The reward system is a collection of circuits that reinforce behaviors necessary for survival []. Given the importance of reproduction for survival, actions that promote successful mating induce pleasurable feeling and are positively reinforced []. This principle is conserved in Drosophila, where successful copulation is naturally rewarding to male flies, induces long-term appetitive memories [], increases brain levels of neuropeptide F (NPF, the fly homolog of neuropeptide Y), and prevents ethanol, known otherwise as rewarding to flies [], from being rewarding []. It is not clear which of the multiple sensory and motor responses performed during mating induces perception of reward. Sexual interactions with female flies that do not reach copulation are not sufficient to reduce ethanol consumption [], suggesting that only successful mating encounters are rewarding. Here, we uncoupled the initial steps of mating from its final steps and tested the ability of ejaculation to mimic the rewarding value of full copulation. We induced ejaculation by activating neurons that express the neuropeptide corazonin (CRZ) [] and subsequently measured different aspects of reward. We show that activating Crz-expressing neurons is rewarding to male flies, as they choose to reside in a zone that triggers optogenetic stimulation of Crz neurons and display conditioned preference for an odor paired with the activation. Reminiscent of successful mating, repeated activation of Crz neurons increases npf levels and reduces ethanol consumption. Our results demonstrate that ejaculation stimulated by Crz/Crz-receptor signaling serves as an essential part of the mating reward mechanism in Drosophila.

Results and Discussion

8 Tayler T.D.

Pacheco D.A.

Hergarden A.C.

Murthy M.

Anderson D.J. A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. 9 Pan Y.

Robinett C.C.

Baker B.S. Turning males on: activation of male courtship behavior in Drosophila melanogaster. 10 Pavlou H.J.

Lin A.C.

Neville M.C.

Nojima T.

Diao F.

Chen B.E.

White B.H.

Goodwin S.F. Neural circuitry coordinating male copulation. 11 Inagaki H.K.

Jung Y.

Hoopfer E.D.

Wong A.M.

Mishra N.

Lin J.Y.

Tsien R.Y.

Anderson D.J. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. 12 von Philipsborn A.C.

Liu T.

Yu J.Y.

Masser C.

Bidaye S.S.

Dickson B.J. Neuronal control of Drosophila courtship song. 13 Crickmore M.A.

Vosshall L.B. Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila. 14 Zhang S.X.

Rogulja D.

Crickmore M.A. Dopaminergic Circuitry Underlying Mating Drive. 15 Hoopfer E.D.

Jung Y.

Inagaki H.K.

Rubin G.M.

Anderson D.J. P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila. 16 Keleman K.

Vrontou E.

Krüttner S.

Yu J.Y.

Kurtovic-Kozaric A.

Dickson B.J. Dopamine neurons modulate pheromone responses in Drosophila courtship learning. 17 Huang J.

Liu W.

Qi Y.X.

Luo J.

Montell C. Neuromodulation of Courtship Drive through Tyramine-Responsive Neurons in the Drosophila Brain. 8 Tayler T.D.

Pacheco D.A.

Hergarden A.C.

Murthy M.

Anderson D.J. A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. Mating interactions in Drosophila are orchestrated by neuronal programs that integrate sensory signals into courtship and copulation motor actions [], as well as programs controlling mating drive, the persistence of copulation process, and its timing []. To test the hypothesis that ejaculation induces the rewarding value of mating, we asked whether promoting sperm and seminal fluid release (SSFR) in the absence of a female partner is sufficient to evoke the perception of reward. To uncouple SSFR from the rest of the mating sequence, we focused on male-specific corazonin (Crz) neurons located in the abdominal ganglion (AG), which were previously shown to induce SSFR [], and tested whether ejaculation induced by the activation of Crz neurons is rewarding for male flies.

6 Devineni A.V.

Heberlein U. Preferential ethanol consumption in Drosophila models features of addiction. 7 Kaun K.R.

Azanchi R.

Maung Z.

Hirsh J.

Heberlein U. A Drosophila model for alcohol reward. 18 Tempel B.L.

Bonini N.

Dawson D.R.

Quinn W.G. Reward learning in normal and mutant Drosophila. 19 Liu C.

Plaçais P.Y.

Yamagata N.

Pfeiffer B.D.

