A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila

Article

A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila Highlights d

Subset of PPM3 DA neurons reduces copulation rate of virgin female Drosophila

d

Downstream I1-FFL circuit is composed of GABAergic and cholinergic neurons

d

GABAA receptors in ACh neurons are required for suppressing virgin female rejection

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A retrograde NO signal activated via Glut/NMDAR may potentiate the GABA neurons

Ishimoto & Kamikouchi, 2020, Current Biology 30, 1–12 February 3, 2020 ª 2019 Elsevier Ltd. https://doi.org/10.1016/j.cub.2019.11.065

Authors Hiroshi Ishimoto, Azusa Kamikouchi

Correspondence [email protected] (H.I.), [email protected] (A.K.)

In Brief Ishimoto and Kamikouchi report on a subclass of dopaminergic neurons that drive a feedforward circuit comprising cholinergic and GABAergic neurons that regulates the pre-mating behavior of virgin females in Drosophila. A glutamate/ NMDAR and NO retrograde signal modulates the circuit function to abolish the rejection response toward the males’ attempts to copulate.

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

Current Biology

Article A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila Hiroshi Ishimoto1,2,* and Azusa Kamikouchi1,* 1Graduate

School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan Contact *Correspondence: [email protected] (H.I.), [email protected] (A.K.) https://doi.org/10.1016/j.cub.2019.11.065 2Lead

SUMMARY

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. Here, we show that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/ R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-D-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. Our study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female. INTRODUCTION Mating behavior is an inherent aptitude whereby both internal and external factors determine the propriety of mating partners through multilevel neuromodulatory systems. Dopaminergic (DA) systems play a major role in regulating courtship, sexual motivation, and social bonding across species [1–3], but the underlying molecular, cellular, and neural circuits remain unclear. The female mating behavior of Drosophila melanogaster is a behavioral model used to elucidate how the brain controls the mating process at the resolution of molecular, cellular, and neuronal/network levels [4]. In the early phase of the courtship ritual, the first reaction of female flies is acute rejection of the courting male [4], because female flies need to determine the

nature of the encounter and then further evaluate the potential mating partner to avoid unfavorable mating. These pre-mating rituals of female flies suggest the existence of neural circuits in the female brain that regulate behavioral switching from courtship rejection to acceptance. The receptivity of virgin female fruit flies is regulated by two subsets of sexually dimorphic neurons, pC1 and pCd [5]. The pCd cluster neurons respond to a volatile male pheromone, 11-cis-vaccenyl acetate (cVA), which increases female receptivity [6]. The cVA information is transmitted to a higher-order brain region, the lateral horn, via a third-order olfactory interneuron, aSP-g. This interneuron extends nerve fibers into the superior medial protocerebrum (SMP) [7], where pCd neurites arborize. It is thus highly likely that SMP is a neuronal hub region for the integration of sexual information to determine the proper response—rejection or acceptance. Although accumulation of the pheromonal information in the pCd through the SMP explains the increase in female receptivity, the neuronal pathways controlling the behavioral switching of the pre-mating response remain largely unexplored. Sexual motivation is one of the important factors for successful mating in both males and females. Recently, Zhang et al. reported that DA neurons projecting to the SMP affect the sexual motivation of male flies [8, 9]. In addition, DA modulates female receptivity, potentially by affecting hormonal pathways [10], but the neuronal implications of the involvement of DA are not clear. Given the role of DA in female receptivity, we explored the neural and molecular mechanisms that control pre-mating behavioral switching of virgin females. Here, we propose that two groups of central complex neurons relaying DA signals form an incoherent feedforward circuit motif and are implicated in the regulation of behavioral switching from rejection to acceptance in pre-mating virgin females.

RESULTS DA Neurons Control Female Mating In contrast to the situation in male flies, it has remained unclear whether DA neurons control the mating behavior of virgin females [10]. Thus, we examined the mating behavior of virgin female flies, in which most DA neurons were conditionally suppressed by the expression of the inward-rectifier potassium ion channel Kir2.1 via the Th-Gal4 driver under the control of the temporal and regional gene expression targeting (TARGET) Current Biology 30, 1–12, February 3, 2020 ª 2019 Elsevier Ltd. 1

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

B

NS

Kir2.1(+) (56) Kir2.1(-) (58) 0

1 2 3 Time (x1000 s)

1 2 3 Time (x1000 s) UAS-Kir2.1/+; 3 Ddc-Gal4/tub-Gal80[ts]

2 NS 1

0 Kir2.1: (-) (49)

(+) (42)

Ddc-Gal4 PPM1/2

2

PPL2

***

1

PPM3

PPM1/2 PPL2

PPM3

**

0 Kir2.1: (-) (44)

E Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

UAS-Kir2.1/+; Ddc-Gal4/tub-Gal80[ts]

0

Copulation latency (x1000 s)

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1 2 3 Time (x1000 s)

Th-Gal4

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(+) (50)

WT (61)

c346-Gal4/UAS-Kir2.1; tub-Gal80[ts]/+

***

Kir2.1(+) (50) Kir2.1(-) (50)

1 2 3 Time (x1000 s)

fmn (31)

c346-Gal4/UAS-Kir2.1; tub-Gal80[ts]/+

3

2

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0 Kir2.1: (-) (37)

***

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c346-Gal4/+; UAS-dTRPA1/+

HS(-) (40) HS(+) (48)

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(+) (46)

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1 2 Time (x1000 s)

Copulation latency (x1000 s)

dDAT[fmn] (40) WT (70)

Kir2.1(+)(50) Kir2.1(-) (50)

dDAT [fmn]

Copulation rate 0.2 0.4 0.6 0.8 1.0

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UAS-Kir2.1/+; Th-Gal4/tub-Gal80[ts]

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UAS-Kir2.1/+; Th-Gal4/tub-Gal80[ts]

Copulation latency (x1000 s)

A

3

c346-Gal4/+; UAS-dTRPA1/+

NS

2

1

0 HS(-) (30)

HS(+) (18)

Figure 1. DA Neurons Are Involved in the Control of Female Mating (A and B) Conditional suppression of DA neurons in females (left). The dDAT mutant, fumin (fmn), and the wild-type (WT) control (right). Copulation rate (A) and copulation latency (B) are shown. See also Figure S1. (C) Illustrations depict DA neurons labeled by Th-Gal4 (left) or Ddc-Gal4 (right) (adapted from Kong et al. [12]). Gal4+ (brown/yellow) and Gal4 (gray) cells are indicated. (D) The copulation rate (left) and the copulation latency (right) of females with suppressed subdivisional DA neurons. (E) The copulation rate (left) and the copulation latency (right) of females with suppressed PPM3 DA neurons. (F) The copulation rate (left) and copulation latency (right) of females with (HS(+)) or without (HS( )) neural activation by dTRPA1 in c346 neurons. For the copulation rate, the log rank test is applied. For the copulation latency, the Wilcoxon-Mann-Whitney test is applied. NS, non-significance (p > 0.05), **p < 0.01, ***p < 0.001. The number of animals analyzed is indicated in parentheses.

system [11] to avoid significant effects of the genetic background. Suppressing most of the DA neurons significantly increased the copulation rate (c2 = 14.8, p = 1e 4; Figure 1A). However, a DA transporter (dDAT) mutant allele, fumin (fmn), whose impaired DA reuptake may increase the DA amount at the synaptic cleft [13, 14], exhibited a dramatic reduction in the copulation rate (c2 = 12.4, p = 4e 4; Figure 1A). These disturbances in DA signaling also affected the copulation latency (Figure 1B). Suppressing DA neurons induced a shorter copulation latency (p = 0.006; Figure 1B, left), whereas the fmn mutation induced a longer copulation latency (p = 6.43e 8; Figure 1B, right). These effects were observed strongly for DA and partly for tyramine and octopamine (Tdc2-Gal4; copulation rate, c2 = 2.7, p = 0.1; copulation latency, p = 0.037), but not for GABA (Gad1-Gal4; copulation rate, c2 = 2.1, p = 0.15; copulation latency, p = 0.59) and serotonin (Trh-Gal4; copulation rate, c2 = 1, p = 0.3; copulation latency, p = 0.47) (Figures S1A–S1F). In Drosophila, most DA neurons make up subclusters in the brain [15, 16]. We next asked which DA subclusters are responsible for the mating behavior. Strong candidates were those subclusters projecting to the SMP, the potential integration site for sex-related information in both males [8, 9] and females [7]. Parts of the posterior DA subclusters (PPL2, PPM2, and PPM3) have been described as DA neurons projecting to the SMP (SMP-DAs) [8] (Figure 1C). In addition to this, we found that conditional suppression of subdivisional DA neurons containing 2 Current Biology 30, 1–12, February 3, 2020

