Intermittent Ethanol Exposure Research Articles

Sex differences in anxiety‐like behaviors following chronic intermittent ethanol and/or repeated stress exposures using an adult zebrafish (Danio rerio) model

Studies on ethanol use have found that stress often impacts responses, such that it may promote increased ethanol intake and, therefore, risk for harm. Additional evidence suggests that stress, especially severe stress, may have a greater negative effect from ethanol in females than males. To improve our understanding of these potential interactions, we used an adult zebrafish model to assess behavioral activities in 6 mth old male and female zebrafish following treatment with chronic intermittent ethanol exposure and/or repeated stress exposure. The 4 treatment conditions were: control (tanks moved to the testing bench but no other manipulations), twice‐daily 60 min 0.5% ethanol exposure and/or 3 times daily 40 sec stress exposures for a total of five days. We used a custom light/dark tank to assess behaviors at approximately 16 hr after the final treatments. Zebrafish were tested individually with data collected for a total of 8 min every 10 seconds: (1) light vs. dark preference, (2) crossings between light and dark, (3) preference for tank top vs. bottom and (4) general activity levels (normal, hyperactive or freezing). Time spent in each activity was summated across the testing period for each animal and averaged by sex within treatment condition. Overall, control females spent more time in the dark (20%) compared to males, suggesting a greater sensitivity to the novelty of the testing tank for female than male fish. We observed a large increase in time spent in the dark for both males (34%) and females (59%) compared to control values for the EtOH‐only treatment whereas the stress + EtOH condition values were similar to controls. There was a prominent sex difference in hyperactivity with the stress only males showing a 26% reduction compared to control levels and females having a 38% increase compared to males. Interestingly, we also saw a reduction (17%) in hyperactivity for EtOH only males compared to control values but a large increase (40%) for the ethanol only females. Again, the stress + EtOH condition values were similar to controls for both sexes, suggestive of some resiliency when exposed to both the intermittent EtOH as well as the repeated stress procedure. We also noted less freezing (believed to reflect high levels of fear/anxiety in the fish) in males and females that were treated with the repeated stress, suggesting that this helped the fish adapt to the novelty of the testing tank more than for the other treatment conditions. These data support the utility of zebrafish as an animal model to study the impact of stress on ethanol responses and showed that behavioral responses support an interaction of stress with ethanol by sex.Support or Funding InformationSupported by Pacific University School of Pharmacy RIG (LLD)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Adolescent Intermittent Ethanol on Noradrenergic Development

In clinical populations, adolescent alcohol drinking has been shown to increase stress sensitivity and the likelihood of developing alcohol use disorders (AUDs) in adulthood. These findings suggest pathways mediating stress‐relating behaviors are impacted by developmental exposure to alcohol, contributing to a greater risk for alcoholism and potentially stress‐induced relapse following treatment. Animal models indicate tyrosine hydroxylase (TH) neurons in the A2 region of the nucleus of the tractus solitarius (NTS) contribute to stress‐induced relapse through enhanced norepinephrine signaling, subsequently increasing neuronal activity in the bed nucleus of the stria terminalus (BNST). Previous research suggests NTS TH expression undergoes developmental changes during adolescence that may make this neuronal population susceptible to modulation by alcohol during this age. We hypothesized adolescent intermittent ethanol (AIE) exposure in mice, a standard preclinical model of adolescent binge drinking, will impact NTS TH neuron development, ultimately altering the NTS‐BNST circuitry resulting in a higher risk of AUDs in adulthood. NTS TH mRNA expression in male and female C57Bl6/J mice exposed to AIE (postnatal day [PND] 28–42) or used as air controls was quantified via qRT‐PCR at different time points (PND29, PND36, PND43, PND49, PND70). Immunohistochemistry methods were implemented to determine the impact of AIE on NTS TH neuronal activity following an ethanol challenge in adulthood (PND70). Results showed a significant increase in TH mRNA in males one day after beginning AIE (PND29) relative to air controls, while females exhibited the greatest TH expression after their first week of exposure to ethanol (PND36). In both sexes, heightened TH mRNA expression occurred earlier than expected during normal development. Surprisingly, compared to adult ethanol naïve mice, AIE decreased the ability of ethanol at doses of 2g/kg and 4g/kg to alter NTS TH neuron cFos expression at PND70 compared to control. Together, these findings suggest an enhancement of NTS TH activity early in development and reduction of ethanol sensitivity in adulthood. Studies are in progress to further delineate the mechanisms by which AIE reduces adult ethanol sensitivity and how this may alter stress‐related behaviors.Support or Funding InformationAA22937AA017823This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic Intermittent Ethanol Exposure Induces Upregulation of Matrix Metalloproteinase-9 in the Rat Medial Prefrontal Cortex and Hippocampus.

