Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of ß-arrestin2.

Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. ß-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although ß-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on ß-arrestin expression and ß-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases ß-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). ß-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between ß-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced ß-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting ß-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced ß-arrestin2 levels and delayed agonist-induced ß-arrestin2 recruitment to the membrane. We conclude that ß-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate ß-arrestin as a potential player mediating behavioral responses to acute alcohol exposure.

Adolescent Vulnerability to Alcohol Use Disorder: Neurophysiological Mechanisms from Preclinical Studies.

Adolescent alcohol use in human populations dramatically increases the likelihood of adult alcohol use disorder. This adolescent vulnerability is recapitulated in preclinical models which provide important opportunities to understand basic neurobiological mechanisms. We provide here an overview of GABAergic and glutamatergic neurotransmission and our current understanding of the sensitivity of these systems to adolescent ethanol exposure. As a whole, the preclinical literature suggests that adolescent vulnerability may be directly related to region-specific neurobiological processes that continue to develop during adolescence. These processes include the activity of intrinsic circuits within diverse brain regions (primarily represented by GABAergic neurotransmission) and activity-dependent regulation of synaptic strength at glutamatergic synapses. Furthermore, GABAergic and glutamatergic neurotransmission within regions/circuits that regulate cognitive function, emotion, and their integration appears to be the most vulnerable to adolescent ethanol exposure. Finally, using documented behavioral differences between adolescents and adults with respect to acute ethanol, we highlight additional circuits and regions for future study.

SNARE Complex-Associated Proteins in the Lateral Amygdala of Macaca mulatta Following Long-Term Ethanol Drinking.

BACKGROUND: Recent work with long-term ethanol (EtOH) self-administration in nonhuman primate models has revealed a complex array of behavioral and physiological effects that closely mimic human alcohol abuse. Detailed neurophysiological analysis in these models suggests a myriad of pre- and postsynaptic neurobiological effects that may contribute to the behavioral manifestations of long-term EtOH drinking. The molecular mechanisms regulating presynaptic effects of this chronic EtOH exposure are largely unknown. To this end, we analyzed the effects of long-term EtOH self-administration on the levels of presynaptic SNARE complex proteins in Macaca mulatta basolateral amygdala, a brain region known to regulate both aversive and reward-seeking behaviors. METHODS: Basolateral amygdala samples from control and EtOH-drinking male and female monkeys were processed. Total basolateral amygdala protein was analyzed by Western blotting using antibodies directed against both core SNARE and SNARE-associated proteins. We also performed correlational analyses between protein expression levels and a number of EtOH drinking parameters, including lifetime grams of EtOH consumed, preference, and blood alcohol concentration. RESULTS: Significant interactions or main effects of sex/drinking were seen for a number of SNARE core and SNARE-associated proteins. Across the range of EtOH-drinking phenotypes, SNAP25 and Munc13-1 proteins levels were significantly different between males and females, and Munc13-2 levels were significantly lower in animals with a history of EtOH drinking. A separate analysis of very heavy-drinking individuals revealed significant decreases in Rab3c (females) and complexin 2 (males). CONCLUSIONS: Protein expression analysis of basolateral amygdala total protein from controls and animals following long-term EtOH self-administration suggests a number of alterations in core SNARE or SNARE-associated components that could dramatically alter presynaptic function. A number of proteins or multiprotein components were also correlated with EtOH drinking behavior, which suggest a potentially heritable role for presynaptic SNARE proteins.

Differential Expression of Munc13-2 Produces Unique Synaptic Phenotypes in the Basolateral Amygdala of C57BL/6J and DBA/2J Mice.