two SMP-DAs (PPL2 and PPM2) by using Ddc-Gal4 [12] had no significant effect on female mating behavior (courtship rate, c2 = 1.4, p = 0.232; courtship latency, p = 0.45; Figure 1D). However, the suppression of PPM3 neurons using c346-Gal4 [17], which labels 2 of 6–8 PPM3 DA neurons per hemisphere, induced a significant increase in the copulation rate (c2 = 23.4, p = 1e 6; Figure 1E, left) with a significantly shorter copulation latency (p = 1.73e 7; Figure 1E, right). Activation of c346-Gal4 neurons with a thermo-activated ion channel (dTRPA1) [18] suppressed the copulation rate (c2 = 12.6, p = 4e 4; Figure 1F, left) without a significant impact on the copulation latency (p = 0.376; Figure 1F, right). These results suggested that PPM3 labeled by c346-Gal4, one of the SMP-DAs, likely contributes to the proper kinetics of mating behavior in female flies. Ellipsoid Body Neurons Control Female Mating To identify the polarity of PPM3 neurons, we mapped the putative pre- and postsynaptic sites of PPM3 neurons by expressing neuronal synaptobrevin GFP (nSyb::GFP) and the Da7 nicotinic acetylcholine receptor (Da7::GFP), respectively, in PPM3 neurons labeled by c346-Gal4 (Figure 2A). The nSyb::GFP signals were found mainly at the ellipsoid body (EB) and sparsely at SMP (Figure 2A, left), whereas the Da7::GFP signals were found mainly at SMP and slightly at EB (Figure 2A, right). Thus, PPM3 may transfer sexual information from SMP to EB. We also found slight nSyb::GFP signals in the fan-shaped body

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

c232-Gal4

Post-synapse

SMP

SMP

R4d EB -bulb EB-ring

EB-bulb

nSyb::GFP

R3

EB -bulb EB-bulb

Dα7::GFP

EB-ring

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Kir2.1(+) (70) Kir2.1(-) (60) 0

0

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1

*** 0 Control Dop1R2 RNAi (40) (53)

1 2 3 Time (x1000 s) UAS-dTRPA1/+; c232-Gal4/+

Copulation rate 0.2 0.4 0.6 0.8 1.0

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Wild-type control

HS(-) (24) HS(+)(24)

NS

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HS(-) (32) HS(+)(39)

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Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

Dop1R2 RNAi (60) Control (49)

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1 2 Time (x1000 s)

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ChAT RNAi (48) Control (49)

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UAS-dTRPA1/+; c232-Gal4/+

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UAS-Kir2.1/+; c232-Gal4/ tub-Gal80[ts]

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0 Kir2.1: (-) (29)

1 2 3 Time (x1000 s)

c232-Gal4/ UAS-ChAT RNAi

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Copulation latency (x1000 s)

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

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c232-Gal4/ UAS-Dop1R2 RNAi

Copulation latency (x1000 s)

c232-Gal4/ UAS-Dop1R2 RNAi

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Copulation latency (x1000 s)

Pre-synapse

UAS-Kir2.1/+; c232-Gal4/ tub-Gal80[ts]

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Copulation latency (x1000 s)

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Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

A

3

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0 Control ChAT (40) RNAi (46)

Wild-type control

** 2 NS 1

0 HS(-) (23)

HS(+) (13)

HS(-) HS(+) (27) (33)

Figure 2. EB Neurons Labeled by c232-Gal4 Negatively Regulate Mating Kinetics of Females (A) Synaptic sites of PPM3 neurons labeled by c346-Gal4. nSyb::GFP (pre-synaptic marker; left) and Da7::GFP (postsynaptic marker; right) are expressed. Dashed lines outline the SMP region. Arrows and arrowheads indicate EB ring and EB bulb, respectively. Scale bars, 50 mm. (B) R neurons labeled by c232-Gal4 are depicted. See also Figures S2B and S2C. (C) Mating behavior of females with suppressed c232 neurons (Kir2.1(+)) and the control (Kir2.1( )). The copulation rate (left) and copulation latency (right) are shown. See also Figures S2C–S2F. (D) The copulation rate (left) and copulation latency (right) of females with Dop1R2 knockdown in c232 neurons and the control. See also Figures S2G–S2L. (E) The copulation rate (left) and copulation latency (right) of females with ChAT knockdown in c232 neurons and the control. See also Figures S2M and S2N. (F and G) The copulation rate (F) and copulation latency (G) of females with (HS(+)) or without (HS( )) neural activation by expressing dTRPA1 in c232 neurons (left) and the WT control (right). For the copulation rate, the log rank test is applied. For the copulation latency, the Wilcoxon-Mann-Whitney test is applied. NS, p > 0.05, **p < 0.01, ***p < 0.001. The number of animals analyzed is indicated in parentheses.

(FB), as previously described (Figure S2A) [12, 19]. We therefore evaluated whether suppressing neurons in the EB or FB affected female mating behavior. We used c232-Gal4 and OK348-Gal4 [20] to suppress EB and FB neurons, respectively, by conditionally expressing Kir2.1. The suppression of EB neurons labeled by c232-Gal4 (hereafter referred to as c232 neurons; Figures 2B, S2B, and S2C) significantly increased the copulation rate (c2 = 19.5, p = 1e 5; Figure 2C, left) and decreased the copulation latency (p = 9.2e 3; Figure 2C, right). Similar results were obtained from another strain, c507-Gal4, which labels EB neurons (Figures S2C–S2E). However, suppressing FB neurons labeled by OK348-Gal4 had little effect on the copulation rate (c2 = 3.4,

p = 0.065; Figure S2F). Therefore, EB neurons are the candidates downstream of the PPM3 neurons that are implicated in the regulation of pre-mating behavior. EB neurons reportedly express the D1-type DA receptors Dop1R1 and Dop1R2 [12, 17]. We then tried to identify the responsible DA receptor in EB by knocking down each DA receptor type in the Drosophila genome. Knockdown of Dop1R2 affected both the copulation rate (c2 = 12, p = 5e 4; Figure 2D, left) and the copulation latency (p = 6.45e 8; Figure 2D, right). Knockdown of other DopRs demonstrated little effect on the copulation rate, however, although knockdown of Dop1R1 or D2R showed significant reduction in copulation latency (Figures S2G–S2L). The effects Current Biology 30, 1–12, February 3, 2020 3

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of Dop1R2 knockdown were quite similar to those observed when c232 neurons were suppressed, suggesting that Dop1R2 is likely to elicit neuronal activity in the EB. EB neurons comprise both excitatory cholinergic neurons and inhibitory GABAergic neurons [21]. Therefore, we evaluated which of these neurotransmitters in the EB is required to control the pre-mating kinetics. We first expressed double-stranded RNA for choline acetyltransferase (ChAT) in c232 neurons to suppress acetylcholine synthesis. Female flies with ChAT knockdown in EB exhibited a significantly increased copulation rate (c2 = 12.3, p = 5e 4; Figure 2E, left) and a significantly decreased copulation latency (p = 5.1e 3; Figure 2E, right). In contrast, suppressing GABA production by knocking down glutamic acid decarboxylase 1 (Gad1) had no significant effect on the copulation rate (c2 = 2.3, p = 0.1; Figure S2M), although their copulation latency was significantly shorter (p = 8.3e 4; Figure S2N). These results suggest that the cholinergic subsets in the EB have more impact on the proper control of premating kinetics than the GABAergic subset. Notably, the suppression of excitatory cholinergic neurons facilitated female acceptance. Therefore, c232 neurons possibly promote the rejection response (i.e., the resistance to copulation). To evaluate this hypothesis, we conditionally activated c232 neurons with dTRPA1. As we expected, the copulation rate was dramatically decreased when flies were placed under the activation temperature (HS(+)) (c2 = 22.7, p = 2e 6; Figure 2F, left), while the same temperature condition did not alter that of the wildtype control (c2 = 0.3, p = 0.6; Figure 2F, right). In addition, the activation of c232 neurons resulted in longer copulation latency (p = 1.05e 3; Figure 2G, left), whereas HS(+) itself had little effect on that of wild-type control flies (p = 0.38; Figure 2G, right). These results support our hypothesis that EB neurons could promote the pre-mating rejection response. Distinct Cluster of EB Neurons Reciprocally Regulates Female Mating Kinetics Thus far, we used c232-Gal4 as a representative strain for EB. However, PPM3 neurons labeled by c346-Gal4 actually connect broadly to several EB regions occupied by c232 neurons and to other regions of the EB, which was visualized by trans-Tango (Figure 3A). Therefore, next, we performed a functional dissection of EB neurons using multiple Gal4 drivers that label spatially distinct subsets of EB neurons. The axons of EB neurons form a circumferential ring-like structure composed of anatomically and molecularly different circular laminae layers in the EB [21]. These ring layers comprise R1 (center), R3, R2, R4m, and R4d (outer) layers, each of which comprises the corresponding R neurons. The c232-Gal4 preferentially labels EB neurons, in particular R3 and R4d neurons (Figure 3B) [22, 23]. To clarify which R neurons affect mating behavior, we conditionally suppressed R3 and R4d neurons using 189y-Gal4 [24] and NP1131-Gal4 [21], respectively (Figures 3B and 3C). Conditional expression of Kir2.1 in R3 neurons had little effect on the copulation rate (c2 = 0.1, p = 0.7; Figure 3D, left). However, suppression of the R4d neurons significantly affected the copulation rate and the copulation latency (c2 = 6.7, p = 0.01; Figure 3D, right, and Figure 3E). The activation of NP1131-Gal4 neurons induced significant suppression of the copulation rate (c2 = 33.1, p = 9e 9; Figure 3F, left) and a 4 Current Biology 30, 1–12, February 3, 2020