Matrix metalloproteinase-9 (MMP-9, Gelatinase B), an extracellular-acting Zn2+-dependent endopeptidase, are involved in brain pathologies including ischemia, glioma, and epilepsy. Recent studies suggested that MMP-9 plays an important role in neuronal plasticity, specifically in learning and memory. To determine whether and how MMP-9 plays role in alcohol-related behaviors, male Sprague-Dawley (SD) rats were subjected to chronic intermittent ethanol (CIE) exposure for 4weeks, following which we collected tissue samples from the hippocampus, medial prefrontal cortex (mPFC), and amygdala at different stages (acute and chronic exposure) during alcohol exposure. Real-time PCR and western blot assays were used to detect changes in the mRNA and protein expression of MMP-9. Our results indicated that both acute and chronic alcohol exposure induced up-regulation of MMP-9 mRNA levels in the hippocampus and mPFC, but not in the amygdala. Furthermore, acute and chronic alcohol exposure up regulated the expression of total MMP-9 and active MMP-9 in these two brain regions. Moreover, the increase of active MMP-9 expression was larger than those in total MMP-9 expression. Immunoprecipitation analyses identified potential MMP-9-interacting proteins, including Itgb1, Src, Eef1a2, tubulin, actin, and histone H2B. These results demonstrate that both acute and CIE exposure induced increases in MMP-9 expression in the mPFC and hippocampus, suggesting that MMP-9 plays a key role in chronic alcohol exposure and dependence.

Paternal Preconception Every-Other-Day Ethanol Drinking Alters Behavior and Ethanol Consumption in Offspring.

Alcohol use disorder is a devastating disease with a complex etiology. Recent preclinical studies have revealed that paternal preconception chronic intermittent ethanol (EtOH) exposure via vaporized EtOH altered drinking behaviors and sensitivity to EtOH selectively in male offspring. In the current study, we used a voluntary oral route of paternal preconception EtOH exposure, i.e., intermittent every-other-day two-bottle choice drinking, and tested offspring for behavioral alterations. Fifteen EtOH drinking sires and 10 control sires were mated to EtOH naïve females to produce EtOH-sired and control-sired offspring. These offspring were tested using the elevated plus maze, open field, drinking in the dark, and unlimited access two-bottle choice assays. We found that paternal preconception every-other-day two-bottle choice drinking resulted in reduced EtOH consumption selectively in male offspring in the drinking in the dark assay compared to control-sired offspring. No differences were detected in either sex in the unlimited access two-bottle choice and elevated plus maze assays. Open field analysis revealed complex changes in basal behavior and EtOH-induced behaviors that were sex specific. We concluded that paternal preconception voluntary EtOH consumption has persistent effects that impact the next generation. This study adds to a growing appreciation that one’s behavioral response to EtOH and EtOH drinking behavior are impacted by EtOH exposure of the prior generation.

Emotional Reactivity to Incentive Downshift in Adult Rats Exposed to Binge-Like Ethanol Exposure During Adolescence.