C57BL/6J (B6) and DBA/2J (D2) mice are well known to differentially express a number of behavioral phenotypes, including anxiety-like behavior, fear conditioning, and drug self-administration. However, the cellular mechanisms contributing to these differences remain unclear. Given the basolateral amygdala (BLA) contributes to these behaviors, we characterized strain-dependent differences in presynaptic and postsynaptic function in BLA neurons by integrating electrophysiological, biochemical, and genetic approaches to identify specific molecular mechanisms. We found that D2 glutamatergic synapses expressed enhanced release probability and lower sensitivity to both the inhibitory effects of low extracellular calcium and facilitation by phorbol esters. Furthermore, repetitive stimulation of BLA afferents at low (2 Hz) or high (40 Hz) frequencies revealed that B6 terminals, relative to D2 terminals, were more sensitive to synaptic fatigue principally because of reduced vesicle recycling rates. Additionally, B6 synapses exhibited more robust augmentation of spontaneous release after repetitive stimulation relative to the D2 strain. In silico analysis of the inheritance of synaptic physiology from an array of BXD recombinant inbred strains (Jansen et al., 2011) identified a segment on chromosome 4 containing the gene encoding Munc13-2, which has calcium-/phorbol ester-binding domains and controls presynaptic function. We subsequently found that B6 mice express substantially higher levels of Munc13-2 compared with the D2 strain whereas expression of several release-related proteins, including Munc13-1, was equivalent. We then knocked down the expression of Munc13-2 in B6 mice using a short hairpin RNA and found this recapitulated the presynaptic phenotype of D2 BLA synapses. SIGNIFICANCE STATEMENT: DBA/2J and C57BL/6J mice have been used to understand the genetic mechanisms controlling behaviors related to a number of psychiatric illnesses. However, the fundamental neurobiological mechanisms producing these behavioral characteristics remain unresolved. Here we identify a critical family of presynaptic proteins differentially expressed by these strains that control strain-dependent synaptic physiology. This family of proteins regulates excitation/secretion coupling, vesicle recycling, and short-term plasticity throughout the CNS. Thus, differential inheritance of proteins like Munc13-2 has broad implications for genetic control over a wide variety of pathological behaviors. Importantly, these proteins also contain a large number of modulatory sites, making them attractive potential targets for the development of novel neuropharmaceutical treatments.

Supersensitive Kappa Opioid Receptors Promotes Ethanol Withdrawal-Related Behaviors and Reduce Dopamine Signaling in the Nucleus Accumbens.

BACKGROUND: Chronic ethanol exposure reduces dopamine transmission in the nucleus accumbens, which may contribute to the negative affective symptoms associated with ethanol withdrawal. Kappa opioid receptors have been implicated in withdrawal-induced excessive drinking and anxiety-like behaviors and are known to inhibit dopamine release in the nucleus accumbens. The effects of chronic ethanol exposure on kappa opioid receptor-mediated changes in dopamine transmission at the level of the dopamine terminal and withdrawal-related behaviors were examined. METHODS: Five weeks of chronic intermittent ethanol exposure in male C57BL/6 mice were used to examine the role of kappa opioid receptors in chronic ethanol-induced increases in ethanol intake and marble burying, a measure of anxiety/compulsive-like behavior. Drinking and marble burying were evaluated before and after chronic intermittent ethanol exposure, with and without kappa opioid receptor blockade by nor-binaltorphimine (10mg/kg i.p.). Functional alterations in kappa opioid receptors were assessed using fast scan cyclic voltammetry in brain slices containing the nucleus accumbens. RESULTS: Chronic intermittent ethanol-exposed mice showed increased ethanol drinking and marble burying compared with controls, which was attenuated with kappa opioid receptor blockade. Chronic intermittent ethanol-induced increases in behavior were replicated with kappa opioid receptor activation in naïve mice. Fast scan cyclic voltammetry revealed that chronic intermittent ethanol reduced accumbal dopamine release and increased uptake rates, promoting a hypodopaminergic state of this region. Kappa opioid receptor activation with U50,488H concentration-dependently decreased dopamine release in both groups; however, this effect was greater in chronic intermittent ethanol-treated mice, indicating kappa opioid receptor supersensitivity in this group. CONCLUSIONS: These data suggest that the chronic intermittent ethanol-induced increase in ethanol intake and anxiety/compulsive-like behaviors may be driven by greater kappa opioid receptor sensitivity and a hypodopaminergic state of the nucleus accumbens.

