Impact of adolescent intermittent ethanol exposure in male and female rats on social drinking and neuropeptide gene expression.

BACKGROUND: Alcohol use during adolescence can alter maturational changes that occur in brain regions associated with social and emotional responding. Our previous studies have shown that adult male, but not female rats demonstrate social anxiety-like alterations and enhanced sensitivity to ethanol-induced social facilitation following adolescent intermittent ethanol exposure (AIE). These consequences of AIE may influence adult social drinking in a sex-specific manner. METHODS: To test the effects of AIE on social drinking, male and female Sprague-Dawley rats exposed to water or ethanol (0 or 4 g/kg, intragastrically, every other day, between postnatal day [P] 25 and 45) were tested as adults (P72-83) in a social drinking paradigm (30-minute access to a 10% ethanol solution in supersac or supersac alone in groups of three same-sex littermates across two 4-day cycles separated by 4 days off). Social behavior was assessed during the last drinking session, along with assessment of oxytocin (OXT), oxytocin receptor (OXTR), vasopressin (AVP), and vasopressin receptors 1a and 1b (AVPR1a, AVPR1b) in the hypothalamus and lateral septum. RESULTS: Males exposed to AIE consumed more ethanol than water-exposed controls during the second drinking cycle, whereas AIE did not affect supersac intake in males. AIE-exposed females consumed less ethanol and more supersac than water-exposed controls. Water-exposed females drinking ethanol showed more social investigation and significantly higher hypothalamic OXTR, AVP, and AVPR1b gene expression than their counterparts ingesting supersac and AIE females drinking ethanol. In males, hypothalamic AVPR1b gene expression was affected by drinking solution, with significantly higher expression evident in males drinking ethanol than those consuming supersac. CONCLUSIONS: Collectively, these findings provide new evidence regarding sex-specific effects of AIE on social drinking and suggest that the hypothalamic OXT and AVP systems are implicated in the effects of ingested ethanol on social behavior in a sex- and adolescent-exposure-dependent manner.

Patterns of neuronal activation following ethanol-induced social facilitation and social inhibition in adolescent cFos-LacZ male and female rats.

Motives related to the enhancement of the positive effects of alcohol on social activity within sexes are strongly associated with alcohol use disorder and are a major contributor to adolescent alcohol use and heavy drinking. This is particularly concerning given that heightened vulnerability of the developing adolescent brain. Despite this linkage, it is unknown how adolescent non-intoxicated social behavior relates to alcohol's effects on social responding, and how the social brain network differs in response within individuals that are socially facilitated or inhibited by alcohol. Sex effects for social facilitation and inhibition during adolescence are conserved in rodents in high and low drinkers, respectively. In the current study we used cFos-LacZ transgenic rats to evaluate behavior and related neural activity in male and female subjects that differed in their social facilitatory or social inhibitory response to ethanol. Subjects were assessed using social interaction on postnatal days 34, 36 and 38 after a 0, 0.5 and 0.75 g/kg ethanol challenge, respectively, with brain tissue being evaluated following the final social interaction. Subjects were binned into those that were socially facilitated or inhibited by ethanol using a tertile split within each sex. Results indicate that both males and females facilitated by ethanol display lower social activity in the absence of ethanol compared to socially inhibited subjects. Analyses of neural activity revealed that females exhibited differences in 54% of examined socially relevant brain regions of interest (ROIs) compared to only 8% in males, with neural activity in females socially inhibited by ethanol generally being lower than facilitated subjects. Analysis of socially relevant ROI neural activity to social behavior differed for select brain regions as a function of sex, with the prefrontal cortex and nucleus accumbens being negatively correlated in males, but positively correlated in females. Females displayed additional positive correlations in other ROIs, and sex differences were noted across the rostro-caudal claustrum axis. Importantly, neural activity largely did not correlate with locomotor activity. Functional network construction of social brain regions revealed further sex dissociable effects, with 90% interconnectivity in males socially inhibited by ethanol compared to 38% of facilitated subjects, whereas interconnectivity in females inhibited by ethanol was 10% compared to nearly 60% in facilitated subjects. However, hub analyses converged on similar brain regions in males and females, with the nucleus accumbens being a hub region in socially inhibited subjects, whereas the central amygdala was disconnected in facilitated subjects. Taken together, these findings support unified brain regions that contribute to social facilitation or inhibition from ethanol despite prominent sex differences in the social brain network.

Patterns of neuronal activation following ethanol-induced social facilitation and social inhibition in adolescent cFos-LacZ male and female rats.