Alcohol use in adolescents is often characterized by binge-like ethanol consumption pattern, which is associated with long-term health consequences and even with important harms to his developing brain. Among this, ethanol exposure induces long-lasting alterations in anxiety-related neurobiological systems such as corticotropin releasing factor (CRF) or melanocortin system (MC). Recently, it has been demonstrated that adult rats exposed to adolescent intermittent ethanol (AIE) exposure exhibited anxiogenic-like behavior. Given that it has been demonstrated that negative affective state is relevant to development of addictive behavior, it is tempting to suggest that increased risk of adult abusive alcohol use exhibited in rats exposed to ethanol during adolescence may be related with differences in anxiety-related behavior. We conducted a study investigating the emotional reactivity after a reward devaluation (12-to-1 pellet or 32-to-4% sucrose downshift) in adult rats exposed to binge-like ethanol exposure during adolescence. For this aim, adolescent Sprague-Dawley rats were treated with ethanol (2.5 g/kg ip; AIE) or saline (AIS) for 2 consecutive days at 48-h intervals over a 14-day period (PND30-PND43). Following 25 free-ethanol days, adult rats were trained in consummatory and instrumental successive negative contrast task (cSNC and iSNC). Our data shows that both AIE and AIS groups exhibited suppression of the consummatory and instrumental behavior after reward devaluation relative to unshifthed control. Also, adult rats exposed to alcohol during adolescence exhibited a particularly strong negative affective state (lower sucrose consumption) with regards to the AIS group in the cSNC. This data demonstrated that adolescent binge-like ethanol exposure might trigger a greater emotional reactivity following incentive downshift, which might be linked to higher vulnerability to substance use disorder.

Persistent Alterations of Accumbal Cholinergic Interneurons and Cognitive Dysfunction after Adolescent Intermittent Ethanol Exposure

Adolescent binge drinking renders young drinkers vulnerable to alcohol use disorders in adulthood; therefore, understanding alcohol-induced brain damage and associated cognitive dysfunctions is of paramount importance. Here we investigated the effects of binge-like adolescent intermittent ethanol (AIE) exposure on nonspatial working memory, behavioral flexibility and cholinergic alterations in the nucleus accumbens (NAc) in male and female rats. On postnatal days P25–57 rats were intubated with water or ethanol (at a dose of 5 g/kg) on a 2-day-on/2-day-off cycle and were then tested in adulthood on social recognition and probabilistic reversal learning tasks. During the social recognition task AIE-treated rats spent similar amounts of time interacting with familiar and novel juveniles, indicating an impaired ability to sustain memory of the familiar juvenile. During probabilistic reversal learning, AIE-treated male and female rats showed behavioral inflexibility as indicated by a higher number of trials needed to complete three reversals within a session, longer response latencies for lever selection, and for males, a higher number of errors as compared to water-treated rats. AIE exposure also reduced the number of cholinergic interneurons in the NAc in males and females. These findings indicate AIE-related pathologies of accumbal cholinergic interneurons and long lasting cognitive-behavioral deficits, which may be associated with cortico-striatal hypofunction.

Absence of compulsive drinking phenotype in adult male rats exposed to ethanol in a binge-like pattern during adolescence.

The abuse of alcohol during adolescence is widespread and represents a particular concern, given that earlier age of drinking onset is associated with increased risk for the development of alcohol use disorders (AUDs). Despite this risk, it remains unclear whether binge-like adolescent alcohol exposure facilitates drinking despite aversive consequences, a characteristic common among individuals with AUDs. The present study examined voluntary alcohol consumption and aversion-resistant drinking in adult male Long-Evans rats that had undergone adolescent intermittent ethanol (AIE) exposure by vapor inhalation between postnatal days (PD) 28-44. Ethanol consumption during adulthood was examined using a two-bottle choice (2BC) intermittent access procedure. Rats were tested for aversion-resistant drinking using ethanol adulterated with quinine (10, 30, 100mg/L) after two 7-week periods of 2BC drinking. After completion of the second test of aversion-resistant drinking, rats were trained to operantly self-administer ethanol. The results revealed that both air control (AIR) and AIE-exposed rats exhibited similar ethanol intake and preference in the 2BC paradigm. After 7 weeks of 2BC drinking, quinine adulteration significantly suppressed ethanol intake, but only at the highest concentration examined (100mg/L). However, upon retesting after a total of 17 weeks of 2BC drinking, 30-mg/L quinine suppressed ethanol intake. Notably, AIR- and AIE-exposed rats were equally sensitive to quinine-adulterated ethanol at both time points. In addition, AIR- and AIE-exposed rats responded similarly during operant ethanol self-administration on both fixed and progressive ratio schedules of reinforcement. Finally, both AIR- and AIE-exposed rats exhibited similar preference for sucrose. The results of this study show that binge-like ethanol vapor exposure during adolescence does not alter voluntary ethanol consumption, motivation to operantly respond for ethanol, or promote aversion-resistant ethanol consumption in adulthood. These data, together with previous work reporting conflicting results across various rodent models of adolescent alcohol exposure, underscore the need to further explore the role that exposure to alcohol during adolescence has on the development of heavy and compulsive drinking phenotypes in adulthood.