significant increase in the copulation latency (p = 0.005; Figure 3F, right). Although NP1131-Gal4 also labels mushroom body g (MBg) neurons (Figures S3A and S3B), suppressing only the MBg neurons using a specific Gal4 driver, 201y-Gal4, did not affect female mating behavior (c2 = 2.8, p = 0.0929; Figure S3C). This finding is consistent with that of a previous study, in which MB ablation had no effect on female receptivity [25]. These results suggested that the mating effects with suppressing/activating c232 neurons were due to the impaired/facilitated function of R4d. If this inference is correct, then Dop1R2 should be required in R4d neurons for mating control. As expected, Dop1R2 knockdown in R4d neurons induced a higher copulation rate (c2 = 9.6, p = 0.00191; Figure 3G, left) and a shorter copulation latency (p = 6.43e 8; Figure 3G, right). All of these results indicate that R4d is critically involved in the suppression of female mating. We further investigated the possible involvement of other R neurons—R1, R2, and R4m—for the mating control. Suppressing R1 neurons labeled by c105-Gal4 had no effect on the copulation rate (c2 = 0, p = 0.833; Figures S3D and S3E). In contrast, suppressing R2/R4m neurons labeled by c819-Gal4 [21] (Figures 3H and 3I) significantly decreased the copulation rate, with little effect on the copulation latency (copulation rate, c2 = 13.7, p = 2e 4; copulation latency, p = 0.9148; Figure 3J). We then evaluated whether DA signals contribute to the function of R2/R4m neurons by knocking down particular DA receptors in R2/R4m. RNAi experiments revealed that knockdown of either Dop1R1 or D2R in R2/R4m decreased the copulation rate (Dop1R1, p = 0.011; D2R, p = 8.6e 7; Figure 3K, left) and increased the copulation latency (Dop1R1, p = 0.027; D2R, p = 2.1e 4; Figure 3K, right). In contrast, knockdown of the other types of DA receptors, Dop1R2 and DopEcR, had no effect on the copulation rate, with a limited effect on copulation latency (Figures S3F–S3I). These results, together with the results of suppressing R2/R4m neurons, suggested that R2/R4m neurons most likely receive DA signaling through Dop1R1 and D2R to promote mating acceptance. This potential function of R2/R4m for mating control is exactly opposite of that of R4d, suggesting the possibility that these neighboring neurons communicate with each other to adjust behavioral output to a proper level. Inhibitory R2/R4m Attenuates the Rejection Response toward Mating Males To elucidate the interactions between R2/R4m and R4d neurons, we visualized the synaptic connections from R2/R4m to R4d using synaptic GRASP [26] (GFP reconstitution across synaptic partners) analysis. The reconstituted GFP signals were observed predominantly at the ring structure regions in the EB, indicating that the synaptic terminals of R2/R4m at least connect with the inner R3 and the outer R4d neuropil (Figure 4A). This result recaptured the findings in a previous report [27]. The connection in the inverse direction, from R4d to R2/R4m, has not been detected [27]; thus, there are likely unidirectional connections from R2/R4m to R4d. Particular subsets of R neurons, especially R2/R4m neurons, produce the inhibitory neurotransmitter GABA [21, 28–30]. According to the inhibitory signaling of GABA and the potential function of R2/R4m that opposes R4d, R2/R4m most likely inhibits R4d to attenuate pre-mating rejection and promote copulation at a certain time. We then examined whether

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

UAS-myrGFP.QUAS-mtdTomato-3xHA/+; trans-TANGO/c346-Gal4

A

Merged

mtdTomato

c232-Gal4

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GFP Merged

R3/R4d

189y-Gal4

NP1131-Gal4

Kir2.1(+) (41) Kir2.1(-) (50)

0

*** HS(-) (33) HS(+) (42) 0

3

2

0 HS(-) (31)

R4m

R2

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HS(+) (21)

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NP1131-Gal4/ UAS-dTRPA1

*** Kir2.1(+) (43) Kir2.1(-) (39)

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1 2 3 Time (x1000 s)

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Kir2.1(+) (73) Kir2.1(-) (84)

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NP1131-Gal4/+; UAS-Dop1R2 RNAi/+

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Dop1R2 RNAi (48) Control (50)

3

2

NS 1

0 Kir2.1: (-) (39)

(+) (33)

NP1131-Gal4/UAS-Kir2.1; tub-Gal80[ts]/+ 3

2

***

1

0 Kir2.1: (-) (76)

(+) (69)

H

NP1131-Gal4/+; UAS-Dop1R2 RNAi/+ 3

c819-Gal4 2

1

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R2/R4m

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Control Dop1R2 (40) RNAi (47)

1 2 3 Time (x1000 s)

UAS-Kir2.1/+; c819-Gal4/tub-Gal80[ts] Copulation latency (x1000 s)

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R4d

c819-Gal4/ UAS-DopRs RNAi

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* *** Control (50) Dop1R1 RNAi (60) D2R RNAi (60) 0

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c819-Gal4/ UAS-DopRs RNAi Copulation latency (x1000 s)

R4d

NP1131-Gal4/UAS-Kir2.1; tub-Gal80[ts]/+

Copulation latency (x1000 s)

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189y-Gal4/UAS-Kir2.1; tub-Gal80[ts]/+

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D NP1131-Gal4

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R3

3

2

1

*

***

0 Control Dop1R1 D2R (48) RNAi RNAi (56) (45)

Figure 3. R4d and R2/R4m Oppositely Regulate Female Mating (A) Visualization of downstream neurons by trans-Tango with c346-Gal4 driver. mtdTomato labels postsynaptic neurons (magenta, left). GFP labels c346-Gal4 neurons (green, right). The merged image is located in the center panel (blue signal shows neuropil visualized with anti-Brp antibody). (B) Different subsets of neurons in the EB labeled by Gal4 strains. Expression patterns in the merged image, c232-Gal4 (R3/R4d), 189y-Gal4 (R3), and NP1131Gal4 (R4d) are indicated. (C) R neurons labeled by 189y-Gal4 (left) or NP1131-Gal4 (right) are depicted. (D) The copulation rate of female flies with conditional suppression of R3 (189y-Gal4, left) or R4d (NP1131-Gal4, right). (E) The copulation latency of female flies with conditional suppression of NP1131-Gal4 neurons. See also Figures S3A–S3C. (F) The copulation rate (left) and copulation latency (right) of females with (HS(+)) or without (HS( )) neural activation by dTRPA1 in NP1131 neurons. (G) Knockdown of Dop1R2 in R4d driven by NP1131-Gal4. The copulation rate (left) and copulation latency (right) are plotted. (H) Expression of c819-Gal4 (green) in EB. (I) R neurons labeled by c819-Gal4 are depicted. (J) R2/R4m is conditionally suppressed by Kir2.1. c819-Gal4 is used. The copulation rate (left) and copulation latency (right) are plotted. See also Figures S3D and S3E. (K) Dop1R1 or D2R knockdown in c819-Gal4 neurons. The copulation rate (left) and copulation latency (right) are plotted. For the copulation rate, the log rank test is applied. See also Figures S3F–S3I. Pairwise comparisons are adjusted by the Benjamini-Hochberg method. For the copulation latency, the Wilcoxon-MannWhitney test is applied. The Steel-Dwass test is applied for multiple comparisons. NS, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. The number of animals analyzed is indicated in parentheses. Scale bars, 50 mm (A, B, and H).

