Both rewarding and aversive effects contribute to alcohol consumption. Animals genetically predisposed to be high drinkers show reduced sensitivity to the aversive effects of alcohol, and in some instances, increased sensitivity to alcohol's rewarding effects. The present studies tested the high drinking in the dark (HDID) selected lines, a genetic model of drinking to intoxication, to determine whether intake in these mice was genetically related to sensitivity to alcohol aversion or reward. Male HDID mice from the first and second replicate lines (HDID-1 and HDID-2, respectively) and mice from the heterogeneous progenitor control population (HS/Npt, or HS) were conditioned for a taste aversion to a salt solution using two doses of alcohol, and lithium chloride (LiCl) and saline controls. In separate experiments, male and female HDID-1, HDID-2 and HS mice were conditioned for place preference using alcohol. HDID mice were found to have an attenuated sensitivity to alcohol at a moderate (2 g/kg) dose compared to HS mice, but did not differ on conditioned taste aversion to a high (4 g/kg) dose or LiCl or saline injections. HDID and HS mice showed comparable development of alcohol-induced conditioned place preference. These results indicate that high blood alcohol levels after drinking in the HDID mice is genetically related to attenuated aversion to alcohol, while sensitivity to alcohol reward is not altered in these mice. Thus, HDID mice may find a moderate dose of alcohol to be less aversive than control mice and consequently may drink more because of this reduced aversive sensitivity.

Gene expression studies identified the interleukin-1 receptor type I (IL-1R1) as part of a pathway associated with a genetic predisposition to high alcohol consumption, and lack of the endogenous IL-1 receptor antagonist (IL-1ra) strongly reduced ethanol intake in mice. Here, we compared ethanol-mediated behaviors in mice lacking Il1rn or Il1r1. Deletion of Il1rn (the gene encoding IL-1ra) increases sensitivity to the sedative/hypnotic effects of ethanol and flurazepam and reduces severity of acute ethanol withdrawal. Conversely, deletion of Il1r1 (the gene encoding the IL-1 receptor type I, IL-1R1) reduces sensitivity to the sedative effects of ethanol and flurazepam and increases the severity of acute ethanol withdrawal. The sedative effects of ketamine and pentobarbital were not altered in the knockout (KO) strains. Ethanol intake and preference were not changed in mice lacking Il1r1 in three different tests of ethanol consumption. Recovery from ethanol-induced motor incoordination was only altered in female mice lacking Il1r1. Mice lacking Il1rn (but not Il1r1) showed increased ethanol clearance and decreased ethanol-induced conditioned taste aversion. The increased ethanol- and flurazepam-induced sedation in Il1rn KO mice was decreased by administration of IL-1ra (Kineret), and pre-treatment with Kineret also restored the severity of acute ethanol withdrawal. Ethanol-induced sedation and withdrawal severity were changed in opposite directions in the null mutants, indicating that these responses are likely regulated by IL-1R1 signaling, whereas ethanol intake and preference do not appear to be solely regulated by this pathway.

The IL-1 receptor antagonist (IL-1ra), encoded by the Il1rn gene, is an endogenous antagonist of the IL-1 receptor. Studies of Il1rn knockout (KO) and wild type (WT) mice identified differences in several ethanol-related behaviors, some of which may be mediated by GABAergic transmission in the central nucleus of the amygdala (CeA). In this study we examined phasic (both evoked and spontaneous) and tonic GABAergic transmission in the CeA of Il1rn KO and WT mice and the ethanol sensitivity of these GABAergic synapses. The mean amplitude of baseline evoked GABAA-inhibitory postsynaptic potentials (IPSPs), and the baseline frequency of spontaneous GABAA-inhibitory postsynaptic currents (sIPSCs), but not the frequency of miniature GABAA-IPSCs (mIPSCs), were significantly increased in KO compared to WT mice, indicating enhanced presynaptic action potential-dependent GABA release in the CeA of KO mice. In KO mice, we also found a cell-type specific switch in the ongoing tonic GABAA receptor conductance such that the tonic conductance in low threshold bursting (LTB) neurons is lost and a tonic conductance in late spiking (LS) neurons appears. Notably, the ethanol-induced facilitation of evoked and spontaneous GABA release was lost in most of the CeA neurons from KO compared to WT mice. Ethanol superfusion increased the sIPSC rise and decay times in both KO and WT mice, suggesting ethanol-induced postsynaptic effects. The pretreatment of CeA slices with exogenous IL-1ra (Kineret; 100ng/ml) returned sIPSC frequency in KO mice to the levels found in WT. Importantly, Kineret also restored ethanol-induced potentiation of the sIPSC frequency in the KO mice. These results show that IL-1ra regulates baseline GABAergic transmission in the CeA and is critical for the ethanol effects at these synapses.

