Large conductance calcium-activated potassium (BK) channels play a key role in the control of neuronal activity. Ethanol is a potent activator of BK channel gating, but how this action may impact ethanol drinking still remains poorly understood. Auxiliary β subunits are known to modulate ethanol-induced potentiation of BK currents. In the present study, we investigated whether BK β1 and β4 subunits influence voluntary ethanol consumption using knockout (KO) mice. In a first experiment, mice were first subjected to continuous two-bottle choice (2BC) and were then switched to intermittent 2BC, which progressively increased ethanol intake as previously described in wildtype mice. BK β1 or β4 subunit deficiency did not affect ethanol self-administration under either schedule of access. In a second experiment, mice were first trained to drink ethanol in a limited-access 2BC paradigm. BK β1 or β4 deletion did not affect baseline consumption. Weeks of 2BC were then alternated with weeks of chronic intermittent ethanol (CIE) or air inhalation. As expected, a gradual escalation of ethanol drinking was observed in dependent wildtype mice, while intake remained stable in non-dependent wildtype mice. However, CIE exposure only produced a mild augmentation of ethanol consumption in BK β4 KO mice. Conversely, ethanol drinking increased after fewer CIE cycles in BK β1 KO mice than in wildtype mice. In conclusion, BK β1 or β4 did not influence voluntary ethanol drinking in non-dependent mice, regardless of the pattern of access to ethanol. However, deletion of BK β4 attenuated, while deletion of BK β1 accelerated, the escalation of ethanol drinking during withdrawal from CIE. Our data suggest that BK β1 and β4 subunits have an opposite influence on the negative reinforcing properties of ethanol withdrawal. Modulating the expression, distribution or interactions of BK channel auxiliary subunits may therefore represent a novel avenue for the treatment of alcoholism.

Alcohol use disorders are pervasive in society and their impact affects quality of life, morbidity and mortality, as well as individual productivity. Alcohol has detrimental effects on an individual's physiology and nervous system, and is associated with disorders of many organ and endocrine systems impacting an individual's health, behavior, and ability to interact with others. Youth are particularly affected. Unfortunately, adolescent usage also increases the probability for a progression to dependence. Several areas of research indicate that the deleterious effects of alcohol abuse may be exacerbated by mixing caffeine with alcohol. Some behavioral evidence suggests that caffeine increases alcohol drinking and binge drinking episodes, which in turn can foster the development of alcohol dependence. As a relatively new public health concern, the epidemiological focus has been to establish a need for investigating the effects of caffeinated alcohol. While the trend of co-consuming these substances is growing, knowledge of the central mechanisms associated with caffeinated ethanol has been lacking. Research suggests that caffeine and ethanol can have additive or synergistic pharmacological actions and neuroadaptations, with the adenosine and dopamine systems in particular implicated. However, the limited literature on the central effects of caffeinated ethanol provides an impetus to increase our knowledge of the neuroadaptive effects of this combination and their impact on cognition and behavior. Research from our laboratories indicates that an established rodent animal model of alcoholism can be extended to investigate the acute and chronic effects of caffeinated ethanol.

The objective of this study was to detect changes in gene expression in the ventral tegmental area (VTA) following repeated excessive binge-like ('loss-of-control') alcohol drinking by alcohol-preferring (P) rats. Adult female P rats (n = 7) were given concurrent access to 10, 20, and 30% EtOH for 4 1-h sessions daily for 10 weeks followed by 2 cycles of 2 weeks of abstinence and 2 weeks of EtOH access. Rats were sacrificed by decapitation 3 h after the 4th daily EtOH-access session at the end of the second 2-week relapse period. A water-control group of female P rats (n = 8) was also sacrificed. RNA was prepared from micro-punch samples of the VTA from individual rats; analyses were conducted with Affymetrix Rat 230.2 GeneChips. Ethanol intakes were 1.2-1.7 g/kg per session, resulting in blood levels \textgreater200 mg% at the end of the 4th session. There were 211 unique named genes that significantly differed (FDR = 0.1) between the water and EtOH groups. Bioinformatics analyses indicated alterations in a) transcription factors that reduced excitation-coupled transcription and promoted excitotoxic neuronal damage involving clusters of genes associated with Nfkbia, Fos, and Srebf1, b) genes that reduced cholesterol and fatty acid synthesis, and increased protein degradation, and c) genes involved in cell-to-cell interactions and regulation of the actin cytoskeleton. Among the named genes, there were 62 genes that showed differences between alcohol-naïve P and non-preferring (NP) rats, with 43 of the genes changing toward NP-like expression levels following excessive binge-like drinking in the P rats. These genes are involved in a pro-inflammatory response, and enhanced response to glucocorticoids and steroid hormones. Overall, the results of this study indicate that the repeated excessive binge-like alcohol drinking can change the expression of genes that may alter neuronal function in several ways, some of which may be deleterious.

