise, the average wing extension duration in OCT/TYR-feminized males was significantly longer than in control males (1.31 vs. 0.5 s) (Fig. 2d). The duration of the wing extension directed toward males or females was indistinguishable in hypomorphic Tβh^MF372 and OCT/TYR-feminized males (Fig. 2). One possible explanation may be that control males respond to cues indicating that pursuit will not be fruitful and quickly stop singing, whereas OCT-deficient and TYR/OCT-feminized males are unable to properly process these cues.

We further tested the ability of OCT-deficient males to process courtship-related cues by analyzing male pair courtship latencies. Male courtship behavior is depressed in the presence of a mated female, and this modified behavior is not dependent on female rejection behavior or visual cues received by the male (48, 49). We measured the time after successful copulation by one male before courtship initiation by the second using virgin CS females and pairings of males of the same genotype (nulls and controls). Two courtship behaviors were scored: the first orienting and wing extension and the first abdomen bending (a part of attempted copulation). The latency before orienting and wing extension is significantly reduced to 53 ± 17 s (±SEM) in males without OCT (Tβh^nM18) compared with 186 ± 42 s with control Tβh^M6 males (P < 0.016). In addition, the latency before the next abdomen bending was also significantly shortened in Tβh^nM18 males (115 ± 48 s) compared with control Tβh^M6 pairs (511 ± 140 s) (P < 0.01).

The observed shortened courtship latency could be due to defects in processing courtship-inhibiting cues (49), or alternatively the Tβh^nM18 males that have copulated may not have transferred pheromonal peptides to the now-mated female (50, 51). To distinguish between these possibilities, we paired males of two different genotypes (Tβh^M6 and Tβh^nM18) with CS females under the same multiple male assay conditions. If control Tβh^M6 males copulated first, null Tβh^nM18 males still had a reduced latency before the first wing extension (101 ± 23 s; Kruskal–Wallis ANOVA followed by post hoc tests to identify significantly different groups, P < 0.05). These results suggest that males without OCT were not responding to the wild-type courtship-related pheromonal changes that should lead to a delay in courtship initiation by a second male.

Finally, we examined experimental and control males for the sustained intermale courtship that is manifested by chaining behavior (see SI Materials and Methods). None of the genotypes exhibited any intermale courtship of this type (data not shown), suggesting that the male cues (i.e., pheromones and visual and gustatory cues) in this controlled environment are at adequate levels to be processed sufficiently.

Fru^M Immunostaining in a Small Subset of OCT Neurons in the SOG.

Recent reports demonstrate that sexually dimorphic neural circuitry is specified by sex-specific splicing of fruitless (52). Therefore, we asked whether Fru^M and OCT are coexpressed in a subset of OCT/TYR neurons. We found that Fru^M colocalizes with TYR or OCT in a small number of dTdc2-Gal4;UAS-mCD8:GFP neurons in the SOG (Fig. 3). The SOG neuropil is a first-order sensory-receiving area for gustatory information (53). Fru^M expression is not seen in these neurons in the feminized dTdc2-Gal4;UAS-tra males. As seen in Fig. 3a and as previously described for OCT (29), OCT/TYR neurons project extensively to many brain regions, where they densely innervate the synaptic neuropil. We found Fru^M and GFP-reporter coexpression in three ventral unpaired median (VUM) neurons of the SOG (Fig. 3 b and c). Neurons of the VUM clusters have been designated as unpaired, although several appear as close pairs (29). Two Fru^M–GFP-positive neurons are located in the VUM 1 cluster (Fig. 3b), and one is located in the VUM 2 cluster (Fig. 3c). To verify that these are octopaminergic neurons, 8-μm sections from dTdc2-Gal4;UAS-mCD8:GFP adult brains were labeled with the monoclonal OA1 antibody (68) and anti-GFP (Fig. 3d). Sections containing the VUM clusters were examined from >10 adult male brains, and in all cases we observed complete overlap of GFP-reporter and OCT immunostaining. Our analysis of the VUM neurons using dTdc2-Gal4 driven GFP reporter expression is comparable with the detailed analysis of OCT immunoreactivity previously reported (29).

Fig. 3.

OCT and the male-specific form of Fruitless are coexpressed in VUM neurons of the SOG. (a) Confocal sections of a transgenic dTdc2-Gal4;UAS-mCD8:GFP adult male brain labeled with anti-GFP (green) and mAb nc82 (red labels neuropil regions) antibodies. The SOG region containing neurons coexpressing Fru^M and OCT is highlighted by the white box. The arrow points to the VUM 1 cluster highlighted in b, and the arrowhead points to the VUM 2 cluster in c and d. (b) Confocal sections from a dTdc2-Gal4;UAS-mCD8:GFP transgenic male labeled with anti-Fru^M (red) antiserum and anti-GFP antibody (green). Two VUM 1 cluster neurons (arrows) located in the SOG coexpress Fru^M and the GFP reporter. The GFP expression is weak in the nuclei of these VUM neurons; therefore, overlap does not appear yellow (n = 9). (c, c′, and c″) A VUM 2 cluster neuron from a dTdc2-Gal4;UAS-mCD8:GFP transgenic male coexpressing FruM (red) (c′) and GFP (green) (c″) (n = 9). (d, d′, and d″) An adult male brain section (dTdc2-Gal4;UAS-mCD8:GFP) depicting neurons of the VUM 2 cluster stained with mAb OA1 (red) (d′) and anti-GFP (green) (d″) (n = 10).

