Intermittent voluntary ethanol consumption combined with ethanol vapor exposure during adolescence increases drinking and alters other behaviors in adulthood in female and male rats

Abstract

Epidemiological studies suggest that binge drinking is prevalent among adolescents, and may result in neurobehavioral consequences. Animal models provide the experimental control to investigate the consequences of "binge" alcohol exposure during this neurodevelopmental epoch. The current study used an animal model that combined an intermittent pattern of alcohol vapor exposure with voluntary drinking of 20% unsweetened alcohol in adolescent male and female Wistar rats (postnatal day [PD] 22-62), in order to test for potential differences in behavioral changes, ethanol drinking, and hypocretin/orexin (Hcrt/OX) signaling associated with exposure status. Two weeks after discontinuation of the alcohol vapor exposure and drinking during adolescence, rats were tested in adulthood for anxiety-like behaviors using a modified open-field conflict task, pre-pulse facilitation of startle response, light/dark box, and marble burying test. Adolescent alcohol exposure led to overall decreased startle response and increased behavioral arousal in the light/dark chamber during adulthood. Additionally, male rats demonstrated more disinhibited behavior during the conflict task compared to females, and female rats exhibited more rearing behavior during the light/dark test. Rats were also given a 2-bottle choice test that resulted in adolescent alcohol-exposed rats drinking significantly more alcohol in adulthood. Further, female rats also consumed more alcohol in adulthood compared to males. Estrous cycle phase did not account for any of the sex differences observed in the behavioral measures. Histological results indicated that adolescent alcohol did not alter Hcrt/OX-1 or Hcrt/OX-2 receptor mRNA expression levels in adult rats compared to control adults. However, female rats expressed a higher level of Hcrt/OX-1 and Hcrt/OX-2 receptor mRNA in the frontal cortex compared to males. These data suggest that our current model of intermittent ethanol exposure in adolescence can modestly affect both behavior and future consumption of alcohol and that Hcrt/OX receptor signaling differs between males and females.

Keywords: Adolescence; Alcohol drinking; Female; Hypocretin/orexin; Wistar rats.

Figure 1. Intermittent Ethanol Vapor and Voluntary Consumption in Adolescence

Adolescent ethanol exposure occurred using voluntary intermittent alcohol access occurred 24h before and 24h after each 4-day vapor binge session with 24h in between voluntary alcohol exposures. This exposure period occurred from PD 22–62. After discontinuation of the adolescent ethanol exposure, both male and female rats were tested on various neurobehavioral paradigms followed by 10 days of voluntary 2 bottle ethanol preference testing in adulthood. (A) Ethanol exposure reduced weight gain in male rats during PD 62–83 compared to controls. Exposure did not impact weight gain in female rats. (B) Female adolescent rats consumed more ethanol over the mean (SEM) 24h sessions compared to males. (C) BALs were taken 24h after the start and again at the end of the vapor binge session. While BALs between males and females differed on various days, BALs were consistent on the last 2 exposure periods. *p < 0.05 vs. male rats.

Figure 2. Adolescent Ethanol Exposure on Neurobehavioral Paradigms in Adulthood

(A) There was no significant difference in mean (±SEM) number of line transitions that occurred during the open field conflict test in adulthood. (B) While there was no effect of ethanol exposure, adult male rats had more mean (±SEM) number of entries into the center grid of the open field box compared to female adult rats. (C) The mean (±SEM) amount of appetitive food consumed in the center grid of the open field box was no different between groups. (D) There was a significant interaction between ethanol exposure group and sex with latency (mean±SEM) to enter the dark chamber after being initially placed in the Light chamber during Light/Dark test. Females exposed to ethanol during adolescence demonstrating a quicker latency to enter the dark compared to female controls. (E) There was no significant differences in mean (±SEM) amount of time spent in the dark chamber during the Light/Dark test. (F) While there was no effect of ethanol exposure on mean (±SEM) number of rears in the light chamber during the Light/Dark test, adult female rats demonstrated more rearing behavior compared to male adult rats. (G) Ethanol exposure during adolescence resulted in reduced mean (±SEM) amplitude for motor startle response during ASR task compared to controls. Male rats also had a significantly greater motor startle response compared to female rats. (I) Percent Pre-pulse facilitation response (mean±SEM) was not significantly different between groups. (J) While trending, there was no significant effect of mean (±SEM) number of marbles buried in 30m during the Marble burying test. *p < 0.05 main effect of group, ^#p < 0.05 vs. control group.

Figure 3. Adult Ethanol Consumption After Adolescent Ethanol Exposure

(A) Mean (±SEM) ethanol consumption in adulthood was significantly higher in rats exposed to ethanol during adolescence compared to those who were not exposed. Additionally, female rats also consumed more overall during the 24h period than males in adulthood (B) Further analysis correlating overall 24h ethanol consumption in adolescence to 24h consumption levels in adulthood demonstrate a significant linear regression for both male and female rats. *p < 0.05 main effect of group.

Figure 4. Hcrt/OX Receptor Expression in Adulthood After Adolescent Ethanol Exposure

Real-time qPCR was conducted on frontal cortex tissue samples of adult rats exposed to ethanol in adolescence to determaine whether adolescent exposure to ethanol has lasting effects on Hcrt/OX receptor expression. (A) Ethanol exposure did not impact Hcrt/OX 1 receptor mRNA expression in adulthood, however female adult rats had overall more Hcrt/OX 1 receptor expression compared to males. (B) Hcrt/OX 2 receptor mRNA expression was again not significantly different between exposure groups. Female rats exhibited more mRNA Hcrt/OX 2 compared to male adult rats. *p<0.05 vs. male group.