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.

Adolescent intermittent ethanol exposure does not alter responsiveness to ifenprodil or expression of vesicular GABA and glutamate transporters.

Adolescent intermittent ethanol (AIE) exposure in the rat results in a retention of adolescent-like responsiveness to ethanol into adulthood characterized by enhanced sensitivity to socially facilitating and decreased sensitivity to socially suppressing and aversive effects. Similar pattern of responsiveness to social and aversive effects of the selective glutamate NMDA NR2B receptor antagonist ifenprodil is evident in adolescent rats, suggesting that AIE would also retain this pattern of ifenprodil sensitivity into adulthood. Social (Experiment 1) and aversive (measured via conditioned taste aversion; Experiment 2) effects of ifenprodil were assessed in adult male and female rats following AIE exposure. Sensitivity to the social and aversive effects of ifenprodil was not affected by AIE exposure. Experiment 3 assessed protein expression of vesicular transporters of GABA (vGAT) and glutamate (vGlut2) within the prelimbic cortex and nucleus accumbens in adolescents versus adults and in AIE adults versus controls. vGlut2 expression was higher in adolescents relative to adults within the PrL, but lower in the NAc. AIE adults did not retain these adolescent-typical ratios. These findings suggest that AIE is not associated with the retention of adolescent-typical sensitivity to NR2B receptor antagonism, along with no AIE-induced shift in vGlut2 to vGAT ratios.

General Anesthetic Exposure During Early Adolescence Persistently Alters Ethanol Responses.

BACKGROUND: Adolescent alcohol abuse can lead to behavioral dysfunction and chronic, relapsing alcohol use disorder (AUD) in adulthood. However, not all adolescents that consume alcohol will develop an AUD; therefore, it is critical to identify neural and environmental risk factors that contribute to increases in susceptibility to AUDs following adolescent alcohol (ethanol [EtOH]) exposure. We previously found that adolescent anesthetic exposure led to strikingly similar behavioral and neural effects as adolescent alcohol exposure. Therefore, we tested the hypothesis that general anesthetic exposure during early adolescence would alter EtOH responses consistent with an exacerbation of the adolescent alcohol phenotype. METHODS: To test this hypothesis, early-adolescent male Sprague-Dawley rats were exposed for a short duration to the general anesthetic isoflurane and tested on multiple EtOH-induced behaviors in mid-late adolescence or adulthood. RESULTS: Adolescent rats exposed to isoflurane exhibited decreases in sensitivity to negative properties of EtOH such as its aversive, hypnotic, and socially suppressive effects, as well as increases in voluntary EtOH intake and cognitive impairment. Select behaviors were noted to persist into adulthood following adolescent isoflurane exposure. Similar exposure in adults had no effects on EtOH sensitivity. CONCLUSIONS: This study demonstrates for the first time that early-adolescent isoflurane exposure alters EtOH sensitivity in a manner consistent with an exacerbation of adolescent-typical alcohol responding. These findings suggest that general anesthetic exposure during adolescence may be an environmental risk factor contributing to an enhanced susceptibility to developing AUDs in an already vulnerable population.

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

Inhibitors of Calcium-Activated Anion Channels Modulate Hypnotic Ethanol Responses in Adult Sprague Dawley Rats.

BACKGROUND: Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels (CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. METHODS: We assessed the role of CAACs in ethanol-induced loss of righting reflex (LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. RESULTS: Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. CONCLUSIONS: These results suggest that CAACs are a major target of ethanol in vivo, and the regulation of these channels contributes to select behavioral actions of ethanol.

Overexpression of the steroidogenic enzyme cytochrome P450 side chain cleavage in the ventral tegmental area increases 3α,5α-THP and reduces long-term operant ethanol self-administration.

Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

Brief prenatal ethanol exposure alters behavioral sensitivity to the kappa opioid receptor agonist (U62,066E) and antagonist (Nor-BNI) and reduces kappa opioid receptor expression.