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B

c819-Gal4/ UAS-Gad1 RNAi

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

lexAop-CD4::spGFP11/ GMR44D11-Lexp65; UAS-Syb::spGFP[1-10]/ c819-Gal4 R2/R4m to R4d

*** Gad1 RNAi (50) Control (50)

0

R4d

(-) (27)

Copulation latency (x1000 s)

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

Rdl RNAi (50) Control (50)

0

J

K

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1.5

1.0

**

1

0 Rdl RNAi (33)

NP1131-Gal4/ UAS-Rdl RNAi

***

Control Rdl (40) RNAi (33)

R2

***

0 Control Gad1 (48) RNAi

SS02709-Gal4/ UAS-dTRPA1

2

* 1

0

NP1131-Gal4/ UAS-GABA-B-R2 RNAi

HS(-) (39)

NS

GABA-B-R2 RNAi (50) Control (50)

0

L 1.0

***

0.8 0.6 0.4 0.2 0 Control (40)

Rdl RNAi (33)

Gad1 RNAi (60) Control (47)

3

2

* 1

0 Control Gad1 (45) RNAi (46)

1 2 3 Time (x1000 s)

NP1131-Gal4/ UAS-empty RNAi Control (40)

NP1131-Gal4/ UAS-Rdl RNAi Rdl RNAi (33)

Copulation

Change point

Copulation

Change point

0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (s) Time (s)

1 2 3 Time (x1000 s) NP1131-Gal4/ UAS-Rdl RNAi

SS02709-Gal4/ UAS-Gad1 RNAi

***

0

HS(+) (33)

I

(37) SS02709-Gal4/ UAS-Gad1 RNAi

F

Copulation latency (x1000 s)

3

1 2 3 Time (x1000 s)

H

2

0.5

0

HS(-) (51) HS(+) (35)

3

Control (39)

1 2 3 Time (x1000 s)

***

0

(+) (34) NP1131-Gal4/ UAS-Rdl RNAi

**

R4m

1

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

1

NP1131-Gal4/ UAS-Rdl RNAi

R3 2

Walking Speed (a.u.) 0.0 0.5 1.0 1.5 2.0 2.5 3.0

G

NS

0 Kir2.1:

1 2 3 Time (x1000 s)

Copulation duration (x1000 s)

0

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SS02709-Gal4/ UAS-dTRPA1

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

Kir2.1(-) (31) Kir2.1(+) (54)

Copulation latency (x1000 s)

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

**

3

E

SS2709-Gal4/ UAS-Gad1 RNAi

M Copulation duration (x1000 s)

SS02709-Gal4/ UAS-Kir2.1; tub-Gal80[ts]/+

SS02709-Gal4/UAS-Kir2.1; tub-Gal80[ts]/+

SS02709-Gal4

GMR44D11 Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

Merged

C

3

1 2 3 Time (x1000 s)

R3

Incomplete copulation rate

GRASP D

c819-Gal4/ UAS-Gad1 RNAi Copulation latency (x1000 s)

R2/R4m to R3

Copulation latency (x1000 s)

A

c819-Gal4/ UAS-Gad1 RNAi

1.5

** ***

1.0

0.5

0

Control (39)

Gad1 RNAi (36)

Control (48)

Gad1 RNAi (34)

Figure 4. GABAergic R2/R4m Promotes Copulation by Repressing R4d via the GABAA Receptor (A) Synaptic GRASP from R2/R4m to R4d in female flies (lexAop-CD4::spGFP11/GMR44D11-Lexp65; UAS-Syb::spGFP [1–10]/c819-Gal4). GRASP signals (green, left), GMR44D11-Lexp65 signals (magenta, right), and merged signals (center). Scale bar, 25 mm. (B) Gad1 knockdown in R2/R4m of female flies. c819-Gal4 is used. The copulation rate (left) and copulation latency (right) are plotted. (C) EB neurons (R3/R2/R4m) labeled by SS02709-Gal4 are depicted. See also Figures S4A–S4E. (D) Conditional suppression of SS02709-Gal4 neurons. The copulation rate (left) and copulation latency (right) are plotted. (E) Conditional activation of SS02709-Gal4 neurons by dTRPA1. The copulation rate (left) and copulation latency (right) are plotted. (F) Gad1 knockdown in SS02709-Gal4 neurons. The copulation rate (left) and copulation latency (right) are plotted. (G) The copulation rate (left) and copulation latency (right) are measured from females with gene knockdown for GABAA receptor subunit Rdl in NP1131-Gal4 neurons. (H) Gene knockdown of GABA-B-R2 in NP1131-Gal4 neurons. The copulation latency is plotted. (I) Walking speed of females for 60 s before copulation. Arrowheads represent change points. Change points are detected at 39 and 52 s in the control (left). In Rdl knockdown females, a change point is detected at 56 s (right). Copulation timing is indicated as a dashed line. See also Figures S4F–S4H. (legend continued on next page)

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GABA production in R2/R4m was required for the control of mating kinetics. Knockdown of Gad1 in R2/R4m neurons by c819 > Gad1 RNAi decreased the copulation rate (c2 = 22, p = 3e 6; Figure 4B, left) and increased the copulation latency (p = 6.33e 4; Figure 4B, right). The c819-Gal4 strain has been previously used to investigate R2/R4m functions in some behavioral assays [31–33]; however, its Gal4 expression level in EB was not strong and the expression pattern was comparatively broad, especially in the ventral nerve cord. For a more precise analysis of the R2/R4m neurons, we identified a splitGal4 strain, SS02709-Gal4 [34, 35], which labels R2/R4m neurons more restrictively than that of c819-Gal4 (Figures 4C and S4A–S4E). The suppression of SS02709-Gal4 neurons significantly decreased the copulation rate (c2 = 9.6, p = 0.002), although no significant change in the copulation latency was observed (p = 0.09; Figure 4D). However, conditional activation of R2/R4m neurons labeled by SS02709-Gal4 significantly increased the copulation rate (c2 = 11.6, p = 7e 4) and slightly decreased the copulation latency (p = 0.0473; Figure 4E). Furthermore, Gad1 knockdown in neurons labeled by SS02709-Gal4 decreased the copulation rate (c2 = 14.9, p = 1e 4) and increased the copulation latency (p = 0.02; Figure 4F). These results mostly recapitulated the key findings obtained by c819-Gal4 and strongly suggest an inhibitory function of GABAergic R2/R4m for the pre-mating kinetics. If R4d receives signals from GABAergic R2/R4m, knockdown of GABA receptors in R4d neurons should affect female mating behavior. The fly genome contains two types of GABA receptors: ionotropic type A (GABAA) receptors and metabotropic type B (GABAB) receptors. Previous studies reported the expression of the GABAA receptor subunit Rdl in a large number of R neurons, including R4d [29, 30]. We therefore knocked down Rdl in R4d neurons using NP1131-Gal4. This manipulation decreased the copulation rate (c2 = 8, p = 0.00473; Figure 4G, left) and increased the copulation latency (p = 0.001; Figure 4G, right). However, knockdown of the GABAB receptor had no effect on the copulation rate (c2 = 0, p = 0.982; Figure 4H). These results thus far support our hypothesis that GABAergic R2/R4m neurons promote copulation by suppressing R4d neurons. Because the central complex, including the EB, is the higher center of locomotor control [36], there still is a counterargument that Rdl knockdown in R4d neurons possibly induces hyperactivity, and that could cause escape from male courtship. We addressed this possibility by investigating the walking speed of females just before they accepted copulation. In contrast with the hyperactivity hypothesis, the walking speed of flies with Rdl knockdown in R4d was lower than that of the corresponding control (Figures 4I and S4F–S4H). Whereas the walking speed of control females gradually decreased beginning 20 s before copulation (Figure 4I, left), Rdl knockdown females did not exhibit a decrease in the walking speed until 4 s before

copulation (Figure 4I, right). A time-dependent reduction in walking speed is considered to be a behavioral readout of female mating willingness [37, 38]. Therefore, these results suggested that females with Rdl knockdown had low copulation willingness, which could sustain the pre-mating rejection. This assumption was supported by our observation of the abnormal posture of a male during copulation with Rdl knockdown females (Figure 4J, bottom panel). During this copulation, the male could not grab the female body with his forelegs, so he could only connect with the female by his genitalia (Video S1), likely due to the lack of copulation acceptance of the females. By observing the behavior video, we could confirm that the control female opened her wings to create a space through which the male fly easily accessed the female genitalia during copulation and grabbed the female wings with his forelegs (Figure 4J, top panel; Video S1). In contrast, Rdl knockdown females kept their wings closed during copulation (Figure 4J, bottom panel; Video S1). In addition to the wing phenotype, Rdl knockdown females kept kicking the male fly during copulation, thereby shortening the copulation duration (p = 4.28e 16; Figure 4K; Video S1). These incomplete copulations made up 90% of the copulation events that occurred with Rdl knockdown females (p = 5.89e 15; Figure 4L). The short-duration phenotype for copulation was also observed in virgin females whose Gad1 was knocked down in R2/R4m neurons using SS02709-Gal4 or c819-Gal4 (p = 0.007 for SS02709-Gal4/UAS-Gad1 RNAi; p = 2.2e 16 for c819Gal4/UAS-Gad1 RNAi; Figure 4M). These findings indicate that R4d positively regulates the rejection response to males, and R2/R4m negatively regulates the R4d function via GABA-GABAA receptor signaling. Retrograde Control of R2/R4m Is Required to Abolish the Acute Rejection Response According to the anatomical and functional observations in this study and those of previous studies [12, 17], both R2/R4m and R4d are regulated by DA. However, the behavioral sequence of females during the pre-mating phase and findings in this study suggested that R4d may be activated predominantly at the early phase of the courtship to execute pre-mating rejection and will be suppressed later by GABAergic R2/R4m. Here, we raised a question: what are the molecular mechanisms to control the activation order for R4d and R2/R4m? To address this question, we explored the interaction between R2/R4m and R4d. Glutamate and N-methyl-D-aspartate receptors (NMDAR) may account for the question for the following reasons. First, EB neurons surrounding R3 layers, such as R2/R4m, express vesicular glutamate transporter (VGlut) [28] and, second, it is reported that EB neurons have immunoreactivity for NMDAR (NR1 and NR2) [39]. Therefore, we tested whether VGlut and NMDAR in R2/R4m and R4d, respectively, are involved in the regulation of pre-mating behavior in female flies. We suppressed

