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.

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.

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 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 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.

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.

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.

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.

Effects of chronic intermittent ethanol exposure during early and late adolescence on anxiety-like behaviors and behavioral flexibility in adulthood.

Although both humans and laboratory rodents demonstrate cognitive and affective alterations associated with adolescent alcohol exposure, it is still unknown whether the consequences of early initiation of alcohol use differ from those of later binge drinking within the adolescent developmental period. The present study was designed to assess the effects of early and late AIE on (1) anxiety-like behavior under social (modified social interaction test) and non-social test circumstances (modified light/dark box test, elevated plus maze), and (2) behavioral flexibility, indexed via set shifting in males and females. Early-mid adolescent intermittent exposure (early AIE) occurred between postnatal days (P) 25 and 45, whereas late adolescent intermittent exposure (late AIE) was conducted between P45 and P65, with behavioral testing initiated not earlier than 25 days after repeated exposure to ethanol (4.0 g/kg intragastrically, every other day for a total of 11 exposures). Anxiety-like behavior on the EPM was evident in males and females following early AIE, whereas only males demonstrated non-social anxiety on the EPM following late AIE. Social anxiety-like alterations and deficits in behavioral flexibility were evident only in males following early AIE. Taken together, the results of the present study demonstrate a particular vulnerability of young adolescent males to long-lasting detrimental effects of repeated ethanol and an insensitivity of older adolescent females to the intermittent ethanol exposure paradigm.

General anesthetic exposure in adolescent rats causes persistent maladaptations in cognitive and affective behaviors and neuroplasticity.

Accumulating evidence indicates that exposure to general anesthetics during infancy and childhood can cause persistent cognitive impairment, alterations in synaptic plasticity, and, to a lesser extent, increased incidence of behavioral disorders. Unfortunately, the developmental parameters of susceptibility to general anesthetics are not well understood. Adolescence is a critical developmental period wherein multiple late developing brain regions may also be vulnerable to enduring general anesthetic effects. Given the breadth of the adolescent age span, this group potentially represents millions more individuals than those exposed during early childhood. In this study, isoflurane exposure within a well-characterized adolescent period in Sprague-Dawley rats elicited immediate and persistent anxiety- and impulsive-like responding, as well as delayed cognitive impairment into adulthood. These behavioral abnormalities were paralleled by atypical dendritic spine morphology in the prefrontal cortex (PFC) and hippocampus (HPC), suggesting delayed anatomical maturation, and shifts in inhibitory function that suggest hypermaturation of extrasynaptic GABAA receptor inhibition. Preventing this hypermaturation of extrasynaptic GABAA receptor-mediated function in the PFC selectively reversed enhanced impulsivity resulting from adolescent isoflurane exposure. Taken together, these data demonstrate that the developmental window for susceptibility to enduring untoward effects of general anesthetics may be much longer than previously appreciated, and those effects may include affective behaviors in addition to cognition.

Oxytocin and vasopressin modulation of social anxiety following adolescent intermittent ethanol exposure.

RATIONALE: Adolescent intermittent ethanol exposure (AIE) produces lasting, sex-specific social anxiety-like alterations in male, but not female rats. Oxytocin (OXT) and vasopressin (AVP) brain systems play opposite roles in regulating social preference/avoidance, with OXT increasing approach to, and AVP increasing avoidance of social stimuli. OBJECTIVES: To test the hypothesis that social anxiety-like alterations seen in adult males after AIE are associated with a shift in the balance between OXT and AVP toward AVP, effectiveness of pharmacological activation of the OXT system and blockade of endogenous activity at AVP receptors for reversing AIE-induced social anxiety-like alterations was assessed, along with examination of the effects of AIE on OXT, vasopressin V1a, and V1b receptor (OXT-R, V1a-R, and V1b-R) surface expression in the hypothalamus. METHODS: Sprague-Dawley male and female rats were given 4 g/kg ethanol (AIE) or water intragastrically every 48 h for a total of 11 exposures during postnatal days (P) 25-45. On P70-72, animals were given a social interaction test following administration of a selective OXT-R agonist WAY-267464, selective V1a-R antagonist SR-49059, or V1b-R antagonist SSR-149415, and hypothalamic tissue was collected. RESULTS: Social anxiety-like behavior was induced by AIE in males but not females, and was selectively reversed by the selective OXT-R agonist and V1b-R antagonist, but not V1a-R antagonist. AIE was also found to decrease OXT-R, but increase V1b-R neuronal surface expression relative to water-exposed controls in the hypothalamus of males, but not females. CONCLUSIONS: These findings demonstrate that AIE induces changes in OXT-R and AVP-R surface expression in the hypothalamus along with social anxiety-like alterations in male rats. These social anxiety-like alterations can be reversed either by activation of the OXT system or by suppression of the AVP system, data that support the hypothesis that social anxiety-like alterations induced by adolescent alcohol exposure in male rats are associated at least in part with an OXT/AVP imbalance.