Alcohol-associated social facilitation together with attenuated sensitivity to adverse alcohol effects play a substantial role in adolescent alcohol use and misuse, with adolescent females being more susceptible to adverse consequences of binge drinking than adolescent males. Adolescent rodents also demonstrate individual and sex differences in sensitivity to ethanol-induced social facilitation and social inhibition, therefore the current study was designed to identify neuronal activation patterns associated with ethanol-induced social facilitation and ethanol-induced social inhibition in male and female adolescent cFos-LacZ rats. Experimental subjects were given social interaction tests on postnatal day (P) 34, 36, and 38 after an acute challenge with 0, 0.5 and 0.75 g/kg ethanol, respectively, and ß-galactosidase (ß-gal) expression was assessed in brain tissue of subjects socially facilitated and socially inhibited by 0.75 g/kg ethanol. In females, positive correlations were evident between overall social activity and neuronal activation of seven out of 13 ROIs, including the prefrontal cortex and nucleus accumbens, with negative correlations evident in males. Assessments of neuronal activation patterns revealed drastic sex differences between ethanol responding phenotypes. In socially inhibited males, strong correlations were evident among almost all ROIs (90 %), with markedly fewer correlations among ROIs (38 %) seen in socially facilitated males. In contrast, interconnectivity in females inhibited by ethanol was only 10 % compared to nearly 60 % in facilitated subjects. However, hub analyses revealed convergence of brain regions in males and females, with the nucleus accumbens being a hub region in socially inhibited subjects. Taken together, these findings demonstrate individual and sex-related differences in responsiveness to acute ethanol in adolescent rats, with sex differences more evident in socially inhibited by ethanol adolescents than their socially facilitated counterparts.

Determining the neuronal ensembles underlying sex-specific social impairments following adolescent intermittent ethanol exposure.

Binge drinking during adolescence can have behavioral and neurobiological consequences. We have previously found that adolescent intermittent ethanol (AIE) exposure produces sex-specific social alterations indexed via decreases of social investigation and/or social preference in rats. The prelimbic cortex (PrL) regulates social interaction, and alterations within the PrL resulting from AIE may contribute to social alterations. The current study sought to determine whether AIE-induced PrL dysfunction underlies decreases in social interaction evident in adulthood. We first examined social interaction-induced neuronal activation of the PrL and several other regions of interest (ROIs) implicated in social interaction. Adolescent male and female cFos-LacZ rats were exposed to water (control) or ethanol (4 g/kg, 25% v/v) via intragastric gavage every other day between postnatal day (P) 25 and 45 (total 11 exposures). Since cFos-LacZ rats express ß-galactosidase (ß-gal) as a proxy for Fos, activated cells that express of ß-gal can be inactivated by Daun02. In most ROIs, expression of ß-gal was elevated in socially tested adult rats relative to home cage controls, regardless of sex. However, decreased social interaction-induced ß-gal expression in AIE-exposed rats relative to controls was evident only in the PrL of males. A separate cohort underwent PrL cannulation surgery in adulthood and was subjected to Daun02-induced inactivation. Inactivation of PrL ensembles previously activated by social interaction reduced social investigation in control males, with no changes evident in AIE-exposed males or females. These findings highlight the role of the PrL in male social investigation and suggest an AIE-associated dysfunction of the PrL that may contribute to reduced social investigation following adolescent ethanol exposure.

Determining the neuronal ensembles underlying sex-specific social impairments following adolescent intermittent ethanol exposure.

Binge drinking during adolescence can have behavioral and neurobiological consequences. We have previously found that adolescent intermittent ethanol (AIE) exposure produces a sex-specific social impairment in rats. The prelimbic cortex (PrL) regulates social behavior, and alterations within the PrL resulting from AIE may contribute to social impairments. The current study sought to determine whether AIE-induced PrL dysfunction underlies social deficits in adulthood. We first examined social stimulus-induced neuronal activation of the PrL and several other regions of interest implicated in social behavior. Male and female cFos-LacZ rats were exposed to water (control) or ethanol (4 g/kg, 25% v/v) via intragastric gavage every other day between postnatal day (P) 25 and 45 (total 11 exposures). Since cFos-LacZ rats express ß-galactosidase (ß-gal) as a proxy for cFos, activated cells that express of ß-gal can be inactivated by Daun02. ß-gal expression in most ROIs was elevated in socially tested adult rats relative to home cage controls, regardless of sex. However, differences in social stimulus-induced ß-gal expression between controls and AIE-exposed rats was evident only in the PrL of males. A separate cohort underwent PrL cannulation surgery in adulthood and were subjected to Daun02-induced inactivation. Inactivation of PrL ensembles previously activated by a social stimulus led to a reduction of social behavior in control males, with no changes evident in AIE-exposed males or females. These findings highlight the role of the PrL in male social behavior and suggest an AIE-associated dysfunction of the PrL may contribute to social deficits following adolescent ethanol exposure.

Impact of Adolescent Intermittent Ethanol Exposure on Social Investigation, Social Preference, and Neuronal Activation in cFos-LacZ Male and Female Rats.