(J) Snapshots of copulation with a control (top) or an Rdl knockdown female (bottom). See also Video S1. (K) The copulation duration of control and Rdl knockdown females is plotted. The Student’s t test is applied for the comparison. (L) Incomplete copulation rate of control and Rdl knockdown females. The Fisher’s exact test is applied. (M) The copulation duration of control and Gad1 knockdown females is plotted. The Wilcoxon-Mann-Whitney test is applied for the comparison. For the copulation rate, the log rank test is applied. For the copulation latency, the Wilcoxon-Mann-Whitney test is applied. NS, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. The number of animals analyzed is indicated in parentheses.

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0

Control VGlut (29) RNAi (22) SS2709-Gal4/ UAS-VGlut RNAi NS

1 2 3 Time (x1000 s)

c232-Gal4/ UAS-NR1 RNAi NS

1.5 1.0 0.5 0

Control (28)

H

VGlut RNAi (18)

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

c232-Gal4/ UAS-Nos RNAi

*** Control (49) NOS RNAi(46)

0

1 2 3 Time (x1000 s)

Control (53)

I 3

NR1 RNAi (47)

c232-Gal4/ UAS-Nos RNAi

2

1

***

0 Control (41)

NOS RNAi (32)

Copulation latency (x1000 s)

0

***

Control (119) NR1 RNAi (76)

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189y-Gal4/ UAS-Nos RNAi

J

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Control (50) NOS RNAi(43)

0

***

1

Control (107)

NS

*** Control (84) sGCa99B RNAi(54)

1 2 3 Time (x1000 s)

NR1 RNAi (52)

Control NR2 RNAi (41) (28)

189y-Gal4/ UAS-Nos RNAi

3

2 NS 1

0 Control (43)

1 2 3 Time (x1000 s) SS02709-Gal4/ UAS-sGCa99B RNAi

UAS-NR2 RNAi/+; c232-Gal4/+

2

1 2 3 Time (x1000 s)

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*** 1

Control (49) NR2 RNAi(76)

***

c232-Gal4/ UAS-NR1 RNAi

3

K Copulation latency (x1000 s)

Copulation duration (x 1000 s)

E

2

UAS-NR2 RNAi/+; c232-Gal4/+

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c232-Gal4/ UAS-NR1 RNAi

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0

3

D

C

SS02709-Gal4/ UAS-VGlut RNAi

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

*** Control (35) VGlut RNAi (45)

B

Copulation latency (x1000 s)

Copulation rate 0.0 0.2 0.4 0.6 0.8 1.0

SS02709-Gal4/ UAS-VGlut RNAi

Copulation latency (x1000 s)

A

NOS RNAi (35)

SS02709-Gal4/ 3 UAS-sGCa99B RNAi

2 NS 1

0 Control sGCa99B RNAi (80) (38)

Figure 5. Retrograde NO Signal Controls R2/R4m to Inhibit the Pre-mating Rejection Response (A and B) Knockdown of VGlut expression in R2/R4m neurons. SS02709-Gal4 is used to drive VGlut RNAi. The copulation rate (A) and copulation latency (B) are plotted. (C and D) Knockdown of NR1 (left) and NR2 (right) expression in R4d neurons. c232-Gal4 is used. The copulation rate (C) and copulation latency (D) are plotted. (E) The copulation duration of control and VGat knockdown females. The Wilcoxon-Mann-Whitney test is applied for the comparison. For the copulation rate, the log rank test is applied. (F and G) Knockdown of NOS expression in R3 neurons. 189y-Gal4 is used to drive NOS RNAi. (H and I) Knockdown of NOS expression in R4d neurons using c232-Gal4. (J and K) Knockdown of sGCa99B expression in R2/R4m neurons. SS02709-Gal4 is used. The copulation rate (F, H, and J) and copulation latency (G, I, and K) are plotted. For the copulation rate, the log rank test is applied. For the copulation latency, the Wilcoxon-Mann-Whitney test is applied. NS, p > 0.05, *p < 0.05, ***p < 0.001. The number of animals analyzed is indicated in parentheses.

VGlut expression in R2/R4m, which reduces glutamate release from R2/R4m, and found a significant decrease in the female copulation rate (c2 = 37.3, p = 1e 9; Figure 5A) and an increase in the copulation latency (p = 1.92e 4; Figure 5B). Likewise, knockdown of either the NR1 or NR2 subunit of NMDAR in postsynaptic R4d neurons induced a significant reduction in the copulation rate (c2 = 50.3, p = 1e 12; Figure 5C, left; c2 = 38.2, p = 6e 10; Figure 5C, right) and an increase in the copulation latency (p = 6.11e 8; Figure 5D, left; p = 0.033; Figure 5D, right). All of these results validate the involvement of VGlut and NMDAR in R2/R4m and R4d, respectively, in female pre-mating regulation. It is possible that R2/R4m would activate R4d via glutamate-NMDAR signaling, in parallel with R2/R4m-to-R4d inhibition via GABA-GABAA receptor signaling. There should be functional differences between these two signaling pathways because suppression of glutamate-NMDAR signaling did not 8 Current Biology 30, 1–12, February 3, 2020

affect the copulation duration, whereas GABA-GABAA receptor signaling significantly decreased it (Figure 5E; see also Figures 4J and 4L). How do these two signals of opposite sign lead to a proper mating regulation? Because of the unidirectional synaptic connection from R2/R4m to R4d [27], we assumed that glutamate-NMDAR signaling not only activates R4d neurons but also facilitates a retrograde signal from R4d to R2/R4m to promote the inhibitory function of R2/R4m. It is known that glutamatergic signals acting through NMDAR activate nitric oxide synthase (NOS) [40]. NO is a gaseous second messenger produced at postsynaptic cells that retrogradely facilitates the synaptic transmission of presynaptic cells [41]. It was reported that R neurons have immunoreactivity for NOS [42]. Although R3 neurons express NOS predominantly, NOS knockdown in R3 neurons showed little effect on both the copulation rate (c2 = 0.7, p = 0.4; Figure 5F) and the copulation

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A

Glu/NMDAR NO/sGC signals

R2/R4m neuron PKG

sGC

a

Glu

GABA

b

NO

NMDAR 2+

Ca

PIP2

GABAA (Rdl)

NOS

CaM

R4d neuron

B

SMP PN

aSP-g

DA neurons PPM3

ORN

DA

cVA Male Information

pCd pC1

copulation latency was increased (p = 1.7e 6; Figure 5I). These NOS effects in R4d indicate the possibility that gaseous NO may facilitate the neurotransmission of R2/R4m as a retrograde signal. We then examined whether the a subunit of soluble guanylyl cyclase (sGCa99B) participated in R2/R4m for the mating control because soluble guanylyl cyclase (sGC) is a mediator of the NO-cyclic guanosine monophosphate (cGMP) pathway that facilitates an exoendocytic cycle of neurotransmission [43]. Although knockdown of sGCa99B in R2/R4m neurons had little effect on the copulation latency (p = 0.11; Figure 5K), it significantly decreased the copulation rate (c2 = 14, p = 2e 4; Figure 5J). Therefore, these results support a scenario that activity-dependent signaling from R4d retrogradely facilitates R2/R4m synaptic transmission to abolish the acute rejection response of female flies. DISCUSSION

Ellipsoid Body

Dop1R1

D2R

R2/R4m GABA

Glu/NMDAR NO/sGC signals

GABA A R4d Dop1R2

Copulation

C

Ach

Rejection I1-FFL

PPM3

X

R2/R4m

Y

R4d

Z

Rejection

Output

Figure 6. A Model of the Neural Circuitry Mediating Pre-mating Behavior in Virgin Female Flies (A) The potential retrograde signaling from R4d to R2/R4m. R2/R4m unidirectionally synapses to R4d. (a) Glutamate/NMDAR is likely to activate R4d. (b) NOS/NO/sGC signals may facilitate synaptic transmission. (B) A model depicting potential neural connections and information flow. Pheromonal information of courting males activates aSP-g, which transmits the information to the pCd/pC1. Postsynaptic sites of PPM3 are located in the SMP region. The PPM3 activates R2/R4m and R4d simultaneously, and R2/ R4m inhibits R4d. Glu/NMDAR and NO/sGC signaling exist between R2/R4m and R4d, as described in (A). Activation of R4d promotes pre-mating rejection by virgin females. (C) Analogy between the neural circuitry defined in this study and the incoherent type 1 feedforward loop (I1-FFL). Factor X regulates factor Y, and both coordinately regulate Z. The actions of X and Y are integrated at Z.