Adolescent Alcohol Exposure Epigenetically Suppresses Amygdala Arc Enhancer RNA Expression to Confer Adult Anxiety Susceptibility

BackgroundAdolescent intermittent ethanol (AIE) exposure is an emerging risk factor for adult psychopathology, such as anxiety disorders. Enhancer RNAs (eRNAs) are short noncoding RNAs transcribed from enhancer regions that regulate synaptic plasticity–associated gene expression, including Arc, but their role in AIE-induced susceptibility to anxiety in adulthood is unknown. MethodsRats were exposed to AIE (ethanol exposure 2 days on/off) or intermittent normal saline during postnatal days 28 to 41 and allowed to grow to adulthood for analysis of behavior and biochemical measures. Some AIE rats and rats with intermittent normal saline exposure were exposed to an acute challenge with ethanol in adulthood. Cohorts of alcohol-naïve adult rats were cannulated in the central nucleus of amygdala and infused with either Kdm6b small interfering RNA or an antisense locked nucleic acid oligonucleotide specific to Arc eRNA before behavioral and biochemical analysis. ResultsAIE adult rats displayed heightened anxiety and decreased Arc eRNA expression, which is regulated epigenetically through decreased Kdm6b expression. This triggered condensed chromatin at the synaptic activity response element site and promoter of the Arc gene, facilitating increased negative elongation factor binding to the Arc promoter and decreasing Arc expression in the amygdala. Knockdown of Kdm6b or Arc eRNA expression in the central nucleus of amygdala provoked anxiety in alcohol-naïve adult rats and recapitulated the molecular and epigenetic phenotypes of AIE. ConclusionsThese data suggest that eRNA regulation via epigenetic reprogramming in the amygdala, particularly at the Arc synaptic activity response element site, contributes to adult anxiety after adolescent alcohol exposure.

Aging with alcohol-related brain damage: Critical brain circuits associated with cognitive dysfunction.

Alcoholism is associated with brain damage and impaired cognitive functioning. The relative contributions of different etiological factors, such as alcohol, thiamine deficiency and age vulnerability, to the development of alcohol-related neuropathology and cognitive impairment are still poorly understood. One reason for this quandary is that both alcohol toxicity and thiamine deficiency produce brain damage and cognitive problems that can be modulated by age at exposure, aging following alcohol toxicity or thiamine deficiency, and aging during chronic alcohol exposure. Pre-clinical models of alcohol-related brain damage (ARBD) have elucidated some of the contributions of ethanol toxicity and thiamine deficiency to neuroinflammation, neuronal loss and functional deficits. However, the critical variable of age at the time of exposure or long-term aging with ARBD has been relatively ignored. Acute thiamine deficiency created a massive increase in neuroimmune genes and proteins within the thalamus and significant increases within the hippocampus and frontal cortex. Chronic ethanol treatment throughout adulthood produced very minor fluctuations in neuroimmune genes, regardless of brain region. Intermittent "binge-type" ethanol during the adolescent period established an intermediate neuroinflammatory response in the hippocampus and frontal cortex, that can persist into adulthood. Chronic excessive drinking throughout adulthood, adolescent intermittent ethanol exposure, and thiamine deficiency all led to a loss of the cholinergic neuronal phenotype within the basal forebrain, reduced hippocampal neurogenesis, and alterations in the frontal cortex. Only thiamine deficiency results in gross pathological lesions of the thalamus. The behavioral impairment following these types of treatments is hierarchical: Thiamine deficiency produces the greatest impairment of hippocampal- and prefrontal-dependent behaviors, chronic ethanol drinking ensues mild impairments on both types of tasks and adolescent intermittent ethanol exposure leads to impairments on frontocortical tasks, with sparing on most hippocampal-dependent tasks. However, our preliminary data suggest that as rodents age following adolescent intermittent ethanol exposure, hippocampal functional deficits began to emerge. A necessary requirement for the advancement of understanding the neural consequences of alcoholism is a more comprehensive assessment and understanding of how excessive alcohol drinking at different development periods (adolescence, early adulthood, middle-aged and aged) influences the trajectory of the aging process, including pathological aging and disease.