Adolescence is a sensitive developmental period during which alcohol use is often initiated and consumed in high quantities, often at binge or even high-intensity drinking levels. Our lab has repeatedly found that adolescent intermittent ethanol (AIE) exposure in rats results in long-lasting social impairments, specifically in males, however our knowledge of the neuronal underpinnings to this sex-specific effect of AIE is limited. The present study was designed to test whether social anxiety-like alterations in AIE-exposed males would be accompanied by alterations of neuronal activation across brain regions associated with social behavior, with AIE females demonstrating no social impairments and alterations in neuronal activation. Adolescent male and female cFos-LacZ transgenic rats on a Sprague-Dawley background were exposed to ethanol (4 g/kg, 25% v/v) or water via intragastric gavage every other day during postnatal days (P) 25-45 for a total of 11 exposures (n = 13 per group). Social behavior of adult rats was assessed on P70 using a modified social interaction test, and neuronal activation in brain regions implicated in social responding was assessed via ß-galactosidase (ß-gal) expression. We found that AIE exposure in males resulted in a significantly lower social preference coefficient relative to water-exposed controls, with no effect evident in females. Exposure-specific relationships between social behavior and neuronal activation were identified, with AIE eliminating correlations found in water controls related to social interaction, and eliciting negative correlations mainly in limbic regions in a sex-specific manner. AIE exposure in the absence of social testing was also found to differentially affect neural activity in the orbitofrontal cortex and central amygdala in males and females. These data suggest that AIE produces sex-specific social impairments that are potentially driven by differential neuronal activation states in regions important for social behavior, including the medial prefrontal and orbitofrontal cortices, nucleus accumbens, lateral septum, and central amygdala. Future studies should be focused on identification of specific neuronal phenotypes activated by interaction with a social partner in AIE-exposed subjects and their control counterparts.

Adolescent Ethanol Exposure: Anxiety-Like Behavioral Alterations, Ethanol Intake, and Sensitivity.

Adolescence is a developmental period associated with rapid age-specific physiological, neural, and hormonal changes. Behaviorally, human adolescents are characterized by age-typical increases in novelty-seeking and risk-taking, including the frequent initiation of alcohol and drug use. Alcohol use typically begins during early adolescence, and older adolescents often report high levels of alcohol consumption, commonly referred to as high-intensity drinking. Early-onset and heavy drinking during adolescence are associated with an increased risk of developing alcohol use disorders later in life. Yet, long-term behavioral consequences of adolescent alcohol use that might contribute to excessive drinking in adulthood are still not well understood. Recent animal research, however, using different exposure regimens and routes of ethanol administration, has made substantial progress in identifying the consequences of adolescent ethanol exposure that last into adulthood. Alterations associated with adolescent ethanol exposure include increases in anxiety-like behavior, impulsivity, risk-taking, and ethanol intake, although the observed alterations differ as a function of exposure regimens and routes of ethanol administration. Rodent studies have also shown that adolescent ethanol exposure produces alterations in sensitivity to ethanol, with these alterations reminiscent of adolescent-typical ethanol responsiveness. The goal of this mini-review article is to summarize the current state of animal research, focusing on the long-term consequences related to adolescent ethanol exposure, with a special emphasis on the behavioral alterations and changes to ethanol sensitivity that can foster high levels of drinking in adulthood.

Adolescent Intermittent Ethanol Exposure Effects on Kappa Opioid Receptor Mediated Dopamine Transmission: Sex and Age of Exposure Matter.

Underage alcohol drinking increases the risk of developing alcohol use disorder (AUD). In rodents, adolescent ethanol exposure augments ethanol consumption and anxiety-like behavior while reducing social interaction. However, the underlying mechanisms driving these adaptations are unclear. The dopamine and kappa opioid receptor (KOR) systems in the nucleus accumbens (NAc) are implicated in affective disorders, including AUD, with studies showing augmented KOR function and reduced dopamine transmission in ethanol-dependent adult animals. Thus, here we examine the impact of adolescent intermittent ethanol (AIE) exposure on dopamine transmission and KOR function in the NAc. Rats were exposed to water or ethanol (4 g/kg, intragastrically) every other day during early (postnatal day (PD) 25-45) or late (PD 45-65) adolescence. While AIE exposure during early adolescence (early-AIE) did not alter dopamine release in male and female rats, AIE exposure during late adolescence (late-AIE) resulted in greater dopamine release in males and lower dopamine release in females. To determine the impact of AIE on KOR function, we measured the effect of KOR activation using U50,488 (0.01-1.00 µM) on dopamine release. Early-AIE exposure potentiated KOR-mediated inhibition of dopamine release in females, while late-AIE exposure attenuated this effect in males. Interestingly, no differences in KOR function were observed in early-AIE exposed males and late-AIE exposed females. Together, these data suggest that AIE exposure impact on neural processes is dependent on sex and exposure timing. These differences likely arise from differential developmental timing in males and females. This is the first study to show changes in KOR function following AIE exposure.