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. Author manuscript; available in PMC: 2009 Sep 22.
Published in final edited form as: Neuron. 2009 Feb 26;61(4):519–526. doi: 10.1016/j.neuron.2008.12.021

Control of the Postmating Behavioral Switch in Drosophila Females by Internal Sensory Neurons

Chung-hui Yang 1,3, Sebastian Rumpf 1,3, Yang Xiang 1, Michael D Gordon 2, Wei Song 1, Lily Y Jan 1, Yuh-Nung Jan 1,*
PMCID: PMC2748846  NIHMSID: NIHMS119053  PMID: 19249273

SUMMARY

Mating induces changes in the receptivity and egg-laying behavior in Drosophila females, primarily due to a peptide pheromone called sex peptide which is transferred with the sperm into the female reproductive tract during copulation. Whereas sex peptide is generally believed to modulate fruitless-GAL4-expressing neurons in the central nervous system to produce behavioral changes, we found that six to eight sensory neurons on the reproductive tract labeled by both ppk-GAL4 and fruitless-GAL4 can sense sex peptide to control the induction of postmating behaviors. In these sensory neurons, sex peptide appears to act through Pertussis toxin-sensitive G proteins and suppression of protein kinase A activity to reduce synaptic output. Our results uncover a neuronal mechanism by which sex peptide exerts its control over reproductive behaviors in Drosophila females.

INTRODUCTION

Mating induces significant behavioral changes in the females of many species. In rodents, for example, pregnancy triggers behaviors such as nest-building and enhanced aggression toward intruders (Broida and Svare, 1982; Ogawa and Makino, 1984). The ability to produce these mating- or pregnancy-induced behaviors is likely hardwired into the female nervous system and crucial for the reproductive success of the species. How the female nervous system orchestrates these behavioral alterations in response to changes in reproductive status is an unsolved problem in neurobiology. Because reproductive hormones play an important role in producing these changes (Brunton and Russell, 2008), identifying neuronal targets of reproductive hormones and the molecular and cellular mechanisms by which these hormones signal may provide a critical entry point into dissecting the circuitry of female reproductive behaviors.

It has long been established that Drosophila females exhibit stereotypical behavioral changes after copulation: whereas virgin Drosophila females are receptive toward courting males and lay few eggs, mated females reject males by running away, kicking, and extruding their ovipositor, and they also lay many eggs (Kubli, 2003). These behavioral changes are largely induced by Sex peptide (SP), a peptide pheromone produced in the male accessory gland and cotransferred into female reproductive tract together with the sperm during copulation (Aigaki et al., 1991; Chapman et al., 2003; Chen et al., 1988; Liu and Kubli, 2003). A G protein-coupled receptor called SPR that responds to SP in vitro was recently identified and was shown to be required for SP-mediated postmating responses (Yapici et al., 2008).

The neuroanatomical targets of SP are contained within a group of ~2000 neurons in the female nervous system that are labeled by the marker fruitless-GAL4 (Yapici et al., 2008). fruitless (fru) was originally identified as a critical regulator of male mating behavior in Drosophila (Hall, 1978; Ryner et al., 1996). Analysis of fru-GAL4, created by “knockin” of GAL4 into the fru-P1 locus as a means to uncover the anatomical substrates of male sexual behaviors, revealed that fru-GAL4 is expressed in ~2000 neurons in the central and peripheral nervous system in both sexes (Manoli et al., 2005; Stockinger et al., 2005). Silencing these neurons renders the males unable to court (Manoli et al., 2005; Stockinger et al., 2005). Interestingly, like their homologs in the male, fru-GAL4 neurons apparently also control sexual behaviors in the female: inhibiting their transmitter release with the conditional dominant-negative dynamin allele shibirets (Kitamoto, 2001) causes virgin females to reject courting males and increase their egg-laying rate (Kvitsiani and Dickson, 2006), hallmarks of SP-induced postmating behaviors. In addition, SPR was found to be expressed in many fru-GAL4 neurons in the central nervous system (CNS) and removal of SPR from these neurons causes mated females to behave as virgins, suggesting that SP acts on fru-GAL4 neurons to control postmating behaviors (Yapici et al., 2008).