Aso Y.

Friedrich A.B.

Siwanowicz I.

Rubin G.M.

Preat T.

Tanimoto H. A subset of dopamine neurons signals reward for odour memory in Drosophila. 20 Burke C.J.

Huetteroth W.

Owald D.

Perisse E.

Krashes M.J.

Das G.

Gohl D.

Silies M.

Certel S.

Waddell S. Layered reward signalling through octopamine and dopamine in Drosophila. 21 Aso Y.

Rubin G.M. Dopaminergic neurons write and update memories with cell-type-specific rules. 22 Shao L.

Saver M.

Chung P.

Ren Q.

Lee T.

Kent C.F.

Heberlein U. Dissection of theDrosophilaneuropeptide F circuit using a high-throughput two-choice assay. 23 Ichinose T.

Aso Y.

Yamagata N.

Abe A.

Rubin G.M.

Tanimoto H. Reward signal in a recurrent circuit drives appetitive long-term memory formation. 24 Zer S.

Ryvkin J.

Wilner H.J.

Zak H.

Shmueli A.

Shohat-Ophir G. A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster. 25 Joseph R.M.

Devineni A.V.

King I.F.

Heberlein U. Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila. 26 Ofstad T.A.

Zuker C.S.

Reiser M.B. Visual place learning in Drosophila melanogaster. 27 Putz G.

Heisenberg M. Memories in Drosophila heat-box learning. 28 Yang Z.

Bertolucci F.

Wolf R.

Heisenberg M. Flies cope with uncontrollable stress by learned helplessness. 22 Shao L.

Saver M.

Chung P.

Ren Q.

Lee T.

Kent C.F.

Heberlein U. Dissection of theDrosophilaneuropeptide F circuit using a high-throughput two-choice assay. Figure 1 Ejaculation Induced by the Activation of Crz Neurons Is Pleasurable to Male Flies Show full caption (A) Schematic illustration of the two-choice positional assay. (B) CRZ-Gal4 expression pattern in representative male and female brain and ventral nerve cord (VNC; upper and lower, respectively). (C–K) Flies’ positional preference toward an optogenetic activation zone when both sides of the arena are un-illuminated (dark/dark, left panel in scheme in A and in C, F, and I) and when one side is illuminated (light/dark, right panel in scheme in A and in D, G, and J). The average positional preference (n = 12 per genotype, 10 flies per arena), calculated according to the equation shown in (A), is indicated over a 5-min interval for CRZ > CsChrimson male flies (C and D), CRZ > CsChrimson female flies (F and G) and CRZ-2fa > CsChrimson male flies (I and J). One-sample t tests were performed for the fifth minute against zero with Bonferroni corrections, ∗∗∗p < 0.001 for CRZ > CsChrimson. All other comparisons were not statistically significant. In (E), (H), and (K), a graphical comparison of the average positional preference of the fifth minute in dark/dark versus light/dark conditions is presented for CRZ > CsChrimson male flies (E), CRZ > CsChrimson female flies (H), and CRZ-2fa>CsChrimson male flies (K). (n = 12, statistical significance determined by two-way ANOVA followed by Bonferroni post hoc comparison. Significant difference was documented in (E). F(2,33) = 16.7965, ∗∗p < 0.001, Bonferroni post hoc comparison for CRZ > CsChrimson male flies dark versus light p < 0.05, versus UAS-CsChrimson p < 0.05, and versus CRZ-Gal4 light p < 0.01. All other comparisons in (H) and (K) were not statistically significant. Error bars signify SEM. See also Figures S1 S2 , and S3 Different behavioral paradigms in Drosophila make it possible to study distinct components of reward processing, including the valence of the experience, expression of appetitive memories, and the motivation to obtain reward []. First, we tested whether ejaculation induced by activation of Crz neurons promotes immediate pleasurable response. Rewarding stimuli promote behavioral actions to extend their duration, while aversive stimuli promote actions to reduce their duration []. For example, in a two-choice preference assay, flies prefer to reside in a zone where they receive optogenetic stimulation of reward-encoding neurons []. Using this assay, we analyzed the position of male flies expressing the red-shifted channelrhodopsin CsChrimson in Crz neurons (CRZ > CsChrimson) in a rectangular arena under two conditions lasting 5 min each: (1) both sides of the arena were un-illuminated and (2) only one side of the arena was illuminated by red LEDs ( Figures 1 A and S1 ). We calculated the positional preference toward the illuminated side (see equation in Figure 1 A). The last minute of each phase was used to test whether there are genotype-dependent differences in positional preference and whether the preference is significantly different than zero. None of the genotypes showed a significant preference for the activation zone when both sides of the arena were un-illuminated ( Figure 1 C). CRZ > CsChrimson males, but not genetic controls, showed a strong positional preference for the illuminated zone during the two-choice phase ( Figures 1 D and 1E). Although optogenetic stimulation of CRZ > CsChrimson induced periodic bouts of ejaculation ( Figure S2 ), their preference for the illuminated zone was not due to a motor defect: they walked freely between the two sides of the arena, and their average velocity during walking bouts was similar to the genetic controls ( Figure S3 ). These results imply that activation of Crz neurons, which promote ejaculation, has a positive valence.