To visualize the arborizations of individual Fru^M-positive OCT neurons, we used the FLP-out technique (54, 55). In one of the resulting preparations, a single VUM 1 Fru^M-positive OCT neuron was identified. The primary neurite (Fig. 4a, arrow) of this neuron divides into two symmetrical secondary neurites that in turn generate many layers of extensive arborizations. Arborizations from this single VUM 1 Fru^M-positive OCT neuron extensively ramify throughout several neuropil regions, including the SOG, posterior antennal lobe, and the lateral accessory lobe (ventral body) (Fig. 4 b and c). This pattern of arborization suggests a means of integrating information from multiple sensory modalities: gustatory, olfactory, and possibly visual (69).

Fig. 4.

Arborizations of a single Fru^M OCT VUM 1 SOG neuron. (a) Composite of four 1-μm optical sections containing the cell body of a VUM 1 cluster Fru^M-positive hs-FLP;dTdc2-Gal4;UAS<-CD2, y⁺>CD8-GFP neuron. The arrow identifies the primary neurite. (Inset) Fru^M and GFP-reporter colocalization. (b) Composite of 18 1-μm optical sections of the VUM 1 cluster Fru^M–GFP-positive neuron identified in a. This region of dense arborization is located 12 μm ventrally to the cell body. The primary neurite branches into two symmetrical secondary neurite arbors at the arrow. Arborizations extensively ramify throughout the SOG. (c) Twenty 1-μm sections of a continued group of SOG arborizations from the VUM 1 cluster Fru^M–GFP-positive neuron. This arborization is located ventrally to the clusters observed in b.

Discussion

Males and females react to environmental cues with distinct sex-specific innate behaviors particularly in the areas of courtship/reproduction and aggression/defense. Results from a number of studies have demonstrated that functional and structural sex differences in the brain can influence and direct these behaviors (56–59), but how sensory cues contribute to the appropriate response of one of these two mutually exclusive behaviors remains unclear. Here we present evidence that the neuromodulator OCT functions within a defined circuit to provide at least one means of regulating the choice between courtship and aggression. The results of our aggression studies indicate that male flies require OCT to respond with an appropriate aggressive response to another male. The results of our male–female courtship assays suggest that normal OCT function provides increased behavioral response confidence about the sensory cues being presented.

Identifying a potential mate or opponent relies on discriminating specific stimuli from background and then integrating this information with other sensory modalities. Anatomically, the extensive arrays of OCT-immunoreactive processes that are found throughout the Drosophila brain offer one such overlying integration network that may fine-tune sensory input and activate sex-specific behavioral subcircuits. In Drosophila, male-specific behavioral circuits are specified by the male-specific products of the fruitless gene (33–35). In this study, we demonstrate that three VUM neurons in the male SOG coexpress Fru^M and OCT. The SOG is the primary taste-processing center in the fly (53). The sensory information sent to this neuropil includes the female pheromone recognition cues necessary for male courtship behavior (47). Therefore, an intriguing possibility is that OCT is necessary in the subset of Fru^M-positive SOG neurons to accurately relay contact gustatory pheromone information.

Our morphological results suggest that a single neuron can provide a simple integration network of multisensory cues. We show that the arborizations of one of the VUM 1 Fru^M-positive OCT neurons extensively ramify throughout multiple neuropil regions, including the SOG, posterior antennal lobe, and the lateral accessory lobe (ventral body), suggesting a link between various sensory modalities. Gustatory information from OCT/Fru^M SOG neurons could also be linked to higher-order processing centers through synaptic contacts with the male-specific SOG projections of Fru^M-expressing mAL neurons identified by ref. 52. The superior lateral protocerebrum has been proposed to be the output site of these interneurons (52). Linkages of this type may be of particular significance because Fru^M-expressing neurons play critical roles in two sex-specific social behaviors: aggression and courtship (33–35). Thus, the same circuits may need to integrate the context-specific sensory information necessary to direct the output of appropriate behavioral patterns.

How might OCT modify distinct SOG neurons to regulate behavioral choice by males? In the spider, OCT increases the overall sensitivity of mechanosensory neurons by local release from efferent endings (60). This local release suggests that sensory input from specific sensilla relative to others can be emphasized depending on behavioral circumstances. In the silkworm moth, OCT specifically increases the sensitivity of male pheromone-sensitive receptor neurons but not general odorant-sensitive responses (61). Recent modeling studies in vertebrates suggest that neuromodulators can play a key role at specific times in decision-making tasks by regulating competition between populations of neurons that represent choices (62). This regulation may allow an organism to integrate noisy sensory information and past experience to make optimal decisions.