Despite the finding that SP target neurons are labeled by fru-GAL4, the neuronal mechanisms by which SP causes behavioral changes remain largely unknown. Although the prevailing view is that SP can exit the reproductive tract and diffuse in the circulating hemolymph to reach its target neurons in the CNS so as to modify behaviors (Kubli, 2003; Yapici et al., 2008), we do not know the identity of the SPR- or fru-GAL4-expressing neurons that regulate postmating behaviors. It is also unclear whether SP acts on one set of neurons to produce rejection and on another to produce egg laying or whether it may act on just one set of SP sensors that then produces the ensemble of behavioral changes by regulating other neurons. In order to identify SP targets, we searched for GAL4 lines that produce postmating responses when expressing a membrane-bound, nondiffusible version of SP (Nakayama et al., 1997). We found that, besides fru-GAL4, the sensory neuron driver pickpocket (ppk)-GAL4 (Grueber et al., 2007) labels SP-responsive neurons. Only a few neurons on the female reproductive tract are labeled by both ppk-GAL4 and fru-GAL4, and we demonstrate that SP signaling through these fru/ppk double positive neurons is necessary and largely sufficient to induce egg laying and rejection, suggesting these fru/ppk neurons sense SP to control the full set of postmating behaviors. In addition, we show that inhibiting Pertussis toxin-sensitive trimeric G proteins or activating protein kinase A (PKA) in these fru/ppk neurons suppresses postmating behaviors in mated females, whereas inhibiting PKA activity or preventing transmitter release from these neurons induces postmating behavior in virgins. Our results uncover a critical neuronal substrate and signaling mechanism by which SP exerts its control over female postmating behaviors.

RESULTS AND DISCUSSION

mSP Expression Identifies ppk-gal4 as a Driver that Labels SP Target Neurons

In order to identify SP-responsive neurons, we took advantage of a membrane-bound sex peptide (referred to as mSP) that has been shown to elicit postmating responses when expressed in virgin females. Importantly, generation of postmating behaviors by mSP (UAS-mSP) depends more critically on its expression pattern than diffusible transgenic SP (UAS-SP) (Nakayama et al., 1997), suggesting it may activate SPR in an “autocrine” manner. To test the utility of mSP, we first expressed it under the control of fru-GAL4. In agreement with the requirement of SPR expression in fru neurons for postmating responses (Yapici et al., 2008), expression of mSP under the control of fru-GAL4 in virgin females led to a strong decrease in receptivity—accompanied by ovipositor protrusion and other rejection behaviors—and to an increase in egg laying (Figures 1A and 1B), to a comparable extent to mated wild-type females (see Figure S1 available online). Moreover, we found that expressing GAL80, a GAL4 inhibitor, specifically in neurons effectively abolished postmating responses elicited by fru-GAL4-directed mSP expression (Figures 1A and 1B), indicating that the observed effects were indeed due to neuronal mSP expression. Expression of mSP under Or67d-GAL4 or ILP7-GAL4, two drivers that label subsets of fru neurons involved in pheromone detection and egg laying (Kurtovic et al., 2007; Yang et al., 2008), respectively, did not elicit postmating behaviors (Figures 1A and 1B), suggesting that induction of postmating behaviors in response to mSP expression carries specificity and is not a general property of all fru neurons.

Figure 1. ppk-GAL4 Labels SP-Responsive Neurons.

Figure 1

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(A and B) Behavioral effects of mSP expression in fru neurons or subsets thereof (by means of fru-GAL4, Or67d-GAL4, or ILP7–GAL4) and ppk-GAL4 on mating behavior and egg laying in virgin females. (A) Receptivity of virgin females, with the score for rejection behavior as well as genotype of experimental animals given below the graph. In each assay, one female of the indicated genotype was confronted with two naive males in a small chamber. Females were scored as receptive when they mated within 20 minutes. Numbers in parentheses are the numbers of females tested. For scoring rejection behavior, ++ indicates frequent rejection behaviors (such as ovipositor protrusion, kicking, wing flicking or running away) when the females were courted, +/− indicates some rejection behaviors, – indicates absence of rejection. **p < 0.005, ***p < 0.0005, two-tailed Fisher’s exact test. (B) Egg laying. For each assay, five females of the indicated group were allowed to lay eggs in a vial with grape media at 25°C. Eggs were counted after 24 hr and the number of eggs in each vial was divided by five. n = 15 assays for all genotypes, error bars indicate SEM, *p < 0.05, ***p < 0.0005, Student’s t test.

(C and D) Knockdown of SPR in ppk neurons reduces postmating behaviors in mated females. (C) Effect of SPR RNAi (UAS-SPR-IR) in ppk neurons on receptivity of virgin and mated females. The receptivity of virgins of the indicated genotypes was assayed as in (A), and receptivity of the same females after mating was assessed 24 hr later. **p < 0.005, ***p < 0.0005, Fisher’s exact test. v and m indicate virgin and mated, respectively. (D) Effect of SPR RNAi expression under ppk-GAL4 on egg laying of virgins or mated females. Egg laying was scored as in (B), but with three females per vial, *p < 0.05, **p < 0.005, ***p < 0.0005, Student’s t test. n = 15 for each genotype. In (C) and (D), wild type (w1118) females that were mated to SP mutant males (SP0/D) or control males (SP+/D) were included as additional controls.