Chronic social isolation during adolescence augments catecholamine response to acute ethanol in the basolateral amygdala.

Adolescent social isolation (SI) results in numerous behavioral alterations associated with increased risk of alcoholism. Notably, many of these changes involve the basolateral amygdala (BLA), including increased alcohol seeking. The BLA sends a strong glutamatergic projection to the nucleus accumbens and activation of this pathway potentiates reward-seeking behavior. Dopamine (DA) and norepinephrine (NE) exert powerful excitatory and inhibitory effects on BLA activity and chronic stress can disrupt the excitation-inhibition balance maintained by these catecholamines. Notably, the impact of SI on BLA DA and NE neurotransmission is unknown. Thus the aim of this study was to characterize SI-mediated catecholamine alterations in the BLA. Male Long Evans rats were housed in groups of four (GH) or in SI for 6 weeks during adolescence. DA and NE transporter levels were then measured using Western blot hybridization and baseline and ethanol-stimulated DA and NE levels were quantified using microdialysis. DA transporter levels were increased and baseline DA levels were decreased in SI compared to GH rats. SI also increased DA responses to an acute ethanol (2 g kg(-1)) challenge. While no group differences were noted in NE transporter or baseline NE levels, acute ethanol (2 g kg(-1)) only significantly increased NE levels in SI animals. Collectively, these SI-dependent changes in BLA catecholamine signaling may lead to an increase in BLA excitability and a strengthening of the glutamatergic projection between the BLA and NAc. Such changes may promote the elevated ethanol drinking behavior observed in rats subjected to chronic adolescent stress.

Locomotor sensitization to ethanol impairs NMDA receptor-dependent synaptic plasticity in the nucleus accumbens and increases ethanol self-administration.

Although alcoholism is a worldwide problem resulting in millions of deaths, only a small percentage of alcohol users become addicted. The specific neural substrates responsible for individual differences in vulnerability to alcohol addiction are not known. In this study, we used rodent models to study behavioral and synaptic correlates related to individual differences in the development of ethanol locomotor sensitization, a form of drug-dependent behavioral plasticity associated with addiction vulnerability. Male Swiss Webster mice were treated daily with saline or 1.8 g/kg ethanol for 21 d. Locomotor activity tests were performed once a week for 15 min immediately after saline or ethanol injections. After at least 11 d of withdrawal, cohorts of saline- or ethanol-treated mice were used to characterize the relationships between locomotor sensitization, ethanol drinking, and glutamatergic synaptic transmission in the nucleus accumbens. Ethanol-treated mice that expressed locomotor sensitization to ethanol drank significantly more ethanol than saline-treated subjects and ethanol-treated animals resilient to this form of behavioral plasticity. Moreover, ethanol-sensitized mice also had reduced accumbal NMDA receptor function and expression, as well as deficits in NMDA receptor-dependent long-term depression in the nucleus accumbens core after a protracted withdrawal. These findings suggest that disruption of accumbal core NMDA receptor-dependent plasticity may represent a synaptic correlate associated with ethanol-induced locomotor sensitization and increased propensity to consume ethanol.

Social isolation rearing increases nucleus accumbens dopamine and norepinephrine responses to acute ethanol in adulthood.