Monoamine transporter contributions to l-DOPA effects in hemi-parkinsonian rats.

l-DOPA is the standard treatment for Parkinson's disease (PD), but chronic treatment typically leads to abnormal involuntary movement or dyskinesia (LID) development. Although poorly understood, dyskinetic mechanisms involve a complex interaction between the remaining dopamine system and the semi-homologous serotonin and norepinephrine systems. Serotonin and norepinephrine transporters (SERT and NET, respectively) have affinity for dopamine uptake especially when dopamine transporters (DAT) are scant. Monoamine reuptake inhibitors have been reported to modulate l-DOPA's anti-parkinsonian effects, but DAT, SERT, and NET's contribution to dyskinesia has not been well delineated. The current investigation sought to uncover the differential expression and function of DAT, SERT, and NET in the l-DOPA-treated hemi-parkinsonian rat. Protein analysis of striatal monoamine transporters in unilateral sham or 6-hydroxydopamine-lesioned rats treated with l-DOPA (0 or 6 mg/kg) showed lesion-induced DAT loss and l-DOPA-induced gain in SERT:DAT and NET:DAT ratios in lesioned rats which positively correlated with dyskinesia expression, suggesting functional shifts among monoamine transporters in the dyskinetic state. SERT blockade with citalopram (3, 5 mg/kg) reduced LID while DAT and NET blockade with GBR-12909 (5, 10 mg/kg) and nisoxetine (5, 10 mg/kg), respectively, mildly exacerbated dyskinesia expression. Transporter inhibition did not significantly alter l-DOPA's ability to reverse motor deficit. Overall, DA and DAT loss with l-DOPA treatment appear to precipitate gain in SERT and NET function. Strong correlations with LID and direct behavioral comparisons of selective transporter blockade reveal novel implications for SERT, DAT, and NET as potential biomarkers and therapeutic targets in the hemi-parkinsonian model and dyskinetic PD patients.

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.

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.

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.

Adolescent and adult rat cortical protein kinase A display divergent responses to acute ethanol exposure.

Adolescent rats display reduced sensitivity to many dysphoria-related effects of alcohol (ethanol) including motor ataxia and sedative hypnosis, but the underlying neurobiological factors that contribute to these differences remain unknown. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway, particularly the type II regulatory subunit (RII), has been implicated in ethanol-induced molecular and behavioral responses in adults. Therefore, the current study examined cerebral cortical PKA in adolescent and adult ethanol responses. With the exception of early adolescence, PKA RIIα and RIIß subunit levels largely did not differ from adult levels in either whole cell lysate or P2 synaptosomal expression. However, following acute ethanol exposure, PKA RIIß P2 synaptosomal expression and activity were increased in adults, but not in adolescents. Behaviorally, intracerebroventricular administration of the PKA activator Sp-cAMP and inhibitor Rp-cAMP prior to ethanol administration increased adolescent sensitivity to the sedative-hypnotic effects of ethanol compared to controls. Sp-cAMP was ineffective in adults whereas Rp-cAMP suggestively reduced loss of righting reflex (LORR) with paralleled increases in blood ethanol concentrations. Overall, these data suggest that PKA activity modulates the sedative/hypnotic effects of ethanol and may potentially play a wider role in the differential ethanol responses observed between adolescents and adults.

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.

Molecular and behavioral characterization of adolescent protein kinase C following high dose ethanol exposure.

RATIONALE: Ethanol is commonly used and abused during adolescence. Although adolescents display differential behavioral responses to ethanol, the mechanisms by which this occurs are not known. The protein kinase C (PKC) pathway has been implicated in mediating many ethanol-related effects in adults, as well as gamma-aminobutyric acid (GABA(A)) receptor regulation. OBJECTIVES: The present study was designed to characterize cortical PKC isoform and GABA(A) receptor subunit expression during adolescence relative to adults as well as assess PKC involvement in ethanol action. RESULTS: Novel PKC isoforms were elevated, while PKCγ was lower during mid-adolescence relative to adults. Whole-cell lysate and synaptosomal preparations correlated for all isoforms except PKCδ. In parallel, synaptosomal GABAA receptor subunit expression was also developmentally regulated, with GABA(A)R δ and α4 being lower while α1 and γ2 were higher or similar, respectively, in adolescents compared to adults. Following acute ethanol exposure, synaptosomal novel and atypical PKC isoform expression was decreased only in adolescents. Behaviorally, inhibiting PKC with calphostin C, significantly increased ethanol-induced loss of righting reflex (LORR) in adolescents but not adults, whereas activating PKC with phorbol dibutyrate was ineffective in adolescents but decreased LORR duration in adults. Further investigation revealed that inhibiting the cytosolic phospholipase A2/arachidonic acid (cPLA2/AA) pathway increased LORR duration in adolescents, but was ineffective in adults. CONCLUSIONS: These data indicate that PKC isoforms are variably regulated during adolescence and may contribute to adolescent ethanol-related behavior. Furthermore, age-related differences in the cPLA2/AA pathway may contribute to ethanol's age-related effects on novel and atypical PKC isoform expression and behavior.