5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. The CRZ-Gal4 expression pattern consists of male-specific neurons in the AG, ∼14 non-dimorphic neurons in the central brain, and dispersed expression throughout the optic lobe ( Figure 1 B). To test whether the positional preference observed in males is produced by the activation of male-specific Crz neurons in the AG, we repeated the two-choice preference assay in females that lack these cells. CRZ > CsChrimson females and the genetic controls did not show positional preference for the activation zone in either of the two phases ( Figures 1 F–1H). Although it is not known whether male and female flies respond similarly to hedonic stimuli, the lack of positional preference in females hints that the preference of male flies is mediated by activation of the male-specific AG Crz neurons. To strengthen this conclusion, we used a CRZ driver that is not expressed in the AG (CRZ-2fa-Gal4 []) to drive CsChrimson and assayed the positional preference of male flies. These flies did not show positional preference to the activation zone in either phase of the experiment, again suggesting that the male-specific AG Crz neurons are required ( Figures 1 I–1K).

21 Aso Y.

Rubin G.M. Dopaminergic neurons write and update memories with cell-type-specific rules. Figure 2 Optogenetic Activation of Crz Neurons Is Rewarding to Male Flies Show full caption 21 Aso Y.

Rubin G.M. Dopaminergic neurons write and update memories with cell-type-specific rules. (A) Schematic illustration of the olfactory conditioning paradigm as described previously []. (B and C) C RZ-GAL4 > CsChrimson males flies (B) or CRZ-2fa-GAL4 > CsChrimson males flies (C) were trained to associate an odor with the optogenetic activation of Crz neurons. Conditioning protocol consisted of a single training session: 45 s of exposure to odor 1 (4-methylcyclohexanol [MCH]) coupled with optogenetic activation (constant light, 617-nm LED light intensity, 20 μW/mm2) followed by 1 min of exposure to air and subsequent 45 s of exposure to odor 2 [3-octanol (OCT)]. To exclude innate bias toward the olfactory cues, another group was trained in reciprocal manner (group II). Conditioned odor preference was immediately tested after the end of the training. Conditioned preference index (CPI) is the average between the CPIs of group I and group II. CRZ-Gal4 > CsChrimson male flies (B) expressed appetitive memory to the odor that was previously paired with optogenetic activation. n = 8 for no activation control, n = 13 for the experimental group; mean ± SEM; Mann Whitney; p < 0.005. CRZ-2fa-Gal4 > CsChrimson male flies (C) did not form appetitive memories. See also Figure S4 Next, we performed olfactory conditioning experiments to test whether the positional preference shown upon the activation of Crz neurons is due to the rewarding effects of Crz-induced ejaculation. CRZ > CsChrimson male flies were trained to associate an odor (4-methylcyclohexanol or 3-octanol) with 45 s of Crz neuron activation, and their preference to the paired odor was subsequently tested when both odors were delivered simultaneously [] ( Figure 2 A). As a control, the flies were exposed to the odors in the absence of neuronal activation. CRZ > CsChrimson males showed a positive, illumination-dependent conditioned odor preference to the odor associated with optogenetic activation ( Figure 2 B), implying that activation of Crz neurons induces appetitive memory. CRZ-2fa > CsChrimson male flies that were trained under the same conditions did not show conditioned odor preference ( Figure 2 C), suggesting that the rewarding effects of activating the population of Crz neurons results from the male-specific neurons located in the AG.