(E and F) SPR expression in ppk neurons rescues the behavioral defects of the SPR-deficient line Df(1)Exel6234 (DSPR). (E) Mating behavior of control Df(1)Exel6234 females and Df(1)Exel6234 females expressing SPR in ppk neurons. Mating assays were performed as in (C). (F) Egg-laying behavior of females of the same genotype was determined as in (D).

We next searched for GAL4 drivers that induce postmating behaviors in virgins when directing mSP expression and identified pickpocket (ppk)-GAL4 (Figures 1A and 1B). This driver had previously been shown to label the peripheral sensory class IV dendritic arborization (da) neurons that cover the larval body wall (Grueber et al., 2003, 2007). In order to verify that ppk-GAL4 indeed drives expression in SP target neurons, we used RNAi to specifically knock down the expression of SPR (Yapici et al., 2008) in ppk-expressing neurons. Whereas virgin females with reduced SPR displayed normal receptivity and a low egg-laying rate, mated females expressing SPR RNAi (UAS-SPR-IR) in ppk neurons no longer curtailed their receptivity to courting males (Figure 1C) and did not increase their egg laying (Figure 1D), similar to females that had been mated to SP mutant males (in fact, their receptivity was even higher, probably because SPR can also be activated by the less effective SP homolog DUP99B that remains in SP mutant animals). Furthermore, SPR expression driven by ppk-GAL4 also restored postmating behaviors to mated SPR mutant females (D SPR, Figures 1E and 1F), demonstrating that SPR expression in ppk neurons is necessary and sufficient for SP-induced postmating behaviors. Thus, ppk-GAL4 labels SP target neurons that are critical mediators of SP-induced postmating behaviors.

ppk-gal4 Labels Peripheral Sensory Neurons on the Reproductive Tract

In order to assess the expression pattern of ppk-GAL4 in adult females, we next expressed a membrane-bound green fluorescent protein (UAS-mCD8-GFP) and a nuclear red fluorescent protein (DsRed, UAS-Red stinger) under the control of ppk-GAL4. No cell bodies in either the adult brain or VNC exhibited DsRed-positive nuclei (Figures 2A and 2B), but there were many mCD8-positive neuronal processes projecting from peripheral tissues into the VNC and a smaller number of processes into brain (Figures 2A and 2B). These observations indicate that ppk-GAL4 drives expression exclusively in peripheral neurons in adults. Because the neuronal processes projected into the VNC were particularly dense in the abdominal segment—a likely target site for sensory neurons on the reproductive organs—we next assessed if ppk-GAL4 labels neurons on these organs. We found several groups of ppk-positive sensory neurons on the oviducts and the uterus (Figures 2C and 2D). On each lateral oviduct, there were two ppk neurons which covered the oviduct with their dendrites (Figure 2E). In addition, there were approximately thirty ppk neurons on the uterus (Figure 2F). These neurons were organized in three tightly packed clusters along each side of the uterus (numbered in Figure 2F). The first, most anterior cluster (“1” in Figure 2F) was positioned at the top of the uterus and contained approximately three neurons (two of which are visible on each side in Figure 2F) with long dendrites lining the common oviduct (Figures 2F, 2D, and inset 2D′). The second, slightly more posterior clusters contained approximately seven neurons and the third clusters, positioned close to the ovipositor, contained approximately five neurons. The close proximity of these ppk-positive internal sensory neurons to the entry site of SP made them particularly good candidates as SP sensors.

Figure 2. ppk-GAL4 and fru-GAL4 Expression Overlap in Sensory Neurons on the Female Reproductive Tract.

Figure 2

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In (A–F), cell bodies and cellular processes were visualized by expression of nuclear DsRed (UAS-Red stinger) and membrane bound GFP (UAS-mCD8-GFP) under the control of ppk-GAL4, followed by mCD8 staining.

(A and B) Projections, but no cell bodies of ppk neurons are present in the brain (A) and the ventral nerve chord (VNC) (B).

(C) Schematic diagram of the Drosophila female reproductive tract. Green dots indicate the positions of ppk-positive neurons, yellow dots those of fru/ppk neurons.

(D–F) ppk-positive neurons on the female reproductive tract. (D) Overview of a reproductive tract with stained ppk neurons. The inset (D′) is a magnified picture of the common oviduct that shows dendrites emanating from ppk neurons on the uterus. (E) and (F) show the lateral oviducts and the uterus, respectively. In (E), cell bodies are marked by arrows. Scale bars are 300, 75, and 100 µm in (D), (E), and (F), respectively. In (F), the ppk neuron clusters are numbered as described in the text.