The continuous two-bottle choice test is the most common measure of alcohol consumption but there is remarkably little information about the development of tolerance or dependence with this procedure. We showed that C57BL/6J × FVB/NJ and FVB/NJ×C57BL/6JF1 hybrid mice demonstrate greater preference for and consumption of alcohol than either parental strain. In order to test the ability of this genetic model of high alcohol consumption to produce neuroadaptation, we examined development of alcohol tolerance and dependence after chronic self-administration using a continuous access two-bottle choice paradigm. Ethanol-experienced mice stably consumed about 16-18 g/kg/day of ethanol. Ethanol-induced withdrawal severity was assessed (after 59 days of drinking) by scoring handling-induced convulsions; withdrawal severity was minimal and did not differ between ethanol-experienced and -naïve mice. After 71 days of drinking, the rate of ethanol clearance was similar for ethanol-experienced and -naïve mice. After 77 days of drinking, ethanol-induced loss of righting reflex (LORR) was tested daily for 5 days. Ethanol-experienced mice had a shorter duration of LORR. For both ethanol-experienced and -naïve mice, blood ethanol concentrations taken at gain of righting reflex were greater on day 5 than on day 1, indicative of tolerance. After 98 days of drinking, ethanol-induced hypothermia was assessed daily for 3 days. Both ethanol-experienced and -naïve mice developed rapid and chronic tolerance to ethanol-induced hypothermia, with significant group differences on the first day of testing. In summary, chronic, high levels of alcohol consumption in F1 hybrid mice produced rapid and chronic tolerance to both the sedative/hypnotic and hypothermic effects of ethanol; additionally, a small degree of metabolic tolerance developed. The development of tolerance supports the validity of using this model of high alcohol consumption in genetic studies of alcoholism.

BACKGROUND: Several recent studies implementing the standard "drinking-in-the-dark" (DID) model of short-term binge-like ethanol (EtOH) intake in C57BL/6J mice highlighted a role for the stress-related neuropeptide corticotropin-releasing factor (CRF) and its primary binding partner, the CRF type-1 (CRF1) receptor. METHODS: We evaluated the selectivity of CRF1 involvement in binge-like EtOH intake by interrupting CRF1 function via pharmacological and genetic methods in a slightly modified 2-bottle choice DID model that allowed calculation of an EtOH preference ratio. In addition to determining EtOH intake and preference, we also measured consumption of food and H2 O during the DID period, both in the presence and absence of EtOH and sweet tastant solutions. RESULTS: Treatment with either of the CRF1-selective antagonists CP-376,395 (CP; 10 to 20 mg/kg, i.p.) or NBI-27914 (10 to 30 mg/kg, i.p.) decreased intake of 15% EtOH in male C57BL/6J mice, but did so in the absence of a concomitant decrease in EtOH preference. These findings were replicated genetically in a CRF1 knockout (KO) mouse model (also on a C57BL/6J background). In contrast to effects on EtOH intake, pharmacological blockade of CRF1 with CP increased intake of 10% sucrose, consistent with previous findings in CRF1 KO mice. Finally, pharmacological and genetic disruption of CRF1 activity significantly reduced feeding and/or total caloric intake in all experiments, confirming the existence of nonspecific effects. CONCLUSIONS: Our findings indicate that blockade of CRF1 receptors does not exert specific effects on EtOH intake in the DID paradigm, and that slight modifications to this procedure, as well as additional consummatory control experiments, may be useful when evaluating the selectivity of pharmacological and genetic manipulations on binge-like EtOH intake.

Evidence that the delta opioid receptor (DOR) is an attractive target for the treatment of brain disorders has strengthened in recent years. This receptor is broadly expressed in the brain, binds endogenous opioid peptides, and shows as functional profile highly distinct from those of mu and kappa opioid receptors. Our knowledge of DOR function has enormously progressed from in vivo studies using pharmacological tools and genetic approaches. The important role of this receptor in reducing chronic pain has been extensively overviewed; therefore this review focuses on facets of delta receptor activity relevant to psychiatric and other neurological disorders. Beneficial effects of DOR agonists are now well established in the context of emotional responses and mood disorders. DOR activation also regulates drug reward, inhibitory controls and learning processes, but whether delta compounds may represent useful drugs in the treatment of drug abuse remains open. Epileptogenic and locomotor-stimulating effects of delta agonists appear drug-dependent, and the possibility of biased agonism at DOR for these effects is worthwhile further investigations to increase benefit/risk ratio of delta therapies. Neuroprotective effects of DOR activity represent a forthcoming research area. Future developments in DOR research will benefit from in-depth investigations of DOR function at cellular and circuit levels.

