Conditioned social preference and reward value of activating oxytocin-receptor-expressing ventral tegmental area neurons following repeated daily binge ethanol intake

Abstract

Background: Individuals with alcohol use disorder (AUD) exhibit a disruption of social behavior and dysregulation of oxytocin signaling in the brain, possibly reflecting decreased activation of oxytocin receptors (OxTRs) in reward pathways in response to social stimuli. We hypothesize that daily binge ethanol intake causes a deficit in social reward and oxytocin signaling in the ventral tegmental area (VTA).

Methods: After 9 weeks of daily binge ethanol intake (blood ethanol concentration >80 mg%), OxTR-cre mice underwent conditioned place preference for social reward. Separate groups of mice were tested for the effects of binge ethanol on voluntary social interactions, food reward, locomotion, and anxiety-like behaviors. A subset of mice underwent transfection of OxTR-expressing VTA neurons (VTA^Oxtr ) with a light-sensitive opsin, followed by operant training to respond to light delivered to VTA.

Results: Ethanol-naïve male mice increased the time spent on the side previously paired with novel mice while ethanol-treated mice did not. Binge ethanol did not affect conditioned place preference for food reward in males, but this response was weakened in ethanol-treated females. Ethanol treatment also caused a sex-specific impairment of voluntary social interactions with novel mice. There were minimal differences between groups in measures of anxiety and locomotion. Ethanol-naïve mice had significantly greater operant responding for activation of VTA^Oxtr than sham-transfected mice but ethanol-treated mice did not. There was no difference in the number of VTA^Oxtr after binge ethanol.

Conclusions: Daily binge ethanol causes social reward deficits that cannot be explained by nonspecific effects on other behaviors, at least in males. Only ethanol-naïve mice exhibited positive reinforcement caused by activation of VTA^Oxtr while daily binge ethanol did not alter the number of VTA^Oxtr in either males or females. Thus, subtle dysregulation of VTA^Oxtr function may be related to the social reward deficits caused by daily binge ethanol.

Keywords: binge ethanol; oxytocin receptor; social reward; ventral tegmental area.

Figure 1:. CSP diagram.

Experimental design for conditioned place preference experiments using social reward.

Figure 2:. SI/SN diagram.

Experimental design for quantifying preference for social interaction with novel and familiar mice.

Figure 3:. OxTR-Cre mice consumed binge levels of ethanol that were calorically similar to plain gelatin consumption.

(A) Ethanol consumption in OxTR-Cre mice during 3-h free access sessions for the first nine weeks of gel exposure. (B) Average calorie consumption in male and female OxTR-Cre mice was not different between ethanol or plain gel. * indicates significant sex difference. Shown are means ± SEMs for plain mice: N=34 (females) and 36 (males), and ethanol mice: N=39 (females) and 35 (males).

Figure 4:. Ethanol gel access resulted in >80 mg/dl blood ethanol levels in most OxTR-Cre mice with blood ethanol concentration positively correlated with ethanol intake.

(A) Blood ethanol level at the end of the 3-hr gel access period was positively correlated with dose of ethanol consumed that day in male and female mice. Dashed line represents the minimum level considered binge drinking: 80 mg/dl. (B) Female mice had an average blood ethanol concentration of 131.2±11.7 mg/dl, and male mice had an average blood ethanol concentration of 109.2±7.6 mg/dl which was not statistically different. (C) On the day blood ethanol level was measured, females consumed significantly greater doses of ethanol (5.0±0.2 g/kg) than males (4.2±0.1 g/kg). * indicates significant sex difference. Shown are means ± SEMs for N = 23 (females) and 21 (males).

Figure 5:. Daily binge ethanol intake decreases conditioned place preference for social reward in male mice.

The average amount of time spent on the paired side before (Pretest – solid bars) and after (Post-test – striped bars) 8 conditioning sessions. Only male plain mice show a significant increase in post-test scores. Data were averaged over two pretest sessions and two post-test sessions. * indicates significant difference from Pretest using 1-tailed T-test. Shown are means ± SEMs for N = 11 (males) and 12 (females) per group.