5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. 8 Tayler T.D.

Pacheco D.A.

Hergarden A.C.

Murthy M.

Anderson D.J. A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. Figure 3 Activation of Male-Specific Crz Neurons Induces npf Transcript Levels and Reduces Ethanol Consumption Show full caption (A) Male flies expressing Kir2.1 in Crz neurons and genetic controls were mated with virgin females for 5 hr on four consecutive days, and their npf transcript levels were compared to virgin counterparts (∗∗p < 0.01, ∗p < 0.05, Student’s t test, n = 3 independent experiments with 15–20 fly heads each). Lower panel is schematic illustration of experimental design. (B–D) Flies expressing CsChrimson in Crz neurons (CRZ> CSChrimson) and genetic controls were subjected to spaced 15-min long activation sessions, repeated on four consecutive days, after which the flies were frozen. npf transcript levels were analyzed using RT-PCR analysis in male flies (B), female flies (C), or when activating brain-only Crz neurons in male flies (using 2fa-Gal4) (D). Statistical significance was determined by one-way ANOVA with Tukey post hoc analysis for the fold change in npf-relative levels between light and dark conditions. Error bars signify SEM (B) F(2,6) = 41.0291, ∗∗p < 0.01, n = 3 independent experiments of 15–20 fly heads per sample, non-significant (C and D). (E–G) Ethanol preference of CRZ > CsChrimson virgin males, and the genetic controls, was assayed for flies that were kept in the dark (E) after repeated 15 min optogenetic activation, 3 times a day, for 3 consecutive days (F), or after repeated 15 min optogenetic activation of brain-only Crz neurons in male flies (CRZ-2fa-GAL4) (G). Significant main effect of genotype was documented only in (F) F(2,21) = 30.3923 ∗∗p < 0.01, two-way repeated-measures ANOVA with Bonferroni posttests; n = 8, 8 flies each. Error bars signify SEM. Schematic illustrations of experimental design are shown in bottom panels. Repeated mating events and repeated exposure to intoxicating levels of ethanol are known to induce expression of npf in the brain []. To determine if the function of Crz neurons is necessary for the mating-induced increase in npf transcript levels, Crz neurons were inhibited by expression of the inwardly rectifying potassium channel Kir2.1, and npf levels were compared in mated and virgin male flies. Inhibiting Crz neurons was previously shown to increase copulation duration by 5-fold and to block sperm and seminal fluid transfer, resulting in non-fertile mating interactions []. Male flies expressing Kir2.1 in Crz neurons and genetic control flies experienced five 1-hr-long sessions of mating with multiple receptive virgin females (ratio 1:3) for 4 days ( Figure 3 A [lower panel]). While the genetic controls showed a clear increase in npf levels after mating, experimental flies, which did not accomplish successful copulation, showed similar npf levels as their virgin counterparts ( Figure 3 A), implying that the function of Crz neurons in stimulating ejaculation is necessary for npf induction.

We next asked if repeated activation of Crz neurons can mimic natural copulation and induce npf levels. CRZ > CsChrimson males were exposed to three 15-min-long activation sessions (designed to mimic three mating events) per day for four consecutive days ( Figure 3 B [lower panel]). This activation protocol induced a 4-fold increase in head npf transcript levels as compared to unstimulated flies ( Figure 3 B). This increase in npf was not observed in genetic controls subjected to identical conditions ( Figure 3 B). To exclude the technical possibility that some Crz-expressing neurons also express NPF, potentially causing direct activation of NPF neurons while activating Crz cells, Crz and NPF expression patterns were compared. Co-immunostaining of CRZ-Gal4-driving mCD8-GFP with anti-NPF antibodies revealed that there is no overlap, indicating the brain NPF and Crz neurons represent mutually exclusive populations of neurons ( Figure S4 ). As a control, we performed similar activation experiments in female flies, which lack Crz neurons in the AG, and in male flies that express CsChrimson only in brain Crz neurons using the CRZ-2fa driver. These flies did not show a change in npf levels ( Figures 3 C and 3D). These experiments suggest that activating male-specific Crz neurons in the AG, which induces ejaculation, is sufficient to elevate npf levels and thus mimics the molecular signature of successful copulation.