Increased glutamatergic neurotransmission appears to mediate the reinforcing properties of drugs of abuse, including ethanol (EtOH). We recently reported that the administration of ceftriaxone (CEF), a β-lactam antibiotic known to upregulate glutamate transporter 1 (GLT1) levels/activity, decreased the maintenance of EtOH intake in adult male alcohol-preferring (P) rats. In the present study, we tested whether CEF administration would reduce the acquisition and maintenance of EtOH drinking in adolescent and adult female P rats. The rats were treated with saline or 200mg/kg ceftriaxone for 7 days (starting at 35 or 75 days old, respectively) followed by the EtOH acquisition test. Five weeks later the effects of CEF were examined regarding the maintenance of EtOH intake. For the maintenance test, half of the animals that received CEF during acquisition received CEF for 7 days and the other half received saline for 7 days. Saline-treated acquisition animals were treated similarly. The results indicated that pretreatment with ceftriaxone reduced the maintenance of EtOH intake in both animals that started as adolescents and those that started as adults. However, the beneficial effect of CEF was more pronounced in rats pretreated with CEF as adults compared with rats pretreated as adolescents. Reductions in EtOH intake by ceftriaxone were paralleled by an upregulation of GLT1 protein levels in both the nucleus accumbens (∼25% in rats starting at both ages) and prefrontal cortex (∼50% in rats starting as peri-adolescents and ∼65% in those starting as adults). These findings provide further support for GLT1-associated mechanisms in high alcohol-consuming behavior, and hold promise for the development of effective treatments targeting alcohol abuse and dependence.

Experimental animals offered continuous 24-hour free choice access to ethanol rarely display voluntary ethanol consumption at levels sufficient to induce intoxication or to engender dependence. One of the simplest ways to increase voluntary ethanol intake is to impose temporal limitations on ethanol availability. Escalation of ethanol intake has been observed in both rats and mice under a variety of different schedules of alternating ethanol access and deprivation. Although such effects have been observed in a variety of rat and mouse genotypes, little is known concerning possible genetic correlations between responses to intermittent ethanol access and other ethanol-related phenotypes. In the present study, we examined the effects of intermittent ethanol access in mouse genotypes characterized by divergent responses to ethanol in other domains, including ethanol preference (C57BL/6J and C3H/HeJ mice), binge-like ethanol drinking (High Drinking in the Dark and HS/Npt mice) and ethanol withdrawal severity (Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice). Although intermittent ethanol access resulted in escalated ethanol intake in all tested genotypes, the robustness of the effect varied across genotypes. On the other hand, we saw no evidence that the effects of intermittent access are correlated with either binge-like drinking or withdrawal severity, and only weak evidence for a genetic correlation with baseline ethanol preference. Thus, these different ethanol-related traits appear to depend on largely unique sets of genetic mediators.

Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mouse lines were bidirectionally selectively bred, respectively, to have severe or mild ethanol withdrawal handling-induced convulsions (HICs) after cessation of 3 days of ethanol vapor inhalation. Murine genotypes with severe withdrawal have been found to show low ethanol consumption, and high consumers show low withdrawal. An early drinking study with WSP and WSR mice showed modest evidence consistent with this genetic correlation, but there were several limitations to that experiment. We therefore conducted a thorough assessment of two bottle ethanol preference drinking in both replicate pairs of WSP/WSR selected lines in mice of both sexes. Greater preference drinking of WSR-2 than WSP-2 female mice confirmed the earlier report. However, in the parallel set of selected lines, the WSP-1 mice drank more than the WSR-1s. Naive mice tested for preference for sucrose, saccharin and quinine did not differ markedly for any tastant. Finally, in a test of binge-like drinking, Drinking in the Dark (DID), WSP mice drank more than WSR mice and attained significantly higher (but still modest) blood ethanol concentrations. Tests of acute withdrawal after DID showed a mild, but significant elevation in handling-induced convulsions in the WSP line. These results provide further evidence that 2-bottle ethanol preference and DID are genetically distinguishable traits.