BACKGROUND: Approximately 10 to 15% of women consume alcohol (ethanol [EtOH]) during pregnancy in the United States. Even low amounts of EtOH consumption during pregnancy can elicit long-term consequences. Prenatal experience with as few as 3 drinks has been associated with increase problem drinking in adulthood. Such effects are corroborated in rodents; however, the underlying neural adaptations contributing to this effect are not clear. In the current set of experiments, we investigated whether changes in EtOH responding following prenatal EtOH exposure involved kappa opioid receptor activation and expression. METHODS: Sprague-Dawley rats were prenatally exposed to low levels of alcohol (1.0 g/kg) during late gestation (gestational days 17 to 20 [GD17-20]) via intragastric intubation of pregnant dams. Following birth, EtOH intake, kappa- and mu-opioid-induced place conditioning, and kappa opioid receptor expression in mesolimbic brain regions were assessed in infant rats (postnatal days 14 to 15 [PD14-15]) that were offspring of dams given EtOH, vehicle, or untreated, during pregnancy. RESULTS: Animals exposed to prenatal alcohol drank more alcohol later in life and exhibited significant changes in the kappa opioid system. While control subjects found kappa opioid activation aversive, animals exposed to EtOH prenatally exhibited either no aversion or appetitive responding. Further analysis revealed that synaptosomal kappa opioid receptor expression was significantly decreased in brain areas implicated in responding to EtOH. CONCLUSIONS: Overall, these data suggest that prenatal EtOH affects kappa opioid function and expression and that these changes may be involved in increased drinking later in life.

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.

Sex-dependent effects of ethanol withdrawal from a single- and repeated binge episode exposures on social anxiety-like behavior and neuropeptide gene expression in adolescent rats.

Ethanol withdrawal sensitivity is a risk factor for the development of alcohol use disorder. Heavy episodic drinking during adolescence often encompasses repeated periods of withdrawal. Adolescent intermittent ethanol exposure of laboratory rodents produces several neurobiological deficits that differ between sexes, but the sensitivity to withdrawal as a contributor to the observed sex differences is not clear. The current study assessed the impact of acute withdrawal from a single- and repeated binge ethanol episodes during adolescence as well as protracted abstinence from repeated binge episodes on social anxiety-like behavior (indexed via significant decreases of social investigation) as well as oxytocin (OXT) and vasopressin (AVP) system gene expression in the hypothalamus (HYP) and central amygdala (CeA) in male and female Sprague Dawley rats. Females displayed social anxiety-like behavior during withdrawal from a single binge episode, whereas both sexes showed social anxiety-like changes following acute withdrawal from repeated binge episodes. After a period of protracted abstinence, only males still displayed ethanol-associated social alterations. Analysis of gene expression in separate, non-socially tested subjects revealed that withdrawal from repeated binge episodes during adolescence increased AVP gene expression in the HYP of males and decreased it in females. Males also displayed increased AVP and OXTR gene expression during acute withdrawal from repeated binge episodes in the CeA, with these changes persisting into adulthood. Together, these findings suggest that adolescent females are sensitive to withdrawal from both acute and repeated ethanol exposures, whereas males are sensitive to withdrawal from repeated ethanol exposures, with affective and transcriptional changes persisting into adulthood.

Prelimbic cortex perineuronal net expression and social behavior: Impact of adolescent intermittent ethanol exposure.

Adolescent intermittent ethanol (AIE) exposure in rats leads to social deficits. Parvalbumin (PV) expressing fast-spiking interneurons in the prelimbic cortex (PrL) contribute to social behavior, and perineuronal nets (PNNs) within the PrL preferentially encompass and regulate PV interneurons. AIE exposure increases PNNs, but it is unknown if this upregulation contributes to AIE-induced social impairments. The current study was designed to determine the effect of AIE exposure on PNN expression in the PrL and to assess whether PNN dysregulation contributes to social deficits elicited by AIE. cFos-LacZ male and female rats were exposed every other day to tap water or ethanol (4 g/kg, 25% w/v) via intragastric gavage between postnatal day (P) 25-45. We evaluated neuronal activation by ß-galactosidase expression and PNN levels either at the end of the exposure regimen on P45 and/or in adulthood on P70. In addition, we used Chondroitinase ABC (ChABC) to deplete PNNs following adolescent exposure (P48) and allowed for PNN restoration before social testing in adulthhod. AIE exposure increased PNN expression in the PrL of adult males, but decreased PNNs immediately following AIE. Vesicular glutamate transporter 2 (vGlut2) and vesicular GABA transporter (vGat) near PNNs were downregulated only in AIE-exposed females. Gene expression of PNN components was largely unaffected by AIE exposure. Removal and reestablishment of PrL PNNs by ChABC led to upregulation of PNNs and social impairments in males, regardless of adolescent exposure. These data suggest that AIE exposure in males upregulates PrL PNNs that likely contribute to social impairments induced by AIE.

Ethanol activation of protein kinase A regulates GABAA α1 receptor function and trafficking in cultured cerebral cortical neurons.