latency (p = 0.478; Figure 5G). In contrast, when NOS was knocked down in R4d neurons, the copulation rate was significantly decreased (c2 = 12.9, p = 3e 4; Figure 5H) and the

Overall, the present findings provide evidence for a neural relation that regulates the action selection of pre-mating behaviors in female Drosophila. The PPM3, a subset of DA neurons, forms a circuit with the R neurons R2/R4m and R4d in the EB. These different types of R neurons require different types of DA receptors, Dop1R1/D2R and Dop1R2. The knockdown of each DA receptor type indicates that all of these receptors are required to activate the expressing neurons, although the D2R conventionally inhibits D2R-expressing neurons [44]. R2/R4m and R4d are GABAergic and cholinergic, respectively. Synaptic GRASP analysis revealed a neuronal connection from R2/R4m to R4d. R4d inhibition via the GABAA receptor is required for the proper reduction of pre-mating rejection. In addition to DA regulation of R2/R4m, the potential retrograde regulation may facilitate the GABA transmission of R2/R4m, depending on the R4d activity, with the production of NO via NMDAR/NOS signaling (Figure 6A). This potentiation-like regulation provides the activation order of each R neuron with flexibility for the neural circuit output, and therefore to the rejection response for controlling the pre-mating behavioral kinetics. The pre-mating rejection should continue if the encounter does not match the female’s criteria. Pheromones are important sexual cues provided by the male. Fruit flies produce cuticular hydrocarbons as pheromonal substances [45]. cVA is a malespecific pheromonal cue that elicits female sexual arousal via the olfactory sensory system [46]. This cVA signal activates pCd via a third-order olfactory interneuron, aSP-g [7]. The SMP region contains aSP-g, pCd, and PPM3, although the direct connection between them has not been demonstrated. This aggregation of important components for female mating behavior suggests that pCd potentially integrates and verifies male information to execute the final decision for initiating the copulation. The female must resist the encounter until the evaluation process is complete—that is, the pre-mating rejection response controlled by the circuit found in the present study. These considerations lead to an assumption that the pCd and PPM3/R neuron circuits execute pre-mating computation in parallel (Figure 6B). Many sexually dimorphic behaviors are reportedly mediated by sex-specific neural circuits [47, 48] (e.g., pCd, pC1). PPM3 and R neurons do not express fruitless [49] or dsx [50], and no morphological sexual dimorphism has Current Biology 30, 1–12, February 3, 2020 9

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

been detected [27]. Thus, we propose a non-sexually dimorphic circuit modulating sex-specific pre-mating behavior. Our genetic manipulations of R neurons altered the pre-mating kinetics in virgin female flies, leading to the question of whether the modulation of R neurons also affects post-mating behavior, which is induced by injection of the seminal fluid sex peptide from the male fly and sustained for several days [51]. One of the remarkable characteristics of post-mating rejection is ovipositor extrusion controlled by a distinct class of neurons in post-mating females [51–53], but either suppression of R2/ R4m neurons or activation of R4d neurons, both of which reduced receptivity, rarely induced ovipositor extrusion. Mated females with R4d suppression, which makes virgin females highly receptive, exhibited a large rejection response similar to that in mated control females, and no copulation was observed during the 1-h observation time (data not shown). In addition to these observations, the virgin females with Rdl knockdown in R4d reduced their walking speed with the progression of the male courtship (Figures 4I and S4F–S4H); however, the walking speed of the mated females increases with the progression of the male courtship [54]. Although further studies are required to elucidate the whole picture of rejection control in females, neuronal regulation of the post-mating response is presumably parallel to the pre-mating rejection response modulated by R neurons in the EB. In this study, we found circuit functions that contain PPM3 DA subcluster neurons, and R neurons R2/R4m and R4d, were involved in the regulation of the kinetics of pre-mating behavior in virgin females (Figure 6B). Pre-mating rejection is acutely elicited and then gradually decreased. A circuital feature of PPM3/R neurons may provide a theoretical action for controlling these pre-mating kinetics. PPM3 sends DA signals to both R2/ R4m and R4d, and this recurrent circuit forms a feedforward motif with a repressor, the so-called incoherent type 1 feedforward loop (I1-FFL) [55]. I1-FFL is one of the most common network motifs implicated in gene and protein regulation networks, metabolic pathways, and neural networks from bacteria to humans [56, 57]. In the I1-FFL circuit, an activator X (e.g., PPM3) activates a target Z (e.g., R4d) and simultaneously activates another target Y (e.g., R2/R4m) that inhibits the target Z (Figure 6C). Previous studies demonstrated that the I1-FFL accelerates the response of target Z, with a shorter response time [55, 58]. Moreover, upon input from X, Z activity increases and then, depending on the Y activity, it decreases toward basal levels. The I1-FFL circuit comprising PPM3, R2/R4m, and R4d would thus theoretically generate the acute activation of R4d, which promotes the pre-mating rejection behavior of females that would later be gradually attenuated by GABAergic signals from R2/R4m. This schema is consistent with our findings, in which the circuit of PPM3/R neurons plays an important role in the action selection of pre-mating behavior. Our genetic analysis indicates that retrograde signals from R4d to R2/R4m, mediated by NO and NMDAR, would add some flexibility to the Y-to-Z regulation in the I1-FFL circuit, contributing to progressive attenuation of the rejection response in virgin females. Depolarization of R4d activates NMDAR by the coincident input of glutamate from R2/R4m. The retrograde signals induced by NMDAR/NOS/sGC probably facilitate the GABA transmission of R2/R4m, which progressively suppresses 10 Current Biology 30, 1–12, February 3, 2020

R4d activity via the GABAA receptors. Because NMDARs require simultaneous activation by glutamate and depolarization [59], the NMDAR pore opening may be insufficient until the activity of R4d neurons will reach a sufficient level. This may lead to keeping the GABAergic R2/R4m function at a lower level that is under a certain threshold required for GABA release, and it may induce a continuous pre-mating rejection response. Recently, new subdivision of R neurons (R5 and R6) were classified by clonal morphological analysis and cell lineage analysis [27]. These new subclasses of R neurons may provide a novel function of the putative I1-FFL. To investigate this further, the physiological properties of each R neuron should be analyzed. Kahsai et al. revealed the distribution pattern of neurotransmitters in EB neurons [28]. In the R2/R4m axonal ring structure, the glutamatergic population seems to be smaller than the GABAergic population, suggesting that the neuronal population in the R2/R4m is heterologous and contains few, if any, neurons that co-express both glutamate and GABA. In the vertebrate system, heterosynaptic regulation of GABAergic transmission is incorporated into inhibitory long-term potentiation [60]. Inhibitory long-term potentiation of GABAergic neurons is induced at heterosynaptic sites containing glutamatergic and GABAergic neurons as presynaptic cells [61]. In GABAergic inhibitory long-term potentiation, NO induced by glutamatergic activation of the NMDAR/NOS pathway retrogradely activates sGC, which augments cGMP levels to enhance GABA release [43]. These intriguing analogies between vertebrate findings and our results suggest that a similar molecular machinery potentiates GABAergic subsets of the R2/R4m neurons for attenuating the pre-mating rejection response, and hence for the action selection of pre-mating kinetics. Regarding the action selection for adaptive behaviors, Strausfeld and Hirth and others proposed a correspondence of functions and neural architectures between vertebrate basal ganglia and the insect central complex, which contains EB neurons [62, 63]. In the basal ganglia, DA activates the neurons of the nucleus accumbens with modulation of glutamate and GABA release [64]. The neurochemicals in the nucleus accumbens released by sexual interaction regulate female action selection for the affiliation of monogamous prairie voles (Microtus ochrogaster) [65]. Our genetic manipulations of particular DA, cholinergic, glutamatergic, and GABAergic systems in the central complex significantly affected the action selection for female fruit fly pre-mating behaviors, implying that the mechanisms are similar to those in the vertebrate system, which sheds light on the evolutionary parallels and diversities across the animal kingdom. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Experimental animals B Female mating assay