BACKGROUND: Early-life stress is associated with increased vulnerability to alcohol addiction. However, the neural substrates linking chronic childhood/adolescent stress and increased risk of alcohol addiction are not well understood. In the nucleus accumbens (NAc), dopamine (DA) and norepinephrine (NE) signaling can be profoundly influenced by stress, anxiety, and drugs of abuse, including ethanol (EtOH). Here, we employed a rodent model of early-life stress that results in enduring increases in behavioral risk factors of alcoholism to gain a better understanding of how chronic adolescent stress may impact the EtOH sensitivity of DA and NE release in the NAc. METHODS: Male Long-Evans rats were either group housed (GH; 4 rats/cage) or socially isolated (SI; 1 rat/cage) for 6 weeks beginning on postnatal day 28. SI and GH rats were tested in adulthood for anxiety-like behaviors (elevated plus maze), and the effects of EtOH (1 and 2 g/kg; intraperitoneally.) on NAc DA and NE were assessed by microdialysis. RESULTS: SI animals showed increased anxiety-like behavior compared to GH animals. Although SI had no effect on baseline levels of DA or NE, baseline DA levels were positively correlated with anxiety measures. In addition, while no significant differences were observed with 1 g/kg EtOH, the 2 g/kg dose induced significantly greater DA release in SI animals. Moreover, EtOH (2 g/kg) only elevated NAc NE levels in SI rats. CONCLUSIONS: These results suggest that chronic early-life stress sensitizes accumbal DA and NE release in response to an acute EtOH challenge. A greater EtOH sensitivity of DA and NE release dynamics in the NAc may contribute to increases in behavioral risk factors of alcoholism, like greater EtOH self-administration, that are observed in SI rats.

Adolescent rearing conditions influence the relationship between initial anxiety-like behavior and ethanol drinking in male Long Evans rats.

BACKGROUND: Rodent studies have demonstrated that adolescent social isolation results in many behavioral perturbations, including increases in anxiety-like behaviors. Socially isolated (SI) rats have also been shown to self-administer greater amounts ethanol (EtOH) in some, but not all, studies. Here, we tested whether juvenile social isolation increases EtOH drinking using an intermittent procedure that engenders relatively high intake in normally reared animals. We also compared the behavioral phenotype of rats reared under social isolation or group-housed conditions with adult rats housed under conditions commonly used in EtOH-drinking studies. METHODS: Male Long Evans rats were procured immediately postweaning and were group housed for 1 week. Subjects were then randomly divided into 2 groups: SI rats, housed individually for 6 weeks and group-housed (GH) rats (4/cage). A third group was procured as young adults and was housed individually upon arrival for 1 week (standard housing condition). Rats were then tested in a plus-maze and novelty assay, and then, all subjects were singly housed and EtOH drinking was assessed. RESULTS: SI rats displayed increased anxiety-like behaviors on the plus-maze, a greater locomotor response to a novel environment, and increased EtOH intake, relative to GH rats. Age-matched standard housed (STD) rats exhibited an anxiety-like behavioral profile on the plus-maze that was similar to SI, and not GH rats, and also drank EtOH at levels comparable with SI subjects. In addition, anxiety-like behavior on the plus-maze correlated with intermittent EtOH intake in SI and GH rats. CONCLUSIONS: These data further support the validity of the rodent juvenile social isolation model for studies directed at elucidating behavioral and neurobiological mechanisms linking anxiety and EtOH drinking. These findings further suggest that housing conditions commonly employed in rodent drinking studies may recapitulate the anxiety-like and EtOH-drinking phenotype engendered by a juvenile social isolation procedure.

Using monosodium glutamate to initiate ethanol self-administration in inbred mouse strains.