(G–G" and H–H") Overlap between fru and ppk markers on the lateral oviducts (G) or the uterus (H). ppk promoter activity was visualized with ppk-eGFP and immunofluorescence with anti-GFP antibodies. fru neurons were visualized by fru-GAL4 and UAS-Red stinger (nuclear DsRed). fru/ppk neurons are marked with arrows, ppk neurons by asterisks. The inset in (H) and (H") shows the second ppk cluster on the uterus at higher magnification.

Abbreviations: com. ov., common oviduct, lat. ov., lateral oviduct.

A Subset of Sensory Neurons on the Reproductive Tract Expresses Both fru-gal4 and ppk-gal4

Because the ppk-GAL4 neurons on the reproductive tract were potential SP targets, we wondered whether these neurons were also labeled by fru-GAL4, which had previously been shown to label SP target neurons (Yapici et al., 2008). To address this question, we used fru-GAL4 to drive expression of nuclear DsRed and examined the reproductive tract. We found eight fru-positive sensory neurons in similar positions to those of a subset of the ppk neurons on the female reproductive tract. There was one fru neuron on each of the lateral oviducts (Figure 2G), and three densely packed fru neurons on each side of the uterus, in a similar position as the second ppk neuron cluster (Figure 2H). To determine whether any of these neurons were positive for both fru and ppk expression, we colabeled ppk neurons with a ppk-eGFP transgene (Grueber et al., 2003). All eight fru neurons on the reproductive tract showed both GFP and DsRed signal (Figures 2G–G” and 2H–2H”), indicating coexpression of the fru and ppk markers. Furthermore, we could not find neurons with overlapping fru and ppk expression in any other tissue (Figure S2; Table S1), indicating that these reproductive tract neurons are the only fru/ppk neurons. In keeping with the idea that the fru/ppk neurons could be SP targets, we also found that all eight express SPR (Figures S3A–S3A” and S3–S3B”). Interestingly, in these neurons, SPR localized predominantly to axons rather than the soma or dendrites. This axonal localization of SPR could be recapitulated in a heterologous system, as ectopic SPR expression in larval ppk neurons also yielded enriched expression in axons as compared with dendrites (Figures S3C–S3C”). Finally, fru/ppk neurons might be sexually dimorphic, as we could only find two on the reproductive system in males (Figure S4). This difference in numbers could also reflect a sex-specific function. Taken together, marker expression and anatomical localization suggest that these eight fru/ppk neurons on the reproductive tract could be SP targets.

ppk/fru Double-Positive Neurons Are Crucial for Postmating Behavioral Switch

Next, we asked whether the fru/ppk neurons we identified were required for the behavioral effects of mSP or SPR RNAi expression. To this end, we expressed GAL80 under the control of the ppk promoter (ppk-GAL80). ppk-GAL80, an effective inhibitor of ppk-GAL4, specifically blocked the fru-GAL4-directed expression in the fru/ppk neurons on the reproductive tract, but left intact fru-GAL4-dircted expression in central neurons in the VNC (Figures 3A–3F). We next asked whether its presence would suppress the behavioral phenotypes induced by mSP or SPR RNAi expression via fru-GAL4. Indeed, ppk-GAL80 largely suppressed egg laying and restored receptivity in virgin females with mSP expression driven by fru-GAL4 (Figures 3G and 3H), and it restored rejection and egg laying in mated females expressing SPR RNAi under the control of fru-GAL4 (Figures 3I and 3J). Thus, expression of mSP or SPR RNAi in fru/ppk neurons is necessary for producing the behavioral phenotypes caused by the manipulation with fru-GAL4, indicating that these neurons are SP target neurons necessary for the induction of postmating behaviors.

Figure 3. fru/ppk Double-Positive Neurons Are Important Mediators of Postmating Behaviors.

Figure 3

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(A–F) Characterization of ppk-GAL80. (A and B) ppk-GAL80 suppresses UAS-mCD-8GFP expression driven by ppk-GAL4. VNCs of ppk-GAL4, UAS-mCD8GFP (A) or ppk-GAL4, UAS-mCD8GFP, ppk-GAL80 females (B) were stained with anti-mCD8 antibodies (green) and neurons with anti-HRP (red). (C and D) ppk-GAL80 does not affect general fru-GAL4 activity in the VNC. fru-GAL4 was used to drive UAS-Red stinger in the absence (C) or presence (D) of ppk-GAL80. Neurons were stained with HRP antibodies (green). (E and F) ppk-GAL80 suppresses fru-GAL4 activity on the reproductive tract. Shown are reproductive tracts of fru-GAL4, UAS-Red stinger females (E) or fru-GAL4, UAS-Red stinger, ppk-GAL80 females (F). Positions of Red stinger-positive or -negative neurons are marked by arrows. Neurons were counterstained with anti-HRP antibodies (green).