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

B

Immunohistochemical analysis Confocal microscopy and image processing QUANTIFICATION AND STATISTICAL ANALYSIS B Quantification of female mating behavior B Quantification analysis of female walking speed B Statistical analysis DATA AND CODE AVAILABILITY B

d

d

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. cub.2019.11.065. ACKNOWLEDGMENTS We thank T. Kitamoto, K. Kume, M. Gallio, the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, the Kyoto Stock Center, and FlyLight for the fly stocks. We also thank T. Kitamoto, K. Kimura, T. Awasaki, H. Shiozaki, Y. Ishikawa, R. Tanaka, and X. Li for comments and discussion and M. Kuno, Y. Maki, and Y. Ishikawa for fly maintenance. This work was supported by MEXT KAKENHI Grants-in-Aid for Scientific Research (B) (grant JP16H04655 to A.K.), Scientific Research on Innovative Areas ‘‘Evolinguistics’’ (grant JP18H05069 to A.K.), Systems Science of Bio-navigation (grant 19H04933 to A.K.), Challenging Research (Exploratory) (grant 17K19450 to A.K.), Grant-in Aid for Scientific Research (C) (15K07147 and 18K06332 to H.I.), the Naito Foundation (to A.K.), and the Inamori Foundation Research Grant, Japan (to H.I.). AUTHOR CONTRIBUTIONS H.I. and A.K. conceived and designed the experiments. H.I. carried out the experiments and analyzed the data. H.I. and A.K. wrote the paper. DECLARATION OF INTERESTS The authors declare no competing interests. Received: July 18, 2019 Revised: October 21, 2019 Accepted: November 21, 2019 Published: January 2, 2020 REFERENCES 1. Chen, S.L., Chen, Y.H., Wang, C.C., Yu, Y.W., Tsai, Y.C., Hsu, H.W., Wu, C.L., Wang, P.Y., Chen, L.C., Lan, T.H., et al. (2017). Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons. Sci. Rep. 7, 44595. 2. Pfaus, J.G. (2009). Pathways of sexual desire. J. Sex. Med. 6, 1506–1533. 3. Numan, M., and Young, L.J. (2016). Neural mechanisms of mother-infant bonding and pair bonding: similarities, differences, and broader implications. Horm. Behav. 77, 98–112. 4. Aranha, M.M., and Vasconcelos, M.L. (2018). Deciphering Drosophila female innate behaviors. Curr. Opin. Neurobiol. 52, 139–148. 5. Zhou, C., Pan, Y., Robinett, C.C., Meissner, G.W., and Baker, B.S. (2014). Central brain neurons expressing doublesex regulate female receptivity in Drosophila. Neuron 83, 149–163. 6. 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, 542–546. 7. Kohl, J., Ostrovsky, A.D., Frechter, S., and Jefferis, G.S.X.E. (2013). A bidirectional circuit switch reroutes pheromone signals in male and female brains. Cell 155, 1610–1623. 8. Zhang, S.X., Rogulja, D., and Crickmore, M.A. (2016). Dopaminergic circuitry underlying mating drive. Neuron 91, 168–181.

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12 Current Biology 30, 1–12, February 3, 2020

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit anti-GFP

Invitrogen

Cat# A-11122; RRID: AB_221569

Anti-Bruchpilot (Brp), nc82

Developmental Studies Hybridoma Bank

RRID: AB_528108

mouse anti-GFP monoclonal

Sigma-Aldrich

Cat# G6539; RRID: AB_259941

Antibodies

anti-CD4 polyclonal

Sigma-Aldrich

Cat# HPA004252; RRID: AB_1078466

Rabbit anti-RFP

Rockland

Cat# 600-401-379; RRID: AB_2209751

Rat anti-GFP

Nakarai Tesque

04404-26

Alexa Fluor 555-conjugated anti-rabbit IgG

Invitrogen

Cat# A-21429; RRID: AB_141761

Alexa Fluor 647-conjugated anti-mouse IgG

Invitrogen

Cat# A-21236; RRID: AB_141725

Alexa Fluor 488-conjugated anti-rat IgG

Jackson

Cat# 112-545-167; RRID: AB_2338362

Experimental Models: Organisms/Strains D. melanogaster: UAS-CD8::GFP: w[1118]; P{y[+t7.7] w[+mC] = 10XUAS-IVS-mCD8::GFP}su(Hw)attP1

Bloomington Drosophila Stock Center

BDSC_32187

D. melanogaster: c346-Gal4: P{w[+mW.hs] = GawB} c346, w[*]

Bloomington Drosophila Stock Center

BDSC_30831

D. melanogaster: c232-Gal4: w[*]; P{w[+mW.hs] = GawB}Alp4[c232]

Bloomington Drosophila Stock Center

BDSC_30828

D. melanogaster: 189y-Gal4: w[*]; P{w[+mW.hs] = GawB}lilli[189Y]

Bloomington Drosophila Stock Center

BDSC_30817

D. melanogaster: c819-Gal4: w[*]; P{w[+mW.hs] = GawB}c819

Bloomington Drosophila Stock Center

BDSC_30849

D. melanogaster: NP1131-Gal4: y* w*; P{w+mW.hs = GawB}NP1131 / CyO, P{w- = UAS-lacZ.UW14}UW14

Kyoto Drosophila Stock Center

Kyoto; 103898

D. melanogaster: c507-Gal4: w[*]; P{w[+mW.hs] = GawB}Alp4[c507]

Bloomington Drosophila Stock Center

BDSC_30840

D. melanogaster: 201y-Gal4: w[1118]; P{w[+mW.hs] = GawB}Tab2[201Y]

Bloomington Drosophila Stock Center

BDSC_4440

D. melanogaster: c105-Gal4: w[*] P{w[+mW.hs] = GawB}c105[c105]

Bloomington Drosophila Stock Center

BDSC_30822

D. melanogaster: Th-Gal4: w[*]; P{w[+mC] = ple-GAL4.F}3

Bloomington Drosophila Stock Center

BDSC_8848

D. melanogaster: Tdc2-Gal4: w[*]; P{w[+mC] = Tdc2-GAL4.C}2

Bloomington Drosophila Stock Center

BDSC_9313

D. melanogaster: Trh-Gal4: w[1118]; P{w[+mC] = Trh-GAL4.long}2

Bloomington Drosophila Stock Center

BDSC_38388

D. melanogaster: Ddc-Gal4: w[1118]; P{w[+mC] = Ddc-GAL4.L}Lmpt[4.36]

Bloomington Drosophila Stock Center

BDSC_7009

D. melanogaster: Gad1-Gal4: P{w[+mC] = Gad1-GAL4.3.098}2/CyO

Bloomington Drosophila Stock Center

BDSC_51630

D. melanogaster: FlyLight Split-GAL4 SS02709-Gal4: R20A02-p65ADZp attp40; R93G02-ZpGdbd attp2 slpit Gal4

FlyLight Split-GAL4 Driver Collection

SS02709, Robot ID:3019125

D. melanogaster: OK348-Gal4

Dr. Toshi Kitamoto, Univ. of Iowa

N/A

D. melanogaster: UAS-Kir2.1; tub-Gal80ts

Dr. Toshi Kitamoto, Univ. of Iowa

N/A

D. melanogaster: dDATfmn

Dr. Kazuhiko Kume, Nagoya City Univ.

N/A

D. melanogaster: UAS-dTRPA1: w[*]; P{y[+t7.7] w[+mC] = UAS-TrpA1(B).K}attP16

Bloomington Drosophila Stock Center

BDSC_26263

FBti0131975

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Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

D. melanogaster: UAS-Da7::GFP: P{UASnAChRa7.GFP}3/MKRS

Bloomington Drosophila Stock Center

BDSC_39692

D. melanogaster: UAS-nSyb::GFP: w[*]; P{w[+mC] = UAS-nSyb.eGFP}3

Bloomington Drosophila Stock Center

BDSC_6922

D. melanogaster: Syb:GRASP: lexAop-CD4:: spGFP11/CyO; UAS-Syb::spGFP [1–10]/TM6b

Dr. Marco Gallio, Northwestern Univ.