Chronic Intermittent Ethanol Exposure Selectively Increases Synaptic Excitability in the Ventral Domain of the Rat Hippocampus

Many studies have implicated hippocampal dysregulation in the pathophysiology of alcohol use disorder (AUD). However, over the past twenty years, a growing body of evidence has revealed distinct functional roles of the dorsal (dHC) and ventral (vHC) hippocampal subregions, with the dHC being primarily involved in spatial learning and memory and the vHC regulating anxiety- and depressive-like behaviors. Notably, to our knowledge, no rodent studies have examined the effects of chronic ethanol exposure on synaptic transmission along the dorsal/ventral axis. To that end, we examined the effects of the chronic intermittent ethanol vapor exposure (CIE) model of AUD on dHC and vHC synaptic excitability. Adult male Long–Evans rats were exposed to CIE or AIR for 10 days (12 h/day; targeting blood ethanol levels of 175–225 mg%) and recordings were made 24 h into withdrawal. As expected, this protocol increased anxiety-like behaviors on the elevated plus-maze and successive alleys test. Extracellular recordings revealed marked CIE-associated increases in synaptic excitation in the CA1 region that were exclusively restricted to the ventral domain of the hippocampus. Western blot analysis of synaptoneurosomal fractions revealed that the expression of two proteins that regulate synaptic strength, GluA2 and SK2, were dysregulated in the vHC, but not the dHC, following CIE. Together, these findings suggest that the ventral CA1 region may be particularly sensitive to the maladaptive effects of chronic ethanol exposure and provide new insight into some of the neural substrates that may contribute to the negative affective state that develops during withdrawal.

Sex-dependent differences in ethanol inhibition of mouse lateral orbitofrontal cortex neurons.

Biological differences between males and females likely influence responses to alcohol and the propensity to engage in excessive drinking. In both humans and rodents, females escalate alcohol use and develop addiction-like behaviors faster than males, while males exhibit more severe withdrawal symptoms during abstinence. The mechanisms underlying these differences are not yet known but may reflect fundamental differences in the ethanol sensitivity of neurons in reward and control areas of the brain. To address this question, we recorded current-evoked spiking of lateral orbitofrontal cortex (lOFC) neurons in male and female C57BL/6J mice following acute and chronic exposure to ethanol. Ethanol (11-66mM) reduced firing of lOFC neurons but produced less inhibition in neurons from female mice. As previously reported for male mice, the glycine receptor blocker strychnine blocked ethanol inhibition of spiking of lOFC neurons from female mice and prevented the ethanol-induced increase in tonic current. Following chronic intermittent ethanol (CIE) exposure, current-evoked spiking of lOFC neurons was significantly enhanced with a greater effect observed in males. After CIE treatment, acute ethanol had no effect on spiking in neurons from male mice, while it produced a slight but significant decrease in firing in females. Finally, like male mice, the inhibitory effect of the glycine transport inhibitor sarcosine was blunted in CIE-exposed female mice. Together, these results suggest that while lOFC neurons in male and female mice are similarly affected by ethanol, there are significant differences in sensitivity that may contribute to differences in alcohol actions between males and females.

Adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons and neuroimmune activation are prevented by exercise and indomethacin.

Basal forebrain cholinergic neurons mature in adolescence coinciding with development of adult cognitive function. Preclinical studies using the rodent model of adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-days on/2-days off from postnatal day [P]25 to P55) reveal persistent increases of brain neuroimmune genes that are associated with cognitive dysfunction. Adolescent intermittent ethanol exposure also reduces basal forebrain expression of choline acetyltransferase (ChAT), an enzyme critical for acetylcholine synthesis in cholinergic neurons similar to findings in the post-mortem human alcoholic basal forebrain. We report here that AIE decreases basal forebrain ChAT+IR neurons in both adult female and male Wistar rats following early or late adolescent ethanol exposure. In addition, we find reductions in ChAT+IR somal size as well as the expression of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor p75NTR, both of which are expressed on cholinergic neurons. The decrease in cholinergic neuron marker expression was accompanied by increased phosphorylation of NF-κB p65 (pNF-κB p65) consistent with increased neuroimmune signaling. Voluntary wheel running from P24 to P80 prevented AIE-induced cholinergic neuron shrinkage and loss of cholinergic neuron markers (i.e., ChAT, TrkA, and p75NTR) as well as the increase of pNF-κB p65 in the adult basal forebrain. Administration of the anti-inflammatory drug indomethacin (4.0 mg/kg, i.p prior to each ethanol exposure) during AIE also prevented the loss of basal forebrain cholinergic markers and the concomitant increase of pNF-κB p65. In contrast, treatment with the proinflammatory immune activator lipopolysaccharide (1.0 mg/kg, i.p. on P70) caused a loss of cholinergic neuron markers that was paralleled by increased pNF-κB p65 in the basal forebrain. These novel findings are consistent with AIE causing lasting activation of the neuroimmune system that contributes to the persistent loss of basal forebrain cholinergic neurons in adulthood.

Alcohol-dependent pulmonary inflammation: A role for HMGB-1.

Previous studies have demonstrated that acute alcohol intoxication significantly impairs lung immune responses, which can lead to the tissue being undefended from microbial infection and resulting disease. Data suggest that acute intoxication presents an axis where simultaneously suppressing early pro-inflammatory cytokines while inducing anti-inflammatory signals contributes to alcohol-dependent immune suppression in the lung, and thus undeterred microbial replication. Interestingly, alcoholics and those with alcohol use disorder present with increased pneumonia and acute respiratory diseases (ARDs), suggesting a more active priming of inflammatory responses in the lungs. There is current research evaluating the acute effects of binge ethanol consumption on adolescents, which is of grave concern, though long-term effects of adolescent ethanol binge exposure are less studied. We hypothesize that adolescent binge drinking may prime the individual to severe pulmonary distress, when later challenged by a microbial pathogen. Herein, we evaluate a model of adolescent intermittent ethanol (AIE) exposure to investigate pulmonary pathology after microbial challenge. Ethanol was administered to adolescent mice using a binge exposure schedule, and mice were then rested to early adulthood. These mice were then challenged with a sub-lethal intranasal inoculation of Klebsiella pneumoniae and evaluated for severity of disease. We find that AIE exposure initially activates inflammatory mediators within the lung, which resolves over time. However, when challenged with a microbial pathogen after this resolution period, these animals present with more severity of inflammation, pulmonary tissue damage, and mortality when challenged with a pulmonary microbial infection. Interestingly, our data suggest a role for alcohol-dependent release of the protein HMGB-1 from host cells, for both morbidity and mortality in our model of microbial-dependent pulmonary inflammation.

Sex Differences in Photic Entrainment and Sensitivity to Ethanol-Induced Chronodisruption in Adult Mice After Adolescent Intermittent Ethanol Exposure.