(G–J) ppk-GAL80 suppresses postmating responses induced by fru-GAL4 and UAS-mSP and restores postmating behavior after SPR RNAi under fru-GAL4. (G) Female receptivity was scored as in Figure 1A. ***p < 0.0005, Fisher’s exact test. (H) Egg laying was scored as in Figure 1B (n = 17). ***p < 0.0005, Student’s t test. Error bar indicates SEM. (I and J) UAS-SPR-IR (SPR RNAi) driven by fru-GAL4 suppresses postmating behaviors in mated females, and ppk-GAL80 restores them. (I) Receptivity. Two to three mated females were put in a vial with three to four naive males and allowed to mate for 20 min. Vials were inspected for copulating animals after 5, 10, and 20 min. ***p < 0.0005, two-tailed Fisher’s exact test. (J) Egg laying. Three mated females per vial were allowed to lay eggs in a grape vial for 24 hr. n = 15, ***p < 0.0005, Student’s t test. Error bar indicates SEM.

(K and L) Clonal mSP expression in fru neurons and behavioral analysis. (K) Experimental outline. (L) Pictures of the uterus region of receptive and rejecting flies, respectively. ***p < 0.0005, n = 15, Student’s t test. Errors indicate SEM.

In order to obtain more direct evidence that postmating behavioral switch depends critically on the fru/ppk neurons on the reproductive tract, we also expressed mSP and mCD8-GFP in fru neurons randomly. If mSP expression on the reproductive tract fru/ppk neurons was causal to the induction of postmating behaviors, one would expect that virgin females that display postmating behaviors will show consistent labeling of fru/ppk neurons while virgins that do not display postmating behaviors will have no or less labeling of these cells. To this end, we prevented fru-GAL4-driven mSP and mCD8-GFP expression with a tub-FRT-GAL80-FRT construct (Gordon and Scott, 2009) and then removed GAL80 in random subsets of fru neurons via FLP-mediated recombination. Next, we tested females for their mating behavior and selected two groups: those that mated quickly (within 5 min after contact with males) and those that showed rejection behaviors and did not mate within the duration of the experiment (30 min) (Figures 3K and 3L). We then analyzed mCD8-GFP expression on the reproductive organs and found that receptive females showed little mCD8-GFP expression on the reproductive tract (~0.3 cells out of six on the uterus, n = 15), but the rejecting females consistently showed strong labeling in cells on the uterus (~3.5 cells, n = 15; Figure 3L). In contrast, we did not find consistent expression patterns in the VNC in either set of animals (data not shown). In conclusion, our results indicate that fru/ppk neurons on the reproductive tract are critical SP sensors that control postmating behaviors.

Silencing fru/ppk Neurons Induces Postmating Behaviors in Virgins

Having established fru/ppk neurons on the reproductive tract as sensors for SP, we next asked how SP influences fru/ppk neurons to induce postmating behaviors. Inhibition of neuronal transmission in fru neurons with the temperature-sensitive dominant-negative dynamin mutant (shibirets) that reduces transmitter release has been shown to induce postmating behaviors (Kvitsiani and Dickson, 2006). This specific induction of postmating behavior suggested that only one or a few subgroups of fru neurons might underlie this effect. Therefore, we tested the hypothesis that SP inhibits fru/ppk neuronal activity to generate postmating behaviors. First, we silenced ppk neurons by expressing shibirets. This manipulation induced postmating responses in virgins at the restrictive temperature: a reduction in receptivity (Figure 4A) and an increase in the egg-laying rate (Figure 4B). Silencing ppk neurons with Kir2.1, a hyperpolarizing potassium channel (Baines et al., 2001) also greatly reduced virgin receptivity and increased oocyte production as evidenced by the greatly increased size of the females’ ovaries (not shown) but actually caused a block in egg laying due to eggs getting jammed in the oviducts, perhaps because some of the fru-negative ppk neurons are involved directly in the egg transport process within the oviduct (Figure S5).

Figure 4. Silencing Neuronal Activity in fru/ppk Neurons Induces Postmating Behaviors.

Figure 4

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(A and B) Behavioral effects induced by expression of shibirets in ppk neurons. (A) The receptivity of virgin females at the restrictive (32°C) or permissive (20°C) temperatures was assayed as in Figure 3I, *p < 0.05, ***p < 0.0005, Fisher’s exact test. (B) Virgin egg laying at the restrictive (29°C) or permissive (20°C) temperatures, as in Figure 1B, but with three females per vial, ***p < 0.0005, Student’s t test (n = 15). Error bar indicates SEM.