N/A

D. melanogaster: GMR44D11-LexA: 44D11LexAp65 in attP40

Bloomington Drosophila Stock Center

BDSC_54165

D. melanogaster: RNAi of ChAT: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF01877}attP2

Bloomington Drosophila Stock Center

BDSC_25856

D. melanogaster: RNAi of Dop1R1: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HM04077}attP2

Bloomington Drosophila Stock Center

BDSC_31765

D. melanogaster: RNAi of Dop1R2: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02043}attP2

Bloomington Drosophila Stock Center

BDSC_26018

D. melanogaster: RNAi of D2R: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02025}attP2

Bloomington Drosophila Stock Center

BDSC_26001

D. melanogaster: RNAi of DopEcR: P{KK111211} VIE-260B

Vienna Drosophila Resource Center

VDRC; 103494

D. melanogaster: RNAi of Gad1: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02916}attP2

Bloomington Drosophila Stock Center

BDSC_28079

D. melanogaster: RNAi of Rdl: y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HMC03643}attP40

Bloomington Drosophila Stock Center

BDSC_52903

D. melanogaster: RNAi of GABA-B-R2: P{KK100020}VIE-260B

Vienna Drosophila Resource Center

VDRC; 110268

D. melanogaster: RNAi of Nos: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF03220}attP2

Bloomington Drosophila Stock Center

BDSC_28792

D. melanogaster: RNAi of NR1: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF01961}attP2

Bloomington Drosophila Stock Center

BDSC_25941

D. melanogaster: RNAi of NR2: y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HMS02176}attP40

Bloomington Drosophila Stock Center

BDSC_40928

D. melanogaster: RNAi of VGlut: y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HMS02175}attP40

Bloomington Drosophila Stock Center

BDSC_40927

D. melanogaster: RNAi of sGCa99B: y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF03176}attP2

Bloomington Drosophila Stock Center

BDSC_28748

D. melanogaster: RNAi TRiP attp2 Background: y[1] v[1]; P{y[+t7.7] = CaryP}attP2

Bloomington Drosophila Stock Center

BDSC_36303

D. melanogaster: RNAi TRiP attp40 Background: y[1] v[1]; P{y[+t7.7] = CaryP}attP40

Bloomington Drosophila Stock Center

BDSC_36304

D. melanogaster: RNAi VDRC Background: w[1118]

Vienna Drosophila Resource Center

VDRC; 60000

Software and Algorithms ImageJ

National Institutes of Health

version 1.47v; RRID: SCR_003070

Excel

Microsoft

version 16.16.14

R

https://www.r-project.org

version 3.4.4.

Other LED light box

Too Marker Products

ComicMaster Tracer

Web camera

Logicool

HD Webcam C270

Vectashield

Vector Labs

H-1200

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hiroshi Ishimoto ([email protected]). This study did not generate new unique reagents. e2 Current Biology 30, 1–12.e1–e4, February 3, 2020

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

EXPERIMENTAL MODEL AND SUBJECT DETAILS Fruit flies D. melanogaster were raised on standard yeast-based media at 25 ± 1 C and 40%–60% relative humidity in 12 h/12 h light/ dark cycle, unless otherwise noted. Detailed information on fly crosses, housing, and age for each experiment are indicated in the relevant Method Details. Genotypes are indicated in figure legends. For strain details please see Key Resources Table. METHOD DETAILS Experimental animals All fly lines used in this study are described in the Key Resources Table. Canton-S2202u flies were used as a wild-type. For gene knockdown we used UAS-RNAi strains that have successful record of performance (Table S1; Supplemental Information References). For their controls, we used corresponding background strains provided from Transgenic RNAi Project (TRiP) and Vienna Drosophila Resource Center (VDRC). Female mating assay Males and females were collected during 5 h after eclosion and were kept in a group (up to 15 flies in a vial with fly food) separately. Both sexes of flies were kept as virgin until experiment. Wild-type males (5-7 d old) were used in all the experiments to serve as the mating partners for female flies (5-7 d old). All behavioral experiments were performed between 5 and 10 h after light onset. A pair of male and female flies was placed into a round courtship chamber (15-mm diameter and 3-mm deep) with a plastic lid by gentle aspiration without anesthesia. The courtship chambers were backlit by placing on an LED light box (ComicMaster Tracer, Too Marker Products, Japan). Fly behaviors were recorded for 1 h at 1 Hz interval with a web camera (Logicool HD Webcam C270, Tokyo, Japan). For neural suppression using the TARGET system, female flies were exposed to the restricted temperature (RT; 29 ± 1 C) or permissive temperature (PT; 18 ± 1 C) for 48 h prior to the mating test, then challenged to the mating test with the wild-type male fly after a 1-h resting period at 25 ± 1 C. For neural activation using dTRPA1, the mating behaviors were carried out at 28.5 ± 1.5 C and 19 ± 1 C as experimental and control temperatures, respectively. Control and experimental groups were tested on the same day. Immunohistochemical analysis Adult brains were dissected from 3 to 5-day-old female flies in phosphate-buffered saline (PBS; pH7.4) and fixed for 1 h with 3.7% formaldehyde at 25 C, in PBS containing 0.05% Triton X-100 (PBST). The brains were blocked with PBST containing 0.1% normal goat serum for 1 h. Immunolabeling was performed as described previously [66]. Antibodies were used at the indicated dilutions as following: Rabbit anti-GFP antibody (1:300; Invitrogen, A11122) for detecting CD8::GFP, nSyb::GFP and Da7::GFP. nc82, the mouse anti-Bruchpilot (Brp) antibody (1:20; Developmental Studies Hybridoma Bank, University of Iowa, AB_528108) to counterstain the brain. For the GRASP analysis, mouse anti-GFP monoclonal (1:300, Sigma-Aldrich, G6539) was used for detecting the reconstructed GFP signals, and rabbit anti-CD4 polyclonal (1:300; Sigma-Aldrich, HPA004252) was used for detecting the CD4::spGFP11. The secondary antibodies used were as follows: Alexa Fluor 555-conjugated anti-rabbit IgG (1:300; Invitrogen, A21429) and Alexa 647-conjugated anti-mouse IgG (1:300; Invitrogen, A21236). For the trans-TANGO analysis, rabbit anti-RFP polyclonal (1:600, Rockland, 600-401-379) was used for detecting the post-synaptic RFP signals, and rat anti-GFP monoclonal (1:600; NAKARAI TESQUE, 04404-26) was used for detecting the pre-synaptic GFP. The secondary antibodies used were as follows: Alexa Fluor 555-conjugated anti-rabbit IgG (1:600; Invitrogen, A21429), Alexa 488-conjugated anti-rat IgG (1:600; Jackson, 112-545-167). and Alexa 647-conjugated anti-mouse IgG (1:600; Invitrogen, A21236). Brain samples were mounted on glass slides with bridge coverslips, using Vectashield (Vector Labs, H-1200). Confocal microscopy and image processing Serial optical sections of brains were obtained at 0.84-mm intervals with a resolution of 512 3 512 pixels (0.83 mm/pixel) using an FV-1000D laser-scanning confocal microscope (Olympus) equipped with a silicone-oil immersion 30x objective lens (UPLSAPO30XS, NA = 1.05; Olympus). Confocal image datasets obtained from Gal4 strains, c346, c232, 189y, NP1131, c507, 201y, c105, and SS02709, labeled with CD8::GFP were digitally aligned to a template brain with non-rigid registration using the Computational Morphometry Toolkit [67] (CMTK; RRID: SCR_002234). The size, contrast, and brightness of the images were adjusted using ImageJ. QUANTIFICATION AND STATISTICAL ANALYSIS Quantification of female mating behavior A semi-automatic method to detect copulation timing was performed offline with ImageJ software (version 1.47v, National Institutes of Health; RRID: SCR_003070). We combined ImageJ functions ‘‘Image Calculator’’ and ‘‘Analyze Particles’’ to determine the pixel sizes of individual fly objects (Figures S5A and S5B). We fixed the threshold pixel size at 120 to discriminate noncopulating and copulating flies. The ImageJ data was exported to Excel (Microsoft, Redmond, WA, USA) to detect the timing of copulation, in which the frames indicating ‘‘copulating flies’’ should be identified in 5 min of contiguous frames to exclude flies unsuccessfully attempting to copulate. This semi-automatic method gave virtually the same score as manual detection by an expert (Figures S5C and S5D). Current Biology 30, 1–12.e1–e4, February 3, 2020 e3

Please cite this article in press as: Ishimoto and Kamikouchi, A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.11.065

Courtship latency (time to start courtship) of male flies was not altered whether they challenged to control or experimental female (data not shown). We did not adopt data from male flies that showed no courtship during the test period. To detect copulation manually, we observed the specific features described in Manning [68]. The copulation duration was measured manually. To calculate the copulation latency, we excluded data from non-mated pairs of flies. It should be noted that because of this data exclusion process, low copulation rate would cause underestimation of the copulation latency. The accumulating copulation rate, the copulation latency, and the copulation duration were analyzed using statistical and graphic software, R (https://www.r-project.org). Quantification analysis of female walking speed Female walking speed was measured using the ImageJ plugin ‘‘Manual Tracking’’ from digital images obtained in the female mating assay. The points of change in the walking speed were calculated using the Pruned Exact Linear Time (PELT) algorithm using the R library ‘‘changepoint.’’ Statistical analysis Statistical analyses were performed using R software version 3.4.4. (https://www.r-project.org). For the mating rate, Log-Rank test was applied using cmprsk package of R. For the latency, Wilcoxon-Mann-Whitney test was applied to the mated flies by using coin package of R. For the average mating duration, Student-t test was applied. For the incomplete mating rate, fisher’s exact test was applied. DATA AND CODE AVAILABILITY Raw data supporting the current study have not been deposited in a public repository because of their large size, but are available on reasonable request from the corresponding author.

e4 Current Biology 30, 1–12.e1–e4, February 3, 2020