The ability to use environmental cues to predict rewarding events is essential to survival. The basolateral amygdala (BLA) plays a central role in such forms of associative learning. Aberrant cue-reward learning is thought to underlie many psychopathologies, including addiction, so understanding the underlying molecular mechanisms can inform strategies for intervention. The transcriptional regulator LIM-only 4 (LMO4) is highly expressed in pyramidal neurons of the BLA, where it plays an important role in fear learning. Because the BLA also contributes to cue-reward learning, we investigated the role of BLA LMO4 in this process using Lmo4-deficient mice and RNA interference. Lmo4-deficient mice showed a selective deficit in conditioned reinforcement. Knockdown of LMO4 in the BLA, but not in the nucleus accumbens, recapitulated this deficit in wild-type mice. Molecular and electrophysiological studies identified a deficit in dopamine D2 receptor signaling in the BLA of Lmo4-deficient mice. These results reveal a novel, LMO4-dependent transcriptional program within the BLA that is essential to cue-reward learning.

A quantitative genetic approach, which involves correlation of transcriptional networks with the phenotype in a recombinant inbred (RI) population and in selectively bred lines of rats, and determination of coinciding quantitative trait loci for gene expression and the trait of interest, has been applied in the present study. In this analysis, a novel approach was used that combined DNA-Seq data, data from brain exon array analysis of HXB/BXH RI rat strains and six pairs of rat lines selectively bred for high and low alcohol preference, and RNA-Seq data (including rat brain transcriptome reconstruction) to quantify transcript expression levels, generate co-expression modules and identify biological functions that contribute to the predisposition of consuming varying amounts of alcohol. A gene co-expression module was identified in the RI rat strains that contained both annotated and unannotated transcripts expressed in the brain, and was associated with alcohol consumption in the RI panel. This module was found to be enriched with differentially expressed genes from the selected lines of rats. The candidate genes within the module and differentially expressed genes between high and low drinking selected lines were associated with glia (microglia and astrocytes) and could be categorized as being related to immune function, energy metabolism and calcium homeostasis, as well as glial-neuronal communication. The results of the present study show that there are multiple combinations of genetic factors that can produce the same phenotypic outcome. Although no single gene accounts for predisposition to a particular level of alcohol consumption in every animal model, coordinated differential expression of subsets of genes in the identified pathways produce similar phenotypic outcomes. DATABASE: The datasets supporting the results of the present study are available at http://phenogen.ucdenver.edu.

BACKGROUND: The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. RESULTS: We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with \textasciitilde65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of \textasciitilde3000 and \textasciitilde500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. CONCLUSIONS: Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding the utilization of the CC populations in the study of complex traits. This report is also the first to establish guidelines in defining and identifying strain-specific splicing across different mouse strains.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by β-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ-secretase.

Chronic alcohol consumption changes gene expression, likely causing persistent remodeling of synaptic structures via altered translation of mRNAs within synaptic compartments of the cell. We profiled the transcriptome from synaptoneurosomes (SNs) and paired total homogenates (THs) from mouse amygdala following chronic voluntary alcohol consumption. In SN, both the number of alcohol-responsive mRNAs and the magnitude of fold-change were greater than in THs, including many GABA-related mRNAs upregulated in SNs. Furthermore, SN gene co-expression analysis revealed a highly connected network, demonstrating coordinated patterns of gene expression and highlighting alcohol-responsive biological pathways, such as long-term potentiation, long-term depression, glutamate signaling, RNA processing and upregulation of alcohol-responsive genes within neuroimmune modules. Alterations in these pathways have also been observed in the amygdala of human alcoholics. SNs offer an ideal model for detecting intricate networks of coordinated synaptic gene expression and may provide a unique system for investigating therapeutic targets for the treatment of alcoholism.