(C–F) Behavioral effects of silencing fru neurons with shibirets and influence of ppk-GAL80. (C and D) Virgin receptivity was assayed as in Figure 3G. For the experiments at the restrictive temperature, vials were preincubated for one hour in a water bath, and then put back into the water bath after addition of-males. **p < 0.005, ***p < 0.0005, Fisher’s exact test. (E and F) Virgin egg laying at 20°C (E) or 29°C (F) was determined as above. **p < 0.005, ***p < 0.0005, Student’s t test, (n = 18 for 20°C, n = 19 for 29°C). Error bar indicates SEM.

Could the induction of postmating behaviors again be attributed to inhibition of fru/ppk neurons? To address this question, we silenced fru neurons with shibirets and then examined the effect of ppk-GAL80 suppression of the fru-GAL4-mediated transgene expression in fru/ppk neurons. Virgin females expressing shibirets in fru neurons strongly rejected courting males and displayed a high egg-laying rate at the restrictive temperature. Remarkably, counteracting fru-GAL4 activity in ppk neurons with ppk-GAL80 restored virgin receptivity significantly (Figures 4C and 4D) and suppressed the virgin egg-laying phenotype (Figures 4E and 4F). Moreover, when Kir2.1-GFP was expressed randomly in ppk neurons in virgins (similar as in Figures 3K and 3L), animals that displayed postmating responses also showed consistent Kir2.1-GFP expression in ppk neurons on the uterus but not in other ppk neurons. This procedure also allowed us to show that the axons of the uterus ppk neurons project to the tip of the abdominal ganglion of the VNC (Figure S6).

Taken together, these results show that inhibition of the fru/ppk neurons on the reproductive tract induces postmating behaviors. Moreover, because silencing of fru/ppk neurons in virgins causes behavioral changes that are very similar to those due to mating or mSP expression in the same neurons, SP most likely acts by reducing neuronal activity and/or synaptic outputs of fru/ppk neurons to produce postmating behaviors.

Signaling through Gαi and PKA Is Required for Postmating Behaviors

How might SP reduce fru/ppk neuronal activity? It has been shown that SPR, a G protein-coupled receptor, can activate trimeric G proteins with Gαi or Gαo subunits in vitro (Yapici et al., 2008). To test for the involvement of inhibitory G proteins in the regulation of postmating behaviors in vivo, we expressed under ppk-GAL4 an irreversible inhibitor of Gαi—Pertussis toxin (PTX) (Hildebrandt et al., 1983). PTX expression in ppk neurons did not affect virgin behaviors; however, it strongly suppressed postmating behaviors (both rejection and egg laying) in mated females, causing them essentially to behave like virgins (Figures 5A and 5B). Importantly, despite the low egg-laying rate of mated females expressing PTX under ppk-GAL4, the few laid eggs produced viable offspring (data not shown), indicating that oogenesis or fertilization were not affected. Thus, SP likely triggers postmating behaviors by activating Gαi/o.

Figure 5. Manipulation of G Protein or PKA Signaling in ppk or fru/ppk Neurons Modulates Postmating Behaviors.

Figure 5

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(A and B) Suppression of postmating responses in mated females by inhibition of Gαi or activation of PKA. (A) Effects on mating/remating. Mating assays with flies expressing Pertussis toxin (UAS-PTX) or the active subunit of mouse PKA (UAS-PKAmc*) were carried out as in Figure 1C. ***p < 0.0005, two-tailed Fisher’s exact test. (B) Effects on egg laying of virgin or mated females. Experiments were performed as in Figure 2D. n = 15, *p < 0.05, **p < 0.005, ***p < 0.0005, Student’s t test. Error bar indicates SEM.

(C and D) Inhibition of PKA in fru/ppk neurons induces partial postmating responses in virgins. (C) Receptivity of virgin females was scored as in Figure 5G. **p < 0.005, Fisher’s exact test. (D) Virgin egg laying, as in Figure 1B, but with three females per vial (n = 15), **p < 0.005, Student’s t test. Error bar indicates SEM. (E) Model. fru/ppk neurons are active in virgins and presumably signal to the CNS to maintain virgin behaviors. After mating, SP stops synaptic outputs of fru/ppk neurons and mated behaviors are induced.