5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. 5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. 29 Kacsoh B.Z.

Lynch Z.R.

Mortimer N.T.

Schlenke T.A. Fruit flies medicate offspring after seeing parasites. 30 Gao X.J.

Riabinina O.

Li J.

Potter C.J.

Clandinin T.R.

Luo L. A transcriptional reporter of intracellular Ca(2+) in Drosophila. 5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. 5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. 24 Zer S.

Ryvkin J.

Wilner H.J.

Zak H.

Shmueli A.

Shohat-Ophir G. A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster. 5 Shohat-Ophir G.

Kaun K.R.

Azanchi R.

Mohammed H.

Heberlein U. Sexual deprivation increases ethanol intake in Drosophila. npf mRNA levels serve as a molecular representation of different motivational states; they are increased by rewarding events, such as successful mating and ethanol intoxication [], and are reduced by exposure to parasitic wasps or repeated sexual deprivation in males []. Sexual deprivation also reduces the activity of NPF neurons []. We have previously established a causal link between repeated copulation events, increased npf levels, and reduced motivation to obtain external rewards such as ethanol []. To strengthen the functional link between ejaculation induced by the Crz neuron activation and the rewarding value of mating, we tested whether activating Crz neurons is sufficient to promote the high-reward state induced by mating that deters flies from consuming ethanol. CRZ > CsChrimson males were subjected to 3-day activation as before, and their voluntary ethanol consumption was subsequently measured using a two-choice ethanol consumption assay [] ( Figures 3 E–3G [lower panels]). In the absence of activation, CRZ > CsChrimson male flies and the genetic controls developed similar levels of preference to consume ethanol ( Figure 3 E [upper panel]). Following activation of Crz neurons, CRZ > CsChrimson flies showed an aversion to ethanol-supplemented food compared to the genetic controls, which preferred ethanol-containing food ( Figure 3 F [upper panel]). In contrast, activation of Crz brain neurons using the CRZ-2fa driver did not modulate ethanol consumption ( Figure 3 G), suggesting that AG Crz neurons are responsible for ejaculation-dependent mating reward. These results imply that activating Crz neurons or subsequent SSFR mimic both the molecular and behavioral responses that accompany the reward state that is naturally achieved by mating, where changes in npf levels direct alteration in ethanol self-administration [].

8 Tayler T.D.

Pacheco D.A.

Hergarden A.C.

Murthy M.

Anderson D.J. A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. 8 Tayler T.D.

Pacheco D.A.

Hergarden A.C.

Murthy M.

Anderson D.J. A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. Figure 4 Ejaculation Induced by the Activation of Crz-Receptor Neurons Is Pleasurable to Male Flies and Induces npf Transcript Levels Show full caption (A) CRZ-receptor-GAL4 expression patterns in 3D images of whole-mount brain and VNC of CRZ-receptor-GAL4-driving mCD8 GFP in Crz receptor neurons. (B–D) Positional preference of CRZ-receptor > CSChrimson male flies was tested in the two-choice arena when both sides are un-illuminated for 2 min (B), one side was illuminated for 5 min (C), or both sides were un-illuminated for 2 min (D). Graphs depict positional preference for the last minute of each period. Statistical significance was determined by one sample t tests against zero with Bonferroni corrections (n = 12), ∗∗∗p < 0.001. All other comparisons were not significant. CRZ-receptor > CsCrimson flies showed increased preference to the activation zone that is light dependent (C compared to B and D). n = 12, two-way ANOVA, F(2,33) = 13.0778, ∗∗∗p < 0.001 and F(2,33) = 25.4768, p < 0.01, Bonferroni post hoc comparison for CRZ-receptor > CsCrimson light (C) versus dark (B and D), p < 0.001, and versus genetic controls under all conditions p < 0.01. (E) CRZ-receptor > CsCrimson male flies and genetic controls were subjected to spaced 5-min-long activation sessions repeated on 4 consecutive days, after which the flies were frozen and npf transcript levels were analyzed using RT-PCR analysis in male flies. Statistical significance was determined by one-way ANOVA with Tukey post hoc analysis for the fold change in npf relative levels between light and dark conditions. Error bars signify SEM. F(2,6) = 297.1936, ∗∗p < 0.01, p > 0.05, n = 3 independent experiments of 15–20 fly heads per sample. n.s., non-significant. (F) Model based on the results. Crz and Crz-receptor neurons are activated during copulation-induced ejaculation leading to sperm and seminal fluid transfer (SSFT), which induces npf levels in the brain. Our results so far illustrate a functional link between Crz-induced ejaculation, immediate pleasurable response, and long-term changes in motivational states represented by induction of npf and reduced motivation to obtain drug rewards. To begin to study the circuit downstream of Crz neurons, we tested whether activation of Crz-receptor neurons, which innervate ejaculatory organs and induce ejaculation [], is rewarding. The Crz-receptor Gal4 is expressed in male-specific AG neurons and in numerous fat cells that surround the nervous system ( Figure 4 A). Consistent with previous results [], optogenetic activation of Crz-receptor neurons induces ejaculation in male flies ( Figure S2 ). We tested the preference of male flies to obtain optogenetic stimulation of Crz-receptor neurons in a two-choice arena. The experiment began with 2 min of dark, followed by 5 min where one side is illuminated, then another 2 min of dark. We incorporated a recovery phase after the activation phase to ensure that the activation effects are transient. CRZ-receptor > CsChrimson flies, but not genetic controls, showed a strong light-dependent preference for the activation zone ( Figures 4 B–4D). These results imply that optogenetic activation of Crz-receptor cells confers a positive valence, similar to activation of Crz neurons.