Alcoholism is a serious public health concern that is characterized by the development of tolerance to alcohol's effects, increased consumption, loss of control over drinking and the development of physical dependence. This cycle is often times punctuated by periods of abstinence, craving and relapse. The development of tolerance and the expression of withdrawal effects, which manifest as dependence, have been to a great extent attributed to neuroadaptations within the mesocorticolimbic and extended amygdala systems. Alcohol affects various neurotransmitter systems in the brain including the adrenergic, cholinergic, dopaminergic, GABAergic, glutamatergic, peptidergic, and serotonergic systems. Due to the myriad of neurotransmitter and neuromodulator systems affected by alcohol, the efficacies of current pharmacotherapies targeting alcohol dependence are limited. Importantly, research findings of changes in glutamatergic neurotransmission induced by alcohol self- or experimenter-administration have resulted in a focus on therapies targeting glutamatergic receptors and normalization of glutamatergic neurotransmission. Glutamatergic receptors implicated in the effects of ethanol include the ionotropic glutamate receptors (AMPA, Kainate, and NMDA) and some metabotropic glutamate receptors. Regarding glutamatergic homeostasis, ceftriaxone, MS-153, and GPI-1046, which upregulate glutamate transporter 1 (GLT1) expression in mesocorticolimbic brain regions, reduce alcohol intake in genetic animal models of alcoholism. Given the hyperglutamatergic/hyperexcitable state of the central nervous system induced by chronic alcohol abuse and withdrawal, the evidence thus far indicates that a restoration of glutamatergic concentrations and activity within the mesocorticolimbic system and extended amygdala as well as multiple memory systems holds great promise for the treatment of alcohol dependence.

Alcohol dependence is a heterogeneous psychiatric disorder characterized by high genetic heritability and neuroadaptations occurring from repeated drug exposure. Through an integrated systems approach we observed consistent differences in transcriptome organization within postmortem human brain tissue associated with the lifetime consumption of alcohol. Molecular networks, determined using high-throughput RNA sequencing, for drinking behavior were dominated by neurophysiological targets and signaling mechanisms of alcohol. The systematic structure of gene sets demonstrates a novel alliance of multiple ion channels, and related processes, underlying lifetime alcohol consumption. Coordinate expression of these transcripts was enriched for genome-wide association signals in alcohol dependence and a meta-analysis of alcohol self-administration in mice. Further dissection of genes within alcohol consumption networks revealed the potential interaction of alternatively spliced transcripts. For example, expression of a human-specific isoform of the voltage-gated sodium channel subunit SCN4B was significantly correlated to lifetime alcohol consumption. Overall, our work demonstrates novel convergent evidence for biological networks related to excessive alcohol consumption, which may prove fundamentally important in the development of pharmacotherapies for alcohol dependence.

The endogenous opioid system is expressed throughout the brain reinforcement circuitry, and plays a major role in reward processing, mood control and the development of addiction. This neuromodulator system is composed of three receptors, mu, delta and kappa, interacting with a family of opioid peptides derived from POMC (β-endorphin), preproenkephalin (pEnk) and preprodynorphin (pDyn) precursors. Knockout mice targeting each gene of the opioid system have been created almost two decades ago. Extending classical pharmacology, these mutant mice represent unique tools to tease apart the specific role of each opioid receptor and peptide in vivo, and a powerful approach to understand how the opioid system modulates behavioral effects of drugs of abuse. The present review summarizes these studies, with a focus on major drugs of abuse including morphine/heroin, cannabinoids, psychostimulants, nicotine or alcohol. Genetic data, altogether, set the mu receptor as the primary target for morphine and heroin. In addition, this receptor is essential to mediate rewarding properties of non-opioid drugs of abuse, with a demonstrated implication of β-endorphin for cocaine and nicotine. Delta receptor activity reduces levels of anxiety and depressive-like behaviors, and facilitates morphine-context association. PEnk is involved in these processes and delta/pEnk signaling likely regulates alcohol intake. The kappa receptor mainly interacts with pDyn peptides to limit drug reward, and mediate dysphoric effects of cannabinoids and nicotine. Kappa/dynorphin activity also increases sensitivity to cocaine reward under stressful conditions. The opioid system remains a prime candidate to develop successful therapies in addicted individuals, and understanding opioid-mediated processes at systems level, through emerging genetic and imaging technologies, represents the next challenging goal and a promising avenue in addiction research.