Because dunce mutants, which lack cAMP phosphodiesterase activity, are defective in their response to SP (Chapman et al., 1996) and Gαi/o is known to inhibit the production of cAMP by adenylate cyclase, we next investigated whether increasing the activity of PKA, the most common downstream effector of cAMP, also suppresses postmating behaviors. Indeed, overexpression of the active subunit of murine PKA (UAS-PKAmc*, (Li et al., 1995)) driven by ppk-GAL4 caused mated females to be more receptive toward courting males, though it did not significantly affect their egg laying (Figures 5A and 5B). Conversely, expression of a mutated regulatory PKA subunit that acts as a PKA inhibitor, PKAr* (Li et al., 1995), under the strong fru-GAL4 driver significantly increased virgin egg laying and caused virgin females to reject males more frequently (although not as strong as mSP expression) (Figures 5C and 5D). These phenotypes could be suppressed by ppk-GAL80 and could therefore be attributed to PKA inhibition in fru/ppk neurons. Thus, SP may trigger postmating behaviors by suppressing PKA activity. Taken together, our results show that activation of Gαi and inhibition of cAMP signaling in fru/ppk neurons play important roles in the induction postmating behaviors, suggesting that SP may act via these signaling events to trigger inhibition of neuronal outputs of fru/ppk neurons postmating.

In conclusion, we have identified a group of six to eight fru/ppk neurons on the reproductive tract that are important for the induction of postmating behaviors in response to SP. Several lines of evidence suggest that they are major neuronal mediators of the effect of SP: first, SPR expression under the control of fru (Yapici et al., 2008) and ppk promoters can rescue the spr mutant phenotype and the two markers only overlap in the fru/ppk neurons on the reproductive tract; second, ppk-GAL80 effectively blocks the phenotype of SPR-RNAi expression in fru neurons, suggesting that the presence of SPR in fru/ppk neurons is sufficient for proper induction of postmating behaviors, and third, when mSP was expressed randomly in fru neurons, expression of mSP on the reproductive tract fru/ppk neurons strongly correlated with the induction of postmating behavior. Our data also suggest a mechanism by which SP acts on these fru/ppk neurons to induce postmating behaviors: it likely silences neuronal transmission from these neurons via Gαi and inhibition of PKA (Figure 5).

Our results provide two conceptual advances with respect to SP action. First, contrary to the idea that SP acts directly on central neurons (after diffusing through the hemolymph) to bring about the postmating behavioral switch, we show here that SP can use a different way to control female behaviors by regulating a small set of internal sensory neurons that are located near the site where SP first enters the female reproductive system. Second, our results indicate that these internal sensory neurons function as a “master switch” to instigate multiple postmating behaviors: in principle, the different aspects of postmating behaviors could be separately controlled by distinct neuronal SP targets; however, we showed that manipulation of just these few fru/ppk sensory neurons can induce at least two distinct postmating programs (egg laying and rejection). Such a concerted behavioral control by SP via a small number of strategically located internal sensory neurons could presumably enhance the robustness and coordination of postmating behaviors.

Taken together, these findings represent an important step in delineating the neuronal circuit that controls the reproductive behaviors of Drosophila females. Given the conservation of core components (Yapici et al., 2008; Dottorini et al., 2007), our findings likely extend to other insects such as mosquitoes and may be of value in pest control.

EXPERIMENTAL PROCEDURES

Fly Stocks

For elav-GAL80 and ppk-GAL80, the GAL80 ORF and the elav and ppk promotors were subcloned into pCasper4 vector. PTX-S1 and FRT-CD2-FRT-Kir2.1-GFP were subcloned into pUAST (Brand and Perrimon, 1993). A ppk-GAL80 insertion on the second chromosome, an elav-GAL80 insertion on the X chromosome, an UAS-FRT-CD2-FRT-Kir2.1-GFP on the X chromosome, and an UAS-PTX insertion on the third chromosome were chosen and used in experiments. ppk-GAL4 lines carried either one insertion on the second or an insertion on both the second and third chromosomes (2× ppk-GAL4) were used. For ppk-eGFP, one carrying insertions on the second and third chromosomes was used. Other fly stocks used were fru-GAL4 (gifts from B. Dickson and B. Baker), UAS-dcr2, UAS-SPR-IR (gifts from B. Dickson), UAS-mSP (gift from T. Aigaki), UAS-Kir2.1-GFP, UAS-mCD8-GFP, UAS-Red stinger, UAS-shits, UAS-PKAmc*, UAS-PKAr*, SP0/SPD, and SP+/SPD (gifts from E. Kubli). SPR RNAi (UAS-SPR-IR) was used with UAS-dcr2 (Dietzl et al., 2007) in the background. All fly stocks were kept on standard medium. Crosses were performed at 25°C or at 20°C when UAS-shits was used.

