Volume 28, Issue 9, Pages 1445-1452.e3 (May 2018) Ejaculation Induced by the Activation of Crz Neurons Is Rewarding to Drosophila Males  Shir Zer-Krispil, Hila Zak, Lisha Shao, Shir Ben-Shaanan, Lea Tordjman, Assa Bentzur, Anat Shmueli, Galit Shohat-Ophir  Current Biology  Volume 28, Issue 9, Pages 1445-1452.e3 (May 2018) DOI: 10.1016/j.cub.2018.03.039 Copyright © 2018 The Authors Terms and Conditions

Current Biology 2018 28, 1445-1452.e3DOI: (10.1016/j.cub.2018.03.039) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Ejaculation Induced by the Activation of Crz Neurons Is Pleasurable to Male Flies (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. Current Biology 2018 28, 1445-1452.e3DOI: (10.1016/j.cub.2018.03.039) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Optogenetic Activation of Crz Neurons Is Rewarding to Male Flies (A) Schematic illustration of the olfactory conditioning paradigm as described previously [21]. (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. Current Biology 2018 28, 1445-1452.e3DOI: (10.1016/j.cub.2018.03.039) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Activation of Male-Specific Crz Neurons Induces npf Transcript Levels and Reduces Ethanol Consumption (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. Current Biology 2018 28, 1445-1452.e3DOI: (10.1016/j.cub.2018.03.039) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Ejaculation Induced by the Activation of Crz-Receptor Neurons Is Pleasurable to Male Flies and Induces npf Transcript Levels (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. Current Biology 2018 28, 1445-1452.e3DOI: (10.1016/j.cub.2018.03.039) Copyright © 2018 The Authors Terms and Conditions