Ethanol exposure produces alterations in GABAergic signaling that are associated with dependence and withdrawal. Previously, we demonstrated that ethanol-induced protein kinase C (PKC) γ signaling selectively contributes to changes in GABAA α1 synaptic receptor activity and surface expression. Here, we demonstrate that protein kinase A (PKA) exerts opposing effects on GABAA receptor adaptations during brief ethanol exposure. Cerebral cortical neurons from day 0-1 rat pups were tested after 18 days in culture. Receptor trafficking was assessed by Western blot analysis, and functional changes were measured using whole-cell patch-clamp recordings of evoked and miniature inhibitory postsynaptic current (mIPSC) responses. One-hour ethanol exposure increased membrane-associated PKC and PKA, but steady-state GABAA α1 subunit levels were maintained. Activation of PKA by Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine alone increased GABAA α1 subunit surface expression and zolpidem potentiation of GABA responses, whereas coexposure of ethanol with the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine decreased α1 subunit expression and zolpidem responses. Exposure to the PKC inhibitor calphostin-C with ethanol mimicked the effect of direct PKA activation. The effects of PKA modulation on mIPSC decay τ were consistent with its effects on GABA currents evoked in the presence of zolpidem. Overall, the results suggest that PKA acts in opposition to PKC on α1-containing GABAA receptors, mediating the GABAergic effects of ethanol exposure, and may provide an important target for the treatment of alcohol dependence/withdrawal.

The cytokine mRNA increase induced by withdrawal from chronic ethanol in the sterile environment of brain is mediated by CRF and HMGB1 release.

BACKGROUND: Many neurobiological factors may initiate and sustain alcoholism. Recently, dysregulation of the neuroimmune system by chronic ethanol (CE) has implicated Toll-like receptor 4 (TLR4) activation. Even though TLR4s are linked to CE initiation of brain cytokine mRNAs, the means by which CE influences neuroimmune signaling in brain in the absence of infection remains uncertain. Therefore, the hypothesis is tested that release of an endogenous TLR4 agonist, high-mobility group box 1 (HMGB1) and/or corticotropin-releasing factor (CRF) during CE withdrawal are responsible for CE protocols increasing cytokine mRNAs. METHODS: Acute ethanol (EtOH; 2.75 g/kg) and acute lipopolysaccharide (LPS; 250 µg/kg) dosing on cytokine mRNAs are first compared. Then, the effects of chronic LPS exposure (250 µg/kg for 10 days) on cytokine mRNAs are compared with changes induced by CE protocols (15 days of continuous 7% EtOH diet [CE protocol] or 3 intermittent 5-day cycles of 7% EtOH diet [CIE protocol]). Additionally, TLR4, HMGB1, and downstream effector mRNAs are assessed after CE, CIE, and chronic LPS. To test whether HMGB1 and/or CRF support the CE withdrawal increase in cytokine mRNAs, the HMGB1 antagonists, glycyrrhizin and ethyl pyruvate, and a CRF1 receptor antagonist (CRF1RA) are administered during 24 hours of CE withdrawal. RESULTS: While cytokine mRNAs were not increased following acute EtOH, acute LPS increased all cytokine mRNAs 4 hours after injection. CE produced no change in cytokine mRNAs prior to CE removal; however, the CE and CIE protocols increased cytokine mRNAs by 24 hours after withdrawal. In contrast, chronic LPS produced no cytokine mRNA changes 24 hours after LPS dosing. TLR4 mRNA was elevated 24 hours following both CE protocols and chronic LPS exposure. While chronic LPS had no effect on HMGB1 mRNA, withdrawal from CE protocols significantly elevated HMGB1 mRNA. Systemic administration of HMGB1 antagonists or a CRF1RA significantly reduced the cytokine mRNA increase following CE withdrawal. The CRF1RA and the HMGB1 antagonist, ethyl pyruvate, also reduced the HMGB1 mRNA increase that followed CE withdrawal. CONCLUSIONS: By blocking HMGB1 or CRF action during CE withdrawal, evidence is provided that HMGB1 and CRF release are critical for the CE withdrawal induction of selected brain cytokine mRNAs. Consequently, these results clarify a means by which withdrawal from CE exposure activates neuroimmune function in the sterile milieu of brain.

Striatal serotonin transporter gain-of-function in L-DOPA-treated, hemi-parkinsonian rats.