Behavioral Assays

Mating

All flies were aged for 4–5 days after eclosion and then analyzed in behavioral assays. For receptivity assays, females and males were housed in small groups of 3–4 flies. In order to assess female receptivity, one female was put into a small (1 cm × 1 cm) grape agar chamber together with two naive Canton S males. The female was scored as receptive if it mated within 20 min. In the remating assays (Figure 5), a female was mated in a receptivity assay and then again examined in another receptivity assay 24 hr later. In experiments involving mating with SP0/SPD and SP+/SPD males, 10 females were mated with 20 SP mutant males for 2–3 hr 24 hr prior to each experiment. Assays were usually performed within the first three hours of the subjective day, except the 20°C controls for shits experiments which were done at 9–11 hr of the same day as the experiments at 30°C. Significance levels for mating behavior were calculated with a two-tailed Fisher’s exact test (similar to a χ2 test) at www.graphpad.com/quickcalcs/. Rejection was scored qualitatively as follows: ++, females repeatedly exhibiting rejecting behavior such as ovipositor extrusion to courting males, preventing or strongly delaying copulation; +/−, females exhibiting rejecting behavior but do so less consistently, often leading to copulation in a later attempt by the male; −, female exhibiting virgin-like behavior, with virtually no display of rejection behavior and early encounters with males already leading to copulation.

Egg Laying

For egg laying, 10 females of the appropriate genotype were aged in vials for 4–5 days. Then three or five (as indicated in the figure legends) females were transferred to a vial with grape media and allowed to lay eggs for 24 hr at 25°C (or at 20°C or 30°C, respectively, when UAS-shits was used). The number of eggs was divided by the number of flies in the vial to give a measure of egg laying. For assays of egg laying by mated females, females of the respective genotype were mated with Canton S or SP0/SPD and SP+/SPD males for 2–3 hr on the day before the experiment, with 10 females and 20 males per vial. All data are given as average ± SEM, significance levels were calculated with the Student’s t test.

Clonal mSP and Kir2.1 Expression and Phenotypic Selection

In order to express mSP in subsets of fru neurons, flies of the genotype MKRS hs-FLP, tub-FRT-GAL80-FRT, fru-GAL4, UAS-mSP, UAS-mCD8-GFP were heat-shocked at the pupal stage. Females were aged for seven days and then tested for mating behavior. Clearly receptive and clearly rejecting females were selected and their reproductive tracts dissected and stained for mCD8. The numbers of labeled cells were counted, and statistical significance was calculated by Student’s t test. For clonal Kir2.1-GFP expression, flies of the genotype hs-FLP, ppk-GAL4, UAS-FRT-CD2stop-FRT-KIR2.1-GFP were treated as described above. Kir2.1-GFP was visualized with anti-GFP antibodies and the remaining ppk neurons not expressing Kir2.1 were stained with anti-CD2 antibodies.

Dissection and Immunocytochemistry

Tissues from flies that were at least 8 days old were dissected under PBS, fixed in 4% formaldehyde in PBS for 40 min at room temperature and permeabilized with PBS 0.3% Triton X-100. Tissues were stained with rat anti-mCD8 (1:200), rabbit anti-GFP (1:3000), and rabbit anti-SPR (1:500, a gift from B. Dickson) and goat-anti-HRP antibodies coupled to Cy2 or Rhodamine. Primary antibodies were detected with Cy2-conjugated goat anti-rat, Alexa488- or Cy2-conjugated goat anti-rabbit and Rhodamine-conjugated goat anti-rabbit secondary antibodies. Images were taken on a Leica TCS SP2 confocal microscope.

Supplementary Material

Suppl. Fig.1-6. SUPPLEMENTAL DATA.

The Supplemental Data for this article include six figures and one table and can be found with this article online at http://www.neuron.org/supplemental/S0896-6273(08)01093-3.

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ACKNOWLEDGMENTS

We thank Barry Dickson, Bruce Baker, Toshiro Aigaki, Eric Kubli, John Kiger, and the Bloomington stock center for fly lines and antibodies. We thank Devenand Manoli, Yuan Quan, and members of the Jan lab for reagents and discussions. We also thank Gary Struhl for providing an easy cloning strategy for the UAS-FRT-CD2-FRT-Kir2.1GFP construct. Requests for tub-FRT-GAL80-FRT should be directed to K. Scott (kscott@berkeley.edu). This work was supported by a Jane Coffins Child postdoctoral fellowship to C.-h.Y., a German Academic Exchange Service (DAAD) postdoctoral fellowship to S.R., a Damon Runyon fellowship to M.D.G., a Human Frontier Science Program fellowship to Y.X., and an NIH grant (R01 NS40929) to Y.-N.J. L.Y.J. and Y.-N.J. are Howard Hughes Medical Institute Investigators.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl. Fig.1-6. SUPPLEMENTAL DATA.

The Supplemental Data for this article include six figures and one table and can be found with this article online at http://www.neuron.org/supplemental/S0896-6273(08)01093-3.

NIHMS119053-supplement-Suppl__Fig_1-6.pdf (455.1KB, pdf)
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NIHMS119053-supplement-01.pdf (144KB, pdf)

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