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Science 335 (6074): 1351-1355

Copyright © 2012 by the American Association for the Advancement of Science

Sexual Deprivation Increases Ethanol Intake in Drosophila

G. Shohat-Ophir*,{dagger}, K. R. Kaun{dagger}, R. Azanchi{dagger}, H. Mohammed, and U. Heberlein*,{dagger}

Department of Anatomy, University of California, San Francisco, CA 94143-2822, USA.


Figure 1
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Fig. 1. Mating and chronic sexual deprivation have opposite effects on voluntary ethanol consumption. (A) Schematic of the behavioral assay. Virgin wild-type males were allowed to mate with virgin females (groups of 4 males and 20 females) for 6 hours daily ("mated-grouped"; green blocks) or were subjected to courtship conditioning for 1 hour, three times daily ("rejected-isolated"; blue squares). Training was repeated for 4 days, after which males were placed in vials where they could choose to feed from capillaries containing food solutions with (red) or without (brown) 15% ethanol (10). Ethanol consumption was measured on days 6 to 8. (B) Rejected-isolated males exhibited higher ethanol preference than mated-grouped males (**P < 0.005, n = 12). (C) Mated-grouped males showed lower ethanol preference than "virgin-grouped" males (*P < 0.05, n = 12). (D) Males conditioned with decapitated virgins showed enhanced ethanol preference compared to mated-grouped males (**P < 0.01, n = 12). (E) Mating reversed the effects of rejection on ethanol preference. Rejected-isolated males that were allowed to mate after the end of the last conditioning session showed lower ethanol preference than similarly conditioned males that were left undisturbed (**P < 0.001, n = 8). Statistical analysis was carried out by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni post tests; comparisons are between treatment groups across all days of the assay. Data shown are the mean + SEM or mean – SEM.

 

Figure 2
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Fig. 2. Sexual experience regulates levels of NPF and NPF mRNA. (A) Total RNA extracted from heads of virgin, rejected, and mated males was analyzed for NPF mRNA levels by quantitative polymerase chain reaction (qPCR). NPF mRNA levels were reduced by sexual rejection and increased by mating (***P < 0.001 compared to virgin control, Dunnett’s test, n = 3 independent experiments). NPF transcript levels were normalized to rp49 mRNA. (B to D) Effect of rejection on NPF protein abundance as determined by immunohistochemistry. (B) Quantitative analysis of overall NPF staining intensity in brains of rejected and mated males (***P < 0.001, t test). (C and D) Differential NPF staining in rejected and mated males was observed in all major regions of NPF expression (arrowheads). Asterisks denote the positions of NPF-expressing cell bodies (which are obscured by high levels of expression in mated males). FSB, fan-shaped body.

 

Figure 3
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Fig. 3. NPF signaling regulates ethanol preference. (A and B) Expression of an NPFR RNA interference (RNAi) transgene (UAS-NPFRRNAi) using a pan-neuronal driver (elav-GAL4) increased ethanol preference in mated males compared to the genetic controls carrying either transgene alone (B) (*P < 0.05, n = 12), but not in virgin males (A) (P > 0.5). (C and D) Activating NPF neurons reduced ethanol preference. Virgin males expressing dTRPA1 in NPF neurons (NPF-GAL4 + UAS-dTRPA1), and the genetic controls carrying either transgene alone, developed similar levels of ethanol preference at 20°C (C) when dTRPA1 is not active (P > 0.05, n = 8), but developed aversion to ethanol containing food at 29°C (D), when dTRPA1 is active (***P < 0.001, n = 8). Statistical analysis was carried out by two-way repeated-measures ANOVA with Bonferroni post tests; comparisons are between treatment groups across all days of the assay. Data are the mean + or mean – SEM (for clarity purposes).

 

Figure 4
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Fig. 4. Mating and NPF cell activation are rewarding and reduce ethanol reward. (A) Mating is rewarding to male flies. Males trained to associate an odor with mating (presence of virgin females) develop preference for that odor. P values were calculated by Wilcoxon analysis against zero. Mating against zero was **P = 0.001; each reciprocal group against zero was P = 0.004 for one odor (IAA) plus mating and P = 0.02 for the reciprocal odor (EA) plus mating. CPI, conditioned preference index (calculated by averaging the odor preference indexes for reciprocally trained males). (B and C) NPF cell activation is rewarding. Males expressing dTRPA1 in NPF neurons (NPF-GAL4 + UAS-dTRPA1) and the genetic controls carrying either transgene alone were exposed to three 1-hour training sessions at 29°C in the presence of odor [red rectangles in (B)] that were spaced by 1-hour rest periods at 18°C in the absence of odor [blue rectangles in (B)]. Testing for odor preference was performed 24 hours after training at 21°C. Experimental males, but not the genetic controls, showed preference for the odor that was associated with dTRPA1 activation in NPF neurons. Data are averages of three independent experiments. Statistical analysis was carried out by two-way ANOVA with Bonferroni post tests; comparisons are between treatment groups (**P < 0.001, n = 24). (D and E) Activation of NPF neurons abolishes ethanol reward. Activation of NPF neurons using dTRPA1 (NPF-GAL4 + UAS-dTRPA1) eliminated conditioned ethanol preference compared to the singly transgenic controls when tested 24 hours after training (*P < 0.01, one-way ANOVA with Wilcoxon/Kruskal-Wallis post-hoc tests, n = 22). (F) NPF transcript levels are induced by ethanol intoxication. Males were exposed to moderately intoxicating levels of ethanol vapor (three 10-min ethanol exposures spaced by 1 hour), collected, and frozen 1 or 24 hours later. NPF mRNA levels, measured by qPCR, were elevated 1 hour after ethanol exposure and returned to basal level after 24 hours (**P < 0.001 compared to air-exposed controls, Dunnett’s test, n = 3 independent experiments with 30 males each).

 


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