L-DOPA is the standard treatment for Parkinson's disease (PD), but chronic treatment typically leads to L-DOPA-induced dyskinesia (LID). LID involves a complex interaction between the remaining dopamine (DA) system and the semi-homologous serotonin (5-HT) system. Since serotonin transporters (SERT) have some affinity for DA uptake, they may serve as a functional compensatory mechanism when DA transporters (DAT) are scant. DAT and SERT's functional contributions in the dyskinetic brain have not been well delineated. The current investigation sought to determine how DA depletion and L-DOPA treatment affect DAT and SERT transcriptional processes, translational processes, and functional DA uptake in the 6-hydroxydopamine-lesioned hemi-parkinsonian rat. Rats were counterbalanced for motor impairment into equally lesioned treatment groups then given daily L-DOPA (0 or 6 mg/kg) for 2 weeks. At the end of treatment, the substantia nigra was processed for tyrosine hydroxylase (TH) and DAT gene expression and dorsal raphe was processed for SERT gene expression. The striatum was processed for synaptosomal DAT and SERT protein expression and ex vivo DA uptake. Nigrostriatal DA loss severely reduced DAT mRNA and protein expression in the striatum with minimal changes in SERT. L-DOPA treatment, while not significantly affecting DAT or SERT alone, did increase striatal SERT:DAT protein ratios. Using ex vivo microdialysis, L-DOPA treatment increased DA uptake via SERT when DAT was depleted. Overall, these results suggest that DA loss and L-DOPA treatment uniquely alter DAT and SERT, revealing implications for monoamine transporters as potential biomarkers and therapeutic targets in the hemi-parkinsonian model and dyskinetic PD patients.

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.

Ethanol-induced conditioned taste aversion and associated neural activation in male rats: Impact of age and adolescent intermittent ethanol exposure.

Individuals that initiate alcohol use at younger ages and binge drink during adolescence are more susceptible to developing alcohol use disorder. Adolescents are relatively insensitive to the aversive effects of alcohol and tend to consume significantly more alcohol per occasion than adults, an effect that is conserved in rodent models. Adolescent typical insensitivity to the aversive effects of alcohol may promote greater alcohol intake by attenuating internal cues that curb its consumption. Attenuated sensitivity to the aversive effects of alcohol is also retained into adulthood following protracted abstinence from adolescent intermittent ethanol (AIE) exposure. Despite these effects, much remains unknown regarding the neural contributors. In the present study, we used a conditioned taste aversion (CTA) paradigm to investigate neuronal activation in late-developing forebrain structures of male adolescents and adult cFos-LacZ transgenic rats as well as in AIE adults following consumption of 0.9% sodium chloride previously paired with an intraperitoneal injection of 0, 1.5 or 2.5 g/kg of ethanol. Adults that were non-manipulated or received water exposure during adolescence showed CTA to both ethanol doses, whereas adolescents displayed CTA only to the 2.5 g/kg ethanol dose. Adults who experienced AIE did not show CTA. Adults displayed increased neuronal activation indexed via number of ß-galactosidase positive (ß-gal+) cells in the prefrontal and insular cortex that was absent in adolescents, whereas adolescents but not adults had a reduced number of ß-gal+ cells in the central amygdala. Adults also displayed greater cortical-insular functional connectivity than adolescents as well as insular-amygdalar and prefrontal cortex-accumbens core functional connectivity. Like adolescents, adults previously exposed to AIE displayed reduced prefrontal-insular cortex and prefrontal-accumbal core functional connectivity. Taken together, these results suggest that attenuated sensitivity to the aversive effects of ethanol is related to a loss of an insular-prefrontal cortex-accumbens core circuit.

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.

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons.

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18 days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4 h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCß isoform with PKCß pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.

The role of sex in the persistent effects of adolescent alcohol exposure on behavior and neurobiology in rodents.

Alcohol drinking is often initiated during adolescence, and this frequently escalates to binge drinking. As adolescence is also a period of dynamic neurodevelopment, preclinical evidence has highlighted that some of the consequences of binge drinking can be long lasting with deficits persisting into adulthood in a variety of cognitive-behavioral tasks. However, while the majority of preclinical work to date has been performed in male rodents, the rapid increase in binge drinking in adolescent female humans has re-emphasized the importance of addressing alcohol effects in the context of sex as a biological variable. Here we review several of the consequences of adolescent ethanol exposure in light of sex as a critical biological variable. While some alcohol-induced outcomes, such as non-social approach/avoidance behavior and sleep disruption, are generally consistent across sex, others are variable across sex, such as alcohol drinking, sensitivity to ethanol, social anxiety-like behavior, and induction of proinflammatory markers.