Evidence supports a role for the circadian system in alcohol use disorders, but the impact of adolescent alcohol exposure on circadian timing later in life is unknown. Acute ethanol (EtOH) attenuates circadian photic phase-resetting in adult, but not adolescent, rodents. However, nearly all studies have focused on males and it is unknown whether this adolescent-typical insensitivity to EtOH persists into adulthood after adolescent drinking. Circadian activity was monitored in C57BL/6J mice receiving adolescent intermittent EtOH (AIE) exposure (15% EtOH and water every other day throughout adolescence) or water alone followed by 24days wherein EtOH was not available (washout). Mice then received a challenge dose of EtOH (1.5g/kg, intraperitoneal) or saline 15minutes prior to a 30-minute phase-delaying light pulse and then were released into constant darkness (DD). To control for possible phase-shifting by EtOH challenge alone, a separate group of mice underwent AIE exposure (or water-only) and washout and then received an EtOH or saline injection, but did not receive a light pulse prior to DD. Striking sex differences in nearly all measures of circadian photic entrainment were observed during adolescence but AIE effects were subtle and few. Only EtOH-naïve adult male mice showed attenuated photic phase-shifts with EtOH challenge, while all other groups showed normal phase-resetting responses to light. AIE-exposed females showed a persistent delay in activity offset. Adult male AIE-exposed mice retained adolescent-like insensitivity to EtOH-induced suppression of photic phase-resetting, suggesting AIE-induced "lock-in" of an adolescent behavioral phenotype. Adult AIE-exposed females showed delayed initiation of the rest phase. Our results also indicate that intermittent EtOH drinking has subtle effects on circadian activity in mice during adolescence that differ from previously reported effects on adult males. The observed sex differences in circadian activity, EtOH consumption and preference, and responses to EtOH challenge merit future mechanistic study.

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

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. 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. Sprague-Dawley male and female rats were given 4g/kg ethanol (AIE) or water intragastrically every 48h 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. 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. 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.

Increasing Brain-Derived Neurotrophic Factor (BDNF) in medial prefrontal cortex selectively reduces excessive drinking in ethanol dependent mice

The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) has been implicated in a number of neuropsychiatric disorders, including alcohol use disorder. Studies have shown that BDNF activity in cortical regions, such as the medial prefrontal cortex (mPFC) mediates various ethanol-related behaviors. We previously reported a significant down-regulation in Bdnf mRNA in mPFC following chronic ethanol exposure compared to control mice. The present study was conducted to extend these findings by examining whether chronic ethanol treatment reduces BDNF protein expression in mPFC and whether reversing this deficit via direct injection of BDNF or viral-mediated overexpression of BDNF in mPFC alters voluntary ethanol consumption in dependent and nondependent mice. Repeated cycles of chronic intermittent ethanol (CIE) exposure was employed to model ethanol dependence, which produces robust escalation of ethanol intake. Results indicated that CIE treatment significantly increased ethanol intake and this was accompanied by a significant decrease in BDNF protein in mPFC that lasted at least 72 h after CIE exposure. In a separate study, once dependence-related increased drinking was established, bilateral infusion of BDNF (0, 0.25, 0.50 μg) into mPFC significantly decreased ethanol intake in a dose-related manner in dependent mice but did not affect moderate drinking in nondependent mice. In a third study, viral-mediated overexpression of BDNF in mPFC prevented escalation of drinking in dependent mice but did not alter intake in nondependent mice. Collectively, these results provide evidence that adaptations in cortical (mPFC) BDNF activity resulting from chronic ethanol exposure play a role in mediating excessive ethanol drinking associated with dependence.

Selective Blockade of T-Type Ca2+ Channels is Protective Against Alcohol-Withdrawal Induced Seizure and Mortality.