Alcohol and drug abuse take a large toll on society and affected individuals. However, very few effective treatments are currently available to treat alcohol and drug addiction. Basic and clinical research has begun to provide some insights into the underlying neurobiological systems involved in the addiction process. Several neurotransmitter pathways have been implicated and distinct reward neurocircuitry have been proposed-including the mesocorticolimbic dopamine (MCL-DA) system and the extended amygdala. The serotonin (5-HT) neurotransmitter system is of particular interest and multiple 5-HT receptors are thought to play significant roles in alcohol and drug self-administration and the development of drug dependence. Among the 5-HT receptors, the 5-HT7 receptor is currently undergoing characterization as a potential target for the treatment of several psychiatric disorders. Although this receptor has received only limited research regarding addictive behaviors, aspects of its neuroanatomical, biochemical, physiological, pharmacological, and behavioral profiles suggest that it could play a key role in the addiction process. For instance, genomic studies in humans have suggested a link between variants in the gene encoding the 5-HT7 receptor and alcoholism. Recent behavioral testing using high-affinity antagonists in mice and preliminary tests with alcohol-preferring rats suggest that this receptor could mediate alcohol consumption and/or reinforcement and play a role in seeking/craving behavior. Interest in the development of new and more selective pharmacological agents for this receptor will aid in examining the 5-HT7 receptor as a novel target for treating addiction.

Alcohol dependence is a complex condition with clear genetic factors. Some of the leading candidate genes code for subunits of the inhibitory GABAA and glycine receptors. These and related ion channels are also targets for the acute actions of alcohol, and there is considerable progress in understanding interactions of alcohol with these proteins at the molecular and even atomic levels. X-ray structures of open and closed states of ion channels combined with structural modeling and site-directed mutagenesis have elucidated direct actions of alcohol. Alcohol also alters channel function by translational and post-translational mechanisms, including phosphorylation and protein trafficking. Construction of mutant mice with either deletion of key proteins or introduction of alcohol-resistant channels has further linked specific proteins with discrete behavioral effects of alcohol. A combination of approaches, including genome wide association studies in humans, continues to advance the molecular basis of alcohol action on receptor structure and function.

BACKGROUND: Two features of alcohol addiction that have been widely studied in animal models are relapse drinking following periods of alcohol abstinence and the escalation of alcohol consumption after chronic continuous or intermittent alcohol exposure. The genetic contribution to these phenotypes has not been systematically investigated. METHODS: HXB/BXH recombinant inbred (RI) rat strains were given access to alcohol sequentially as follows: alcohol (10%) as the only fluid for 1 week; alcohol (10%) and water in a 2-bottle choice paradigm for 7 weeks ("pre-alcohol deprivation effect [ADE] alcohol consumption"); 2 weeks of access to water only (alcohol deprivation); and 2 weeks of reaccess to 10% alcohol and water ("post-ADE alcohol consumption"). The periods of deprivation and reaccess to alcohol were repeated 3 times. The ADE was defined as the amount of alcohol consumed in the first 24 hours after deprivation minus the average daily amount of alcohol consumed in the week prior to deprivation. Heritability of the phenotypes was determined by analysis of variance, and quantitative trait loci (QTLs) were identified. RESULTS: All strains showed increased alcohol consumption, compared to the predeprivation period, in the first 24 hours after each deprivation (ADE). Broad-sense heritability of the ADEs was low (ADE1, 9.1%; ADE2, 26.2%; ADE3, 16.3%). Alcohol consumption levels were relatively stable over weeks 2 to 7. Post-ADE alcohol consumption levels consistently increased in some strains and were decreased or unchanged in others. Heritability of pre- and post-ADE alcohol consumption was high and increased over time (week 2, 38.5%; week 7, 51.1%; week 11, 56.8%; week 15, 63.3%). QTLs for pre- and post-ADE alcohol consumption were similar, but the strength of the QTL association with the phenotype decreased over time. CONCLUSIONS: In the HXB/BXH RI rat strains, genotypic variance does not account for a large proportion of phenotypic variance in the ADE phenotype (low heritability), suggesting a role of environmental factors. In contrast, a large proportion of the variance across the RI strains in pre- and post-ADE alcohol consumption is due to genetically determined variance (high heritability).