Lastly, we tested whether activation of Crz-receptor cells in male flies increases npf levels. CRZ-receptor > CsChrimson males were exposed to three 5-min-long activation sessions for 4 consecutive days. This activation protocol induced a 5-fold increase in head npf transcript levels as compared to flies that were kept in the dark ( Figure 4 E). The increase in npf levels was not observed in the genetic controls subjected to either activation or non-activation conditions ( Figure 4 E). Thus, artificial activation of Crz-receptor cells, which promotes SSFR, is sufficient to induce the immediate perception of reward, as well as long-term molecular changes in neurons that process natural and drug rewards. Since the CRZ-receptor Gal4 is not expressed in brain neurons, these results further support our claim that the rewarding properties associated with the activation of the entire population of Crz neurons in male flies originate from the male-specific neurons located in the AG. In addition, these results suggest that CRZ release is not necessary for mating-induced reward and that reward stimulus is not the activation of Crz neurons per se, but probably other downstream events that take place along the pathway that induces or perceives SSFR.

Our work demonstrates that activation of Crz neurons, which induces SSFR, is sufficient to mimic all the rewarding aspects of successful copulation: it carries positive valence, induces npf transcript levels, drives appetitive memories, and reduces the motivation to consume ethanol as a drug reward. The former two effects are also induced by activation of Crz-receptor neurons. Considering the causal link between activation of Crz/Crz-receptor neurons and ejaculation, it is impossible to distinguish whether these rewarding effects are directly induced by ejaculation or result from Crz/Crz receptor neuron activation independent of ejaculation. Overall, our data suggest a model in which Crz neurons are activated during courtship encounters that culminate in copulation, causing the activation of Crz-receptor neurons. Activation of Crz-receptor neurons induces SSFR, which is sensed by some yet unidentified sensory neurons, and the information is subsequently relayed to the NPF neurons in the brain, leading to increase in npf transcription ( Figure 4 F).

22 Shao L.

Saver M.

Chung P.

Ren Q.

Lee T.

Kent C.F.

Heberlein U. Dissection of theDrosophilaneuropeptide F circuit using a high-throughput two-choice assay. 22 Shao L.

Saver M.

Chung P.

Ren Q.

Lee T.

Kent C.F.

Heberlein U. Dissection of theDrosophilaneuropeptide F circuit using a high-throughput two-choice assay. 29 Kacsoh B.Z.

Lynch Z.R.

Mortimer N.T.

Schlenke T.A. Fruit flies medicate offspring after seeing parasites. 30 Gao X.J.

Riabinina O.

Li J.

Potter C.J.

Clandinin T.R.

Luo L. A transcriptional reporter of intracellular Ca(2+) in Drosophila. 31 Beshel J.