Although the mouse neural pathways that mediate the output of two sex-specific behaviors, reproduction and defense, are anatomically segregated (63), a recent study identified a hypothalamic point of convergence that may function as a choice selection mechanism for sensory activation of defensive responses over reproduction (64). The results suggest that whether an individual male mouse responds with the appropriate behavior depends on the coordinated activation of the appropriate subcircuits by amygdalo–hypothalamic projections. Likewise the different behavioral outputs of Drosophila males and females could be generated through the activation of sex-specific segregated neural ensembles. However, behavioral differences also could emerge through sex-specific modulation of circuits that are common to both sexes. In males, Fru^M proteins are expressed in small groups of neurons throughout the CNS, and eliminating Fru^M expression in a neuronal subset has profound effects on the progression of male courtship behaviors (42). At the gross level almost all of the Fru^M-producing neurons have counterparts in the female and in terms of function, a recent report indicates that the sex-specific reproductive behaviors of females and males involve shared neural circuits (65). The splicing of fru^M-specific transcripts have been proposed to modify neurons common in both sexes for male-specific functions through differences in neuron morphology and/or physiology (33, 34, 52, 66).

In addition to changing the activity of OCT neurons, we feminized OCT/TYR neurons in an otherwise masculine brain and demonstrated altered male behavioral choice. Our results from OCT immunostaining do not indicate any sex-dependent changes in SOG neuron numbers (S.J.C. and E.A.K., unpublished data), nor were any sex-specific changes described in ref. 29. The identification of a sex-independent marker for the Fru^M-positive OCT neurons should allow us to determine whether feminizing these neurons changes either their branching patterns, their synaptic connections, or their OCT-related biochemical properties. Further examination of these OCT/Fru^M SOG neurons should offer a behaviorally relevant ensemble with which to address questions of sex-specific morphology and function-related physiology.

Materials and Methods

Fly Stocks.

The following strains were used in this study: the Tyramine β-hydroxylase-null allele, Tβh^nM18, the control Tβh^M6, the hypomorphic allele Tβh^MF372 from M. Monastirioti (Institute of Molecular Biology, Crete, Greece) (38), the Canton-S and UAS-tra lines from the Bloomington Stock Center (Bloomington, IN), and the dTdc2-Gal4 line from J. Hirsh (University of Virginia, Charlottesville, VA) (27). The Tβh^nM18 null allele and the Tβh^M6 control allele were previously generated (38) as a result of P-element manipulations on the same chromosome. Both the original Tβh^nM18 and the Tβh^M6 stock carried an additional mutation in the w (white) gene. To eliminate any behavioral performance effects caused by different eye pigmentation and to avoid possible amine deficiencies resulting from the w background, the lines were individually crossed with CS and recombinant w⁺ Tβh^nM18 and w⁺ Tβh^M6 chromosomes were isolated. More specific information on isolating these recombinant chromosomes can be found in SI Materials and Methods.

Immunohistochemistry.

Staged pupal and adult male and female dissected brains were labeled by using a previously described protocol (67). The following primary antibodies were used: rabbit anti-GFP (1:500) (Molecular Probes, Eugene, OR), mouse anti-GFP (1:100) (Molecular Probes), rabbit anti-Fru^M (1:3000) (66), mAb OA1 (1:1,000) (68), mAb nc82 (1:100) (A. Hofbauer, Universitaet Regensburg, Regensburg, Germany). Secondary antibodies include Alexa Fluor 488- and Alexa Fluor 594-conjugated cross-adsorbed antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). A detailed description of the adult brain section labeling can be found in SI Materials and Methods.

Behavioral Assays.

Flies were raised on standard cornmeal medium and kept on a 12-h/12-h day/night cycle at 25°C in 50% relative humidity. To collect socially naïve adults, pupae were isolated in individual 16- × 100-mm glass vials containing 1.5 ml of food medium. Upon eclosion, flies were anesthetized with CO₂, painted on the thorax with acrylic paint for identification, and returned to their isolation vials. Flies were maintained for 2–3 additional days to age and allow for recovery from anesthesia before testing. All flies were 3–5 days old at the time of testing, and each pair of flies used in a courtship or aggression assay were the same age. Previous experiments (40) and references therein demonstrated that reliable fighting can be observed after 3 days of isolation.

Aggression and Courtship Assays.

Fights between pairs of males were conducted by using a chamber and experimental conditions as previously described (39). All courtship assays were performed in a 12-well polystyrene plate (catalog no. 82050-930; VWR, San Francisco, CA) with minor modifications from previously described conditions (46). Additional assay details are described in SI Materials and Methods.

Statistical Analyses.

For the data that were not normally distributed, means were statistically compared by using the Wilcoxon rank sum test for nonparametric data. For groups of data that were not normally distributed, the nonparametric Kruskal–Wallis ANOVA for unpaired groups was used followed by a post hoc Wilcoxon rank sum test to identify the significantly different groups. Normally distributed data were analyzed by using Student's t test. All statistical analyses were performed with JMP IN software, Version 5.1 (SAS Institute, Cary, NC) or VasserStat (http://faculty.vassar.edu/lowry/VassarStats.html).

Abbreviations

OCT

octopamine

TYR

tyramine

SOG

suboesophageal ganglion

CS

Canton-S

VUM

ventral unpaired median.