Voluntary oral ethanol consumption in rodents is generally limited by strong taste-aversion in these species. Historically, this has been overcome by combining ethanol with a sweetener, typically sucrose or saccharine, and then slowly 'fading' away the sweetener. While useful in most instances, this approach has not proven as successful for some inbred strains of mice (e.g. DBA/2J) despite consistent evidence in the literature that these same strains express strong conditioned place preference for intraperitoneal- or intragastric-administered ethanol. Importantly, DBA/2J mice express a polymorphism in a 'sweet' taste receptor subunit gene that reduces the potency of sweet substances in these mice. We hypothesized that the presence of this polymorphism might help explain the contrasting behavioral findings of weak voluntary oral ethanol consumption following sucrose-fade yet robust conditioned place preference for ethanol in this strain. To test this, we compared ethanol consumption initiated by either a 'traditional' sucrose-fade or a fade from an alternative tastant, monosodium glutamate (MSG). We found that in both C57BL/6J and DBA/2J mice, the MSG-fade produced robust increases in home cage ethanol consumption relative to the traditional sucrose-fade. This increased ethanol intake following MSG-fade was evident across a range of ethanol concentrations. Our findings suggest the potential utility of the MSG-fade to establish stable voluntary oral ethanol consumption in mice, particularly ethanol 'non-preferring' strains such as DBA/2J and lend additional support to the notion that ethanol consumption in DBA/2J mice is limited by pronounced taste aversion.

Chronic Alcohol Dysregulates Glutamatergic Function in the Basolateral Amygdala in a Projection-and Sex-Specific Manner.

Chronic intermittent ethanol and withdrawal (CIE/WD) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections to the nucleus accumbens (NAC) and bed nucleus of the stria terminalis (BNST), respectively. Previous studies revealed that CIE/WD induces input- and sex-specific adaptations to glutamatergic function in the BLA. The BLA receives information from two distinct input pathways. Glutamatergic afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE/WD increases presynaptic glutamatergic function at stria terminalis synapses and postsynaptic function at external capsule synapses. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative "reward" and "aversion" BLA neurons, and showed that CIE/WD alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE/WD increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE/WD increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE/WD enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE/WD-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.

Structural, functional, and behavioral significance of sex and gonadal hormones in the basolateral amygdala: A review of preclinical literature.

The basolateral amygdala (BLA) is intimately involved in the development of neuropsychiatric disorders such as anxiety and alcohol use disorder (AUD). These disorders have clear sex biases, with women more likely to develop an anxiety disorder and men more likely to develop AUD. Preclinical models have largely confirmed these sex-specific vulnerabilities and emphasize the effects of sex hormones on behaviors influenced by the BLA. This review will discuss sex differences in BLA-related behaviors and highlight potential mechanisms mediated by altered BLA structure and function, including the composition of GABAergic interneuron subpopulations, glutamatergic pyramidal neuron morphology, glutamate/GABA neurotransmission, and neuromodulators. Further, sex hormones differentially organize dimorphic circuits during sensitive developmental periods (organizational effects) and initiate more transient effects throughout adulthood (activational effects). Current literature indicates that estradiol and allopregnanolone, a neuroactive progestogen, generally reduce BLA-related behaviors through a variety of mechanisms, including activation of estrogen receptors or facilitation of GABAA-mediated inhibition, respectively. This enhanced GABAergic inhibition may protect BLA pyramidal neurons from the excitability associated with anxiety and alcohol withdrawal. Understanding sex differences and the effects of sex hormones on BLA structure and function may help explain sex-specific vulnerabilities in BLA-related behaviors and ultimately improve treatments for anxiety and AUD.

Chronic Intermittent Ethanol Exposure Dysregulates Nucleus Basalis Magnocellularis Afferents in the Basolateral Amygdala.

Nucleus basalis magnocellularis (NBM) cholinergic projections to the basolateral amygdala (BLA) regulate the acquisition and consolidation of fear-like and anxiety-like behaviors. However, it is unclear whether the alterations in the NBM-BLA circuit promote negative affect during ethanol withdrawal (WD). Therefore, we performed ex vivo whole-cell patch-clamp electrophysiology in both the NBM and the BLA of male Sprague Dawley rats following 10 d of chronic intermittent ethanol (CIE) exposure and 24 h of WD. We found that CIE exposure and withdrawal enhanced the neuronal excitability of NBM putative "cholinergic" neurons. We subsequently used optogenetics to directly manipulate NBM terminal activity within the BLA and measure cholinergic modulation of glutamatergic afferents and BLA pyramidal neurons. Our findings indicate that CIE and withdrawal upregulate NBM cholinergic facilitation of glutamate release via activation of presynaptic nicotinic acetylcholine receptors (AChRs). Ethanol withdrawal-induced increases in NBM terminal activity also enhance BLA pyramidal neuron firing. Collectively, our results provide a novel characterization of the NBM-BLA circuit and suggest that CIE-dependent modifications to NBM afferents enhance BLA pyramidal neuron activity during ethanol withdrawal.

