Sexual deprivation increases ethanol intake in Drosophila

Abstract

The brain's reward systems reinforce behaviors required for species survival, including sex, food consumption, and social interaction. Drugs of abuse co-opt these neural pathways, which can lead to addiction. Here, we used Drosophila melanogaster to investigate the relationship between natural and drug rewards. In males, mating increased, whereas sexual deprivation reduced, neuropeptide F (NPF) levels. Activation or inhibition of the NPF system in turn reduced or enhanced ethanol preference. These results thus link sexual experience, NPF system activity, and ethanol consumption. Artificial activation of NPF neurons was in itself rewarding and precluded the ability of ethanol to act as a reward. We propose that activity of the NPF-NPF receptor axis represents the state of the fly reward system and modifies behavior accordingly.

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 h daily (“mated-grouped”, green blocks) or were subjected to courtship conditioning for 1 h, 3 times daily (“rejected-isolated”; blue squares). Training was repeated for four 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 6-8. (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 ANOVA with Bonferroni post-tests; comparisons are between treatment groups across all days of the assay. Data shown is mean+SEM or mean–SEM.

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 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-D) Effect of rejection on NPF protein expression 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, 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.

Fig. 3. NPF signaling regulates ethanol preference

(A, B) Expression of an NPFR RNAi transgene (UAS-NPFR^RNAi) 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, 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 is mean + or – SEM (for clarity purposes).

Fig. 4. Mating and NPF cell activation is rewarding and reduces 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, 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 one-hour training sessions at 29°C in the presence of odor (red rectangles in B) that where spaced by one-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 repeated-measures ANOVA with Bonferroni post-tests; comparisons are between treatment groups (**P<0.001, n=24). (D, 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-tests, n=22). F. NPF transcript levels are induced by ethanol intoxication. Males were exposed to moderately intoxicating levels of ethanol vapor (three 10-minute ethanol exposures spaced by one hour), collected, and frozen one or 24 hours later. NPF mRNA levels, measured by qPCR, were elevated one 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).