BACKGROUND: Chronic alcohol exposure can change splice variant expression. The gamma-aminobutyric acid type B (GABAB) receptor undergoes splicing and is an alcoholism treatment target, but there is little information about splicing changes in this receptor in alcoholics. We studied GABAB receptor subunit 1 (GABAB1) splicing in alcoholic postmortem brains. METHODS: To maximize GABAB1 splice junction identification, we combined gene specific libraries with RNA-seq. Splice junctions and mapped reads were also found from intronic and intergenic regions. We compared GABAB1 splice junctions in prefrontal cortices from 14 alcoholic and 15 control subjects and introduced new strategies, reads per kilobase of splice junction model per million mapped reads and reads per kilobase of gene model per million mapped reads, for quantitating splice junction and gene expression. RESULTS: Novel splice junction detection indicated that the GABAB1 gene is at least two times longer than the previously reported gene length. GABAB1 exon and intron expression data showed low expression at the 5' end exons and exon grouping. This indicated that there are short splicing variants in addition to GABAB receptor subunit GABAB1a, the longest known major transcript. We found that chronic alcohol altered exon/intron expression and splice junction levels. Decreased expression of the gamma-aminobutyric acid binding site, a transmembrane domain and a microRNA binding site may decrease normal GABAB1 transcript population and thereby decrease normal signal transduction in alcoholics. CONCLUSIONS: We discovered novel, complex splicing of GABAB1 in human brain and showed that chronic alcohol produces additional splicing complexity.

{Thiamine (vitamin B1) deficiency, associated with a variety of conditions, including chronic alcoholism and bariatric surgery for morbid obesity, can result in the neurological disorder Wernicke's encephalopathy (WE). Recent work building upon early observations in animal models of thiamine deficiency has demonstrated an inflammatory component to the neuropathology observed in thiamine deficiency. The present, multilevel study including in vivo magnetic resonance imaging (MRI) and spectroscopy (MRS) and postmortem quantification of chemokine and cytokine proteins sought to determine whether a combination of these in vivo neuroimaging tools could be used to characterize an in vivo MR signature for neuroinflammation. Thiamine deficiency for 12days was used to model neuroinflammation; glucose loading in thiamine deficiency was used to accelerate neurodegeneration. Among 38 animals with regional brain tissue assayed postmortem for cytokine/chemokine protein levels, three groups of rats (controls+glucose

BACKGROUND: Use of in silico bioinformatics analyses has led to important leads in the complex nature of alcoholism at the genomic, epigenomic, and proteomic level, but has not previously been successfully translated to the development of effective pharmacotherapies. In this study, a bioinformatics approach led to the discovery of neuroimmune pathways as an age-specific druggable target. Minocycline, a neuroimmune modulator, reduced high ethanol (EtOH) drinking in adult, but not adolescent, mice as predicted a priori. METHODS: Age and sex-divergent effects in alcohol consumption were quantified in FVB/NJ × C57BL/6J F1 mice given access to 20% alcohol using a 4 h/d, 4-day drinking-in-dark (DID) paradigm. In silico bioinformatics pathway overrepresentation analysis for age-specific effects of alcohol in brain was performed using gene expression data collected in control and DID-treated, adolescent and adult, male mice. Minocycline (50 mg/kg i.p., once daily) or saline alone was tested for an effect on EtOH intake in the F1 and C57BL/6J (B6) mice across both age and gender groups. Effects of minocycline on the pharmacokinetic properties of alcohol were evaluated by comparing the rates of EtOH elimination between the saline- and minocycline-treated F1 and B6 mice. RESULTS: Age and gender differences in DID consumption were identified. Only males showed a clear developmental increase difference in drinking over time. In silico analyses revealed neuroimmune-related pathways as significantly overrepresented in adult, but not in adolescent, male mice. As predicted, minocycline treatment reduced drinking in adult, but not adolescent, mice. The age effect was present for both genders, and in both the F1 and B6 mice. Minocycline had no effect on the pharmacokinetic elimination of EtOH. CONCLUSIONS: Our results are a proof of concept that bioinformatics analysis of brain gene expression can lead to the generation of new hypotheses and a positive translational outcome for individualized pharmacotherapeutic treatment of high alcohol consumption.