Chronic ethanol and withdrawal differentially modulate lateral/basolateral amygdala paracapsular and local GABAergic synapses.

Withdrawal-related anxiety is cited as a major contributor to relapse in recovering alcoholics. Changes in lateral/basolateral amygdala (BLA) neurotransmission could directly influence anxiety-like behaviors after chronic ethanol exposure and withdrawal. We have shown that these treatments enhance BLA glutamatergic function and neurotransmission. However, the BLA GABAergic system tightly controls the expression of anxiety-like behavior, and additional neuroadaptations in this system are potentially important as well. The intrinsic BLA GABAergic system consists of at least two populations of interneurons: local feed-back interneurons scattered throughout the region and feed-forward interneurons concentrated within groups found in the lateral/paracapsular region of the BLA. In the present study, we found that withdrawal from chronic ethanol robustly decreased presynaptic function at feed-forward GABA synapses but did not alter neurotransmitter release from local interneurons. Differential presynaptic changes at these synapses were complemented by decreased zolpidem sensitivity at feed-forward synapses and decreased midazolam sensitivity at local synapses. Consistent with this, chronic ethanol/withdrawal decreased expression of GABA α1-subunit total protein and increased surface expression of α4-subunit protein. We also found transient increases in GABA-receptor-associated protein levels and persistent increases in γ2-subunit and gephyrin proteins that would suggest alterations in GABA(A) receptor trafficking that might help regulate changes in α4-subunit localization. These data together suggest that chronic ethanol and withdrawal differentially modulate local and lateral paracapsular cell GABAergic synapses via distinct presynaptic and postsynaptic mechanisms. These findings extend our understanding of the neurobiological mechanisms governing changes in anxiety-like behavior after chronic ethanol exposure and withdrawal.

Ethanol modulation of cortico-basolateral amygdala circuits: Neurophysiology and behavior.

This review highlights literature relating the anatomy, physiology, and behavioral contributions by projections between rodent prefrontal cortical areas and the basolateral amygdala. These projections are robustly modulated by both environmental experience and exposure to drugs of abuse including ethanol. Recent literature relating optogenetic and chemogenetic dissection of these circuits within behavior both compliments and occasionally challenges roles defined by more traditional pharmacological or lesion-based approaches. In particular, cortico-amygdala circuits help control both aversive and reward-seeking. Exposure to pathology-producing environments or abused drugs dysregulates the relative 'balance' of these outcomes. Modern circuit-based approaches have also shown that overlapping populations of neurons within a given brain region frequently govern both aversion and reward-seeking. In addition, these circuits often dramatically influence 'local' cortical or basolateral amygdala excitatory or inhibitory circuits. Our understanding of these neurobiological processes, particularly in relation to ethanol research, has just begun and represents a significant opportunity. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Chronic Ethanol Exposure Potentiates Cholinergic Neurotransmission in the Basolateral Amygdala.