We have previously demonstrated that blockade of T-type calcium channels by the non-selective antagonist, ethosuximide (ETX), is effective at reducing electrographical and behavioral correlates of alcohol-withdrawal (WD) seizure. Here, we investigated whether blockade of these calcium channels with the selective antagonist TTA-P2 also reduces alcohol-WD seizure. The non-specific T-type calcium channel antagonist, ETX, is protective against alcohol-WD seizure. However, the mechanism of this effect is unclear. Here, we provide evidence that further suggests selective blockade of T-type calcium channels are protective against alcohol-WD seizure and WD-related mortality. We used an intermittent ethanol exposure model to produce WD-induced hyperexcitability in DBA/2 J mice. Seizure severity was intensified with the chemoconvulsant pentylenetetrazole (PTZ). TTA-P2 (10 mg/kg) reduced seizure severity in mice undergoing alcohol WD with concurrent PTZ treatment (20 mg/kg). Moreover, TTA-P2 (20 and 40 mg/kg) was also protective against PTZ-induced (40 mg/kg) seizure and mortality. These results are consistent with prior results using ETX, and suggest that the protective effects of ETX and TTA-P2 against EtOH WD seizures are mediated by T-type calcium channels.

Adolescent intermittent ethanol exposure: Effects on pubertal development, novelty seeking, and social interaction in adulthood

Alcohol use initiated early in adolescence is a major predictor for the development of alcohol use disorders. This risk may be increased when drinking is initiated around the time of puberty, given evidence of bidirectional relationships between alcohol and gonadal hormones. The current study examined the effects of adolescent intermittent ethanol exposure (AIE) on pubertal timing and expression of novelty-seeking and peer-directed behaviors as well as neural correlates of these behaviors. AIE did not affect pubertal timing or the later expression of novelty-seeking and peer-directed behaviors. AIE increased corticosterone (CORT) levels in females not tested behaviorally in adulthood or tested in the novel-object exploration paradigm, whereas social interaction blunted CORT levels in AIE females. Delays in pubertal timing and decreases in CORT levels were correlated, however, with increased novelty seeking in adult males - a phenotype associated with increased addiction vulnerability. In females, social testing elevated oxytocin receptor (OXTR) mRNA expression in the central amygdala (CeA), with this social testing-associated elevation evident in the lateral septum (LS), regardless of sex. Vasopressin receptor 1a (AVP-1aR) mRNA expression in the CeA was enhanced by social testing in females, but not males, with expression of this gene suppressed by social testing in the LS in males, but not females. Together, these data demonstrate that behavioral and neural alterations that may serve as risk factors in later drug vulnerabilities are likely not the result of a single insult, but may reflect interactions among several variables including sex, pubertal timing, stress reactivity, and test circumstances.

Calcium chloride mimics the effects of acamprosate on cognitive deficits in chronic alcohol-exposed mice.

Acamprosate (calcium-bis N-acetylhomotaurinate) is the leading medication approved for the maintenance of abstinence, shown to reduce craving and relapse in animal models and human alcoholics. Acamprosate can improve executive functions that are impaired by chronic intermittent ethanol (CIE) exposure. Recent work has suggested that acamprosate's effects on relapse prevention are due to its calcium component, which raises the question whether its pro-cognitive effects are similarly mediated by calcium. This study examined the effects of acamprosate on alcohol-induced behavioral deficits and compared them with the effects of the sodium salt version of N-acetylhomotaurinate or calcium chloride, respectively. We exposed mice to alcohol via three cycles of CIE and measured changes in alcohol consumption in a limited-access paradigm. We then compared the effects of acamprosate and calcium chloride (applied subchronically for 3days during withdrawal) in a battery of cognitive tasks that have been shown to be affected by chronic alcohol exposure. CIE-treated animals showed deficits in attentional set-shifting and deficits in novel object recognition. Alcohol-treated animals showed no impairments in social novelty detection and interaction, or delayed spontaneous alternation. Both acamprosate and calcium chloride ameliorated alcohol-induced cognitive deficits to comparable extents. In contrast, the sodium salt version of N-acetylhomotaurinate did not reverse the cognitive deficits. These results add evidence to the notion that acamprosate produces its anti-relapse effects through its calcium moiety. Our results also suggest that improved regulation of drug intake by acamprosate after withdrawal might at least in part be related to improved cognitive function.

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

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