Chronic exposure to ethanol produces a number of detrimental effects on behavior. Neuroadaptive changes in brain structure or function underlie these behavioral effects and may be transient or persistent in nature. Central to the functional changes are alterations in the biology of neuronal and glial cells of the brain. Recent data show that ethanol induces glial cells of the brain to produce elevated levels of neuroimmune factors including CCL2, a key innate immune chemokine. Depending on the conditions of ethanol exposure, the upregulated levels of CCL2 can be transient or persistent and outlast the period of ethanol exposure. Importantly, results indicate that the upregulated levels of CCL2 may lead to CCL2-ethanol interactions that mediate or regulate the effects of ethanol on the brain. Glial cells are in close association with neurons and regulate many neuronal functions. Therefore, effects of ethanol on glial cells may underlie some of the effects of ethanol on neurons. To investigate this possibility, we are studying effects of chronic ethanol on hippocampal synaptic function in a transgenic mouse model that expresses elevated levels of CCL2 in the brain through enhanced glial expression, a situation know to occur in alcoholics. Both CCL2 and ethanol have been reported to alter synaptic function in the hippocampus. In the current study, we determined if interactions are evident between CCL2 and ethanol at the level of hippocampal synaptic proteins. Two ethanol exposure paradigms were used; the first involved ethanol exposure by drinking and the second involved ethanol exposure in a paradigm that combines drinking plus ethanol vapor. The first paradigm does not produce dependence on ethanol, whereas the second paradigm is commonly used to produce ethanol dependence. Results show modest effects of both ethanol exposure paradigms on the level of synaptic proteins in the hippocampus of CCL2 transgenic mice compared with their non-transgenic littermate controls, consistent with ethanol-CCL2 interactions. No evidence of toxic effects of CCL2 or CCL2-ethanol interactions was observed. Taken together, these results support the idea that ethanol induced astrocyte production of CCL2 can result in neuroadaptive changes that interact with the actions of ethanol.

Synaptic alterations in the nucleus accumbens (NAc) are crucial for the aberrant reward-associated learning that forms the foundation of drug dependence. Altered glutamatergic synaptic plasticity, in particular, is thought to be a vital component of the neurobiological underpinnings of addictive behavior. The development of bacterial artificial chromosome-eGFP (enhanced green fluorescent protein) transgenic mice that express eGFP driven by endogenous D1 dopamine receptor (D1R) promoters has now allowed investigation of the cell type-specific synaptic modifications in the NAc in response to drugs of abuse. In this study, we used whole-cell ex vivo slice electrophysiology in Drd1-eGFP mice to investigate cell type-specific alterations in NAc synaptic plasticity following ethanol exposure. Electrophysiological recordings were made from eGFP-expressing medium spiny neurons (D1+ MSNs) and non-eGFP-expressing (putative D2 receptor-expressing) (D1- MSNs) from the shell subregion of the NAc. We observed low frequency-induced long-term depression (1Hz-LTD) of α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-mediated excitatory postsynaptic currents (EPSCs) solely in D1+ MSNs. However, 24h following four consecutive days of in vivo chronic intermittent ethanol (CIE) vapor exposure, 1-Hz LTD was conversely observed only in D1- MSNs, and now absent in D1+ MSNs. Complete recovery of the baseline plasticity phenotype in both cell types required a full 2 weeks of withdrawal from CIE vapor exposure. Thus, we observed a cell type specificity of synaptic plasticity in the NAc shell, as well as, a gradual recovery of the pre-ethanol exposure plasticity state following extended withdrawal. These changes highlight the adaptability of NAc shell MSNs to the effects of ethanol exposure and may represent critical neuroadaptations underlying the development of ethanol dependence.

The objective of this study was to determine changes in gene expression within the extended amygdala following binge-like alcohol drinking by male adolescent alcohol-preferring (P) rats. Starting at 28 days of age, P rats were given concurrent access to 15 and 30% ethanol for 3 one-h sessions/day for 5 consecutive days/week for 3 weeks. Rats were killed by decapitation 3 h after the first ethanol access session on the 15th day of drinking. RNA was prepared from micropunch samples of the nucleus accumbens shell (Acb-sh) and central nucleus of the amygdala (CeA). Ethanol intakes were 2.5-3.0 g/kg/session. There were 154 and 182 unique named genes that significantly differed (FDR=0.2) between the water and ethanol group in the Acb-sh and CeA, respectively. Gene Ontology (GO) analyses indicated that adolescent binge drinking produced changes in biological processes involved with cell proliferation and regulation of cellular structure in the Acb-sh, and in neuron projection and positive regulation of cellular organization in the CeA. Ingenuity Pathway Analysis indicated that, in the Acb-sh, there were several major intracellular signaling pathways (e.g., cAMP-mediated and protein kinase A signaling pathways) altered by adolescent drinking, with 3-fold more genes up-regulated than down-regulated in the alcohol group. The cAMP-mediated signaling system was also up-regulated in the CeA of the alcohol group. Weighted gene co-expression network analysis indicated significant G-protein coupled receptor signaling and transmembrane receptor protein kinase signaling categories in the Acb-sh and CeA, respectively. Overall, the results of this study indicated that binge-like alcohol drinking by adolescent P rats is differentially altering the expression of genes in the Acb-sh and CeA, some of which are involved in intracellular signaling pathways and may produce changes in neuronal function.