Chronic intermittent ethanol (CIE) exposure dysregulates glutamatergic and GABAergic neurotransmission, facilitating basolateral amygdala (BLA) pyramidal neuron hyperexcitability and the expression of anxiety during withdrawal. It is unknown whether ethanol-induced alterations in nucleus basalis magnocellularis (NBM) cholinergic projections to the BLA mediate anxiety-related behaviors through direct modulation of GABA and glutamate afferents. Following 10 days of CIE exposure and 24 h of withdrawal, we recorded GABAergic and glutamatergic synaptic responses in BLA pyramidal neurons with electrophysiology, assessed total protein expression of cholinergic markers, and quantified acetylcholine and choline concentrations using a colorimetric assay. We measured α7 nicotinic acetylcholine receptor (nAChR) dependent modulation of presynaptic function at distinct inputs in AIR- and CIE-exposed BLA coronal slices as a functional read-out of cholinergic neurotransmission. CIE/withdrawal upregulates the endogenous activity of α7 nAChRs, facilitating release at both GABAergic' local' interneuron and glutamatergic synaptic responses to stria terminalis (ST) stimulation, with no effect at GABAergic lateral paracapsular cells (LPCs). CIE caused a three-fold increase in BLA acetylcholine concentration, with no changes in α7 nAChR or cholinergic marker expression. These data illustrate that α7 nAChR-dependent changes in presynaptic function serve as a proxy for CIE-dependent alterations in synaptic acetylcholine levels. Thus, cholinergic projections appear to mediate CIE-induced alterations at GABA/glutamate inputs.

Withdrawal from chronic ethanol exposure increases postsynaptic glutamate function of insular cortex projections to the rat basolateral amygdala.

A key feature of alcohol use disorder (AUD) is negative affect during withdrawal, which often contributes to relapse and is thought to be caused by altered brain function, especially in circuits that are important mediators of emotional behaviors. Both the agranular insular cortex (AIC) and the basolateral amygdala (BLA) regulate emotions and are sensitive to ethanol-induced changes in synaptic plasticity. The AIC and BLA are reciprocally connected; and the effects of chronic ethanol exposure on this circuit have yet to be explored. Here, we use a combination of optogenetics and electrophysiology to examine the pre- and postsynaptic changes that occur to AIC-BLA synapses following withdrawal from 7- or 10-days of chronic intermittent ethanol (CIE) exposure. While CIE/withdrawal did not alter presynaptic glutamate release probability from AIC inputs, withdrawal from 10, but not 7, days of CIE increased AMPA receptor-mediated postsynaptic function at these synapses. Additionally, NMDA receptor-mediated currents evoked by electrical stimulation of the external capsule, which contains AIC afferents, were also increased during withdrawal. Notably, a single subanesthetic dose of ketamine administered at the onset of withdrawal prevented the withdrawal-induced increases in both AMPAR and NMDAR postsynaptic function. Ketamine also prevented the withdrawal-induced increases in anxiety-like behavior measured using the elevated zero maze. Together, these findings suggest that chronic ethanol exposure increases postsynaptic function within the AIC-BLA circuit and that ketamine can prevent ethanol withdrawal-induced alterations in synaptic plasticity and negative affect.

Chronic Ethanol Differentially Modulates Glutamate Release from Dorsal and Ventral Prefrontal Cortical Inputs onto Rat Basolateral Amygdala Principal Neurons.

The medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) have strong reciprocal connectivity. Projections from the BLA to the mPFC can drive innate, anxiety-related behaviors, but it is unclear whether reciprocal projections from the mPFC to BLA have similar roles. Here, we use optogenetics and chemogenetics to characterize the neurophysiological and behavioral alterations produced by chronic ethanol exposure and withdrawal on dorsal mPFC (dmPFC) and ventral mPFC (vmPFC) medial prefrontal cortical terminals in the BLA. We exposed adult male Sprague Dawley rats to chronic intermittent ethanol (CIE) using vapor chambers, measured anxiety-like behavior on the elevated zero maze, and used electrophysiology to record glutamatergic and GABAergic responses in BLA principal neurons. We found that withdrawal from a 7 d CIE exposure produced opposing effects at dmPFC (increased glutamate release) and vmPFC (decreased glutamate release) terminals in the BLA. Chemogenetic inhibition of dmPFC terminals in the BLA attenuated the increased anxiety-like behavior we observed during withdrawal. These data demonstrate that chronic ethanol exposure and withdrawal strengthen the synaptic connections between the dmPFC and BLA but weakens the vmPFC-BLA pathway. Moreover, facilitation of the dmPFC-BLA pathway during withdrawal contributes to anxiety-like behavior. Given the opposing roles of dmPFC-BLA and vmPFC-BLA pathways in fear conditioning, our results suggest that chronic ethanol exposure simultaneously facilitates circuits involved in the acquisition of and diminishes circuits involved with the extinction of withdrawal-related aversive behaviors.