Figure 6:. Duration of social and empty cup interactions during place preference conditioning sessions was not affected by binge ethanol intake.

All mice spent more time interacting with a mouse than with an empty cup, regardless of sex or ethanol intake during the first conditioning sessions. However, during the last conditioning sessions, only male mice spent more time interacting with a mouse vs an empty cup. There were no differences in mouse or empty cup interaction due to ethanol treatment. * indicates significant difference compared to empty cup. Shown are means ± SEMs for N = 11 (males) and N = 12 (females) per group.

Figure 7:. Daily binge ethanol intake decreases conditioned place preference for food reward in female mice.

Conditioning and test sessions were conducted identically to Figure 5 except a graham cracker was used as the reward on the non-preferred side. The average amount of time spent on the paired side before (Pretest – solid bars) and after (Post-test – striped bars) 8 conditioning sessions. ethanol female mice did not increase time spent on the food-paired side during the post-test compared to the other 3 groups. Data were averaged over two pretest sessions and two post-test sessions. * indicates significant difference from Pretest using 1-tailed T-test. Shown are means ± SEMs for N = 6 (males) and 7, 9 (females) per group.

Figure 8:. Social interaction and social novelty are decreased by daily binge ethanol drinking differently in male and female mice.

OxTR-cre mice were tested in the Social Interaction/Social Novelty test after nine weeks of daily plain or ethanol gel consumption. (A) There were no significant differences in time spent on either side during the first 10 min of the test. (B) During the second 10 min (Social Interaction Test), plain female mice and all male mice spent more time with the mouse under the cup than with the empty cup, however ethanol female mice did not distinguish between the two sides. * indicates significant difference from time spent with mouse. (C) During the third 10 min (Social Novelty Test), all female mice and plain male mice spent slightly more time with the novel mouse compared to the familiar mouse, while male ethanol mice spent less time with the novel mouse. * indicates significant difference from time spent with familiar mouse. Shown are means ± SEMs for N = 6-8 per group.

Figure 9:. Effects of short-term withdrawal from binge ethanol treatment on anxiety-like behavior.

(A) There was no effect of prior ethanol consumption on time spent in the open arm of the elevated plus maze. Shown are means ± SEMs for N = 6 (males) and 12 (females) per group. (B) There was no effect of prior ethanol consumption on the number of marbles buried during a 20 min test. Shown are means ± SEMs for N = 8 (males) and 9 (females) per group. (C) Daily binge ethanol consumption significantly decreased time spent in the center of an open field in both male and female mice. * indicates significant difference from plain mice. Shown are means ± SEMs for N = 6 per group.

Figure 10:. Operant responding for optogenetic self-stimulation of OxTR-expressing VTA neurons is decreased by daily binge ethanol.

(A) Diagram of the virus injection sites (red circles) and optic fiber placement (gray cylinder). Diagram is modified from Franklin & Paxinos (2007). (B) OxTR-Cre mice were bilaterally transfected in the VTA with either AAVs containing Cre-dependent opsin (ChR2+) or not (Sham). After 2 weeks, mice were trained to nosepoke in the active port in order to receive 1-sec blue light (473 nm, 15 Hz, 20 msec pulse duration) delivered via the optic fiber. After 5 daily 30-min operant sessions, active port entries (blue) were increased compared to inactive port entries (light gray) for ChR2+ plain mice but not for any Sham mice or any ethanol mice. * indicates significant difference in active and inactive port entries. Shown are means ± SEMs for Ch2R2+ mice (N = 6 for plain and 7 for ethanol) or Sham mice (N = 6 for plain and 7 for ethanol). Groups were comprised of equal proportions of male (gray squares) and female (blue circles) mice.

Figure 11:. Binge ethanol intake does not alter the number of OTR-expressing VTA neurons.

Average number of GFP-stained cells in lateral and medial areas of the VTA across the four treatment groups. GFP-stained cells were quantified in a defined square within either medial or lateral VTA in multiple sections from −3.16 to −3.64 bregma. N = 6 males per group and 4-5 females per group.