Zhong Y. Graded encoding of food odor value in the Drosophila brain. 32 Chung B.Y.

Ro J.

Hutter S.A.

Miller K.M.

Guduguntla L.S.

Kondo S.

Pletcher S.D. Drosophila Neuropeptide F Signaling Independently Regulates Feeding and Sleep-Wake Behavior. 33 Krashes M.J.

DasGupta S.

Vreede A.

White B.

Armstrong J.D.

Waddell S. A neural circuit mechanism integrating motivational state with memory expression in Drosophila. The elevation in npf transcript and the changes in ethanol consumption require several activation events over the course of four days, possibly reflecting different aspects of immediate and long-term accumulation of rewarding experiences. While the activation of Crz neurons promotes immediate reward, it is possible that the observed changes in npf transcription are noticeable only upon repeated rewarding experiences. A recent study demonstrated that activation of NPF neurons induces immediate reward in both male and female flies [], suggesting that the observed changes in npf transcript levels not only encode the valence of the experience, but possibly integrates the magnitude and cumulative changes in internal reward levels. There is substantial evidence to support the proposed role of the NPF/NPF-receptor system as a central player in modulating and encoding motivational states associated with sugar reward, sexual and drug reward, and the homeostatic regulation of motivational responses [].

34 Veenstra J.A. Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. 35 Zhao Y.

Bretz C.A.

Hawksworth S.A.

Hirsh J.

Johnson E.C. Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila. 36 Kapan N.

Lushchak O.V.

Luo J.

Nässel D.R. Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin. 37 Kubrak O.I.

Lushchak O.V.

Zandawala M.

Nässel D.R. Systemic corazonin signalling modulates stress responses and metabolism in Drosophila. 38 McClure K.D.

Heberlein U. A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila. 39 Sha K.

Choi S.H.

Im J.

Lee G.G.

Loeffler F.

Park J.H. Regulation of ethanol-related behavior and ethanol metabolism by the Corazonin neurons and Corazonin receptor in Drosophila melanogaster. 40 Varga K.

Nagy P.

Arsikin Csordás K.

Kovács A.L.

Hegedűs K.

Juhász G. Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila. 39 Sha K.

Choi S.H.

Im J.

Lee G.G.

Loeffler F.

Park J.H. Regulation of ethanol-related behavior and ethanol metabolism by the Corazonin neurons and Corazonin receptor in Drosophila melanogaster. 41 Devineni A.V.

McClure K.D.

Guarnieri D.J.

Corl A.B.

Wolf F.W.

Eddison M.

Heberlein U. The genetic relationships between ethanol preference, acute ethanol sensitivity, and ethanol tolerance in Drosophila melanogaster. 38 McClure K.D.

Heberlein U. A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila. 42 Miyamoto T.

Amrein H. Diverse roles for the Drosophila fructose sensor Gr43a. 43 Miyamoto T.

Slone J.

Song X.

Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain. CRZ is a highly conserved insect neuropeptide that regulates various physiological processes in arthropods [] including response to stress [], ethanol sensitivity and metabolism []. Theoretically, the reduction in voluntarily ethanol consumption in response to the activation of Crz neurons could result from changes in sensitivity to ethanol and its metabolism. Flies deficient in Crz/Crz-receptor neurons show delayed recovery from ethanol-induced sedation due to defective acetaldehyde metabolism [], implying that Crz neurons function to promote recovery form ethanol sedation. Therefore, activating Crz neurons should induce rapid ethanol metabolism, allowing for higher rather than lower ethanol consumption. In addition, previous attempts to identify an association between genes that affect ethanol sensitivity and sedation to genes/pathways that affect ethanol consumption phenotypes failed to show any significant correlation []. Thus, the decreased ethanol consumption in male flies that experienced ejaculation is most likely not related to the role of Crz neurons in promoting ethanol sensitivity []. In addition, brain Crz neurons express the fructose receptor Gr43a that functions as a nutrient sensor for hemolymph fructose levels, assigning positive valence for feeding during starvation and negative valence for feeding when flies are satiated []. Still, the activation of brain Crz/Gr43a neurons in satiated male flies was neutral in all the reward-related assays, suggesting that under the conditions used in this study, they do not function in encoding reward.