Early social isolation in male Long-Evans rats alters both appetitive and consummatory behaviors expressed during operant ethanol self-administration.

BACKGROUND: Postweaning social isolation in rats produces profound and long-lasting cognitive and behavioral deficits in adult animals. Importantly, this housing manipulation alters sensitivity to a number of drugs of abuse including ethanol. However, most studies with ethanol have utilized continuous or limited home-cage access to examine interactions between juvenile social experience and drinking. More recently, social isolation was shown to increased ethanol responding in a "dipper" model of self-administration (Deehan et al., 2007). In the current study, we utilize a "sipper" operant self-administration model to distinguish the effects of isolation rearing on ethanol seeking- and drinking-related behaviors. METHODS: Postweaning juvenile male Long-Evans rats were placed into 2 housing groups for 6 weeks: one group consisted of individually housed animals; the second group was housed 4 animals per cage. Following the isolation period, anxiety-like behavior was assessed to confirm the efficacy of the isolation procedure. In some animals, ethanol drinking in the home cage was assessed using a continuous access, 2-bottle choice paradigm. All animals were then individually housed and trained to lever-press for a sipper tube containing either an ethanol solution or a sucrose solution. RESULTS: Postweaning social isolation increased the expression of anxiety-like behavior in the elevated plus maze but not the light-dark box. Ethanol consumption was also increased during continuous home-cage access with the 2-bottle choice paradigm. During operant self-administration, isolation housing increased the response rate and increased ethanol consumption but did not alter responding for or consumption of sucrose. The housing manipulation did not change the total number of lever responses during extinction sessions. Paired-pulse inhibition deficits that are characteristic of juvenile isolation remained intact after prolonged experience with sucrose self-administration. DISCUSSION: The effects of postweaning social isolation on ethanol drinking in the home cage are also manifest during operant self-administration. Importantly, these alterations in adult operant self-administration are ethanol-specific.

Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability.

Chronic peri-adolescent stress in humans increases risk to develop a substance use disorder during adulthood. Rats reared in social isolation during peri-adolescence (aSI; 1 rat/cage) period show greater ethanol and cocaine intake compared to group housed (aGH; 4 rats/cage) rats. In addition, aSI rats have a heightened dopamine response in the nucleus accumbens (NAc) to rewarding and aversive stimuli. Furthermore, single pulse electrical stimulation in slices containing NAc core elicits greater dopamine release in aSI rats. Here, we further investigated dopamine release kinetics and machinery following aSI. Dopamine release, across a wide range of stimulation intensities and frequencies, was significantly greater in aSI rats. Interestingly, subthreshold intensity stimulations also resulted in measurable dopamine release in accumbal slices from aSI but not aGH rats. Extracellular [Ca2+] manipulations revealed augmented calcium sensitivity of dopamine release in aSI rats. The readily releasable pools of dopamine, examined by bath application of Ro-04-1284/000, a vesicular monoamine transporter 2 (VMAT2) inhibitor, were depleted faster in aGH rats. Western blot analysis of release machinery proteins (VMAT2, Synaptogyrin-3, Syntaxin-1, and Munc13-3) showed no difference between the two groups. Tyrosine hydroxylase (TH) protein expression levels, however, were elevated in aSI rats. The greater dopamine release could potentially be explained by higher levels of TH, the rate-limiting step for dopamine synthesis. This augmented responsivity of the dopamine system and heightened dopamine availability post-aSI may lead to an increased risk of addiction vulnerability.