Chronically available alcohol escalates drinking in mice and a single injection of the immune activator lipopolysaccharide can mimic this effect and result in a persistent increase in alcohol consumption. We hypothesized that chronic alcohol drinking and lipopolysaccharide injections will produce some similar molecular changes that play a role in regulation of alcohol intake. We investigated the molecular mechanisms of chronic alcohol consumption or lipopolysaccharide insult by gene expression profiling in prefrontal cortex and liver of C57BL/6J mice. We identified similar patterns of transcriptional changes among four groups of animals, three consuming alcohol (vs water) in different consumption tests and one injected with lipopolysaccharide (vs. vehicle). The three tests of alcohol consumption are the continuous chronic two bottle choice (Chronic), two bottle choice available every other day (Chronic Intermittent) and limited access to one bottle of ethanol (Drinking in the Dark). Gene expression changes were more numerous and marked in liver than in prefrontal cortex for the alcohol treatments and similar in the two tissues for lipopolysaccharide. Many of the changes were unique to each treatment, but there was significant overlap in prefrontal cortex for Chronic-Chronic Intermittent and for Chronic Intermittent-lipopolysaccharide and in liver all pairs showed overlap. In silico cell-type analysis indicated that lipopolysaccharide had strongest effects on brain microglia and liver Kupffer cells. Pathway analysis detected a prefrontal cortex-based dopamine-related (PPP1R1B, DRD1, DRD2, FOSB, PDNY) network that was highly over-represented in the Chronic Intermittent group, with several genes from the network being also regulated in the Chronic and lipopolysaccharide (but not Drinking in the Dark) groups. Liver showed a CYP and GST centered metabolic network shared in part by all four treatments. We demonstrate common consequences of chronic alcohol consumption and immune activation in both liver and brain and show distinct genomic consequences of different types of alcohol consumption.

The objectives of this study were to determine innate differences in gene expression in 2 regions of the extended amygdala between 5 different pairs of lines of male rats selectively bred for high or low ethanol consumption: a) alcohol-preferring (P) vs. alcohol-non-preferring (NP) rats, b) high-alcohol-drinking (HAD) vs. low-alcohol-drinking (LAD) rats (replicate line-pairs 1 and 2), c) ALKO alcohol (AA) vs. nonalcohol (ANA) rats, and d) Sardinian alcohol-preferring (sP) vs. Sardinian alcohol-nonpreferring (sNP) rats, and then to determine if these differences are common across the line-pairs. Microarray analysis revealed up to 1772 unique named genes in the nucleus accumbens shell (AcbSh) and 494 unique named genes in the central nucleus of the amygdala (CeA) that significantly differed [False Discovery Rate (FDR) = 0.10; fold-change at least 1.2] in expression between the individual line-pairs. Analysis using Gene Ontology (GO) and Ingenuity Pathways information indicated significant categories and networks in common for up to 3 or 4 line-pairs, but not for all 5 line-pairs. However, there were almost no individual genes in common within these categories and networks. ANOVAs of the combined data for the 5 line-pairs indicated 1014 and 731 significant (p \textless 0.01) differences in expression of named genes in the AcbSh and CeA, respectively. There were 4-6 individual named genes that significantly differed across up to 3 line-pairs in both regions; only 1 gene (Gsta4 in the CeA) differed in as many as 4 line-pairs. Overall, the findings suggest that a) some biological categories or networks (e.g., cell-to-cell signaling, cellular stress response, cellular organization, etc.) may be in common for subsets of line-pairs within either the AcbSh or CeA, and b) regulation of different genes and/or combinations of multiple biological systems may be contributing to the disparate alcohol drinking behaviors of these line-pairs.

Advances in next-generation sequencing suggest that RNA-Seq is poised to supplant microarray-based approaches for transcriptome analysis. This article briefly reviews the use of microarrays in the brain-behavior context and then illustrates why RNA-Seq is a superior strategy. Compared with microarrays, RNA-Seq has a greater dynamic range, detects both coding and noncoding RNAs, is superior for gene network construction, detects alternative spliced transcripts, detects allele specific expression and can be used to extract genotype information, e.g. nonsynonymous coding single nucleotide polymorphisms. Examples of where RNA-Seq has been used to assess brain gene expression are provided. Despite the advantages of RNA-Seq, some disadvantages remain. These include the high cost of RNA-Seq and the computational complexities associated with data analysis. RNA-Seq embraces the complexity of the transcriptome and provides a mechanism to understand the underlying regulatory code; the potential to inform the brain-behavior relationship is substantial.

BACKGROUND: Alcohol dependence (AD) is often accompanied by comorbid depression. Recent clinical evidence supports the benefit of subtype-specific pharmacotherapy in treating the population of alcohol-dependent subjects with comorbid major depressive disorder (MDD). However, in many alcohol-dependent subjects, depression is a reactive response to chronic alcohol use and withdrawal and abates with a period of abstinence. Genetic markers may distinguish alcohol-dependent subjects with MDD not tied chronologically and etiologically to their alcohol consumption. In this work, we investigated the association of adenylyl cyclase genes (ADCY1-9), which are implicated in both AD and mood disorders, with alcoholism and comorbid depression. METHODS: Subjects from Vienna, Austria (n = 323) were genotyped, and single nucleotide polymorphisms (1,152) encompassing the genetic locations of the 9 ADCY genes were examined. The Vienna cohort contained alcohol-dependent subjects differentiated using the Lesch Alcoholism Typology. In this typology, subjects are segregated into 4 types. Type III alcoholism is distinguished by co-occurrence of symptoms of depression and by affecting predominantly females. RESULTS: We identified 4 haplotypes associated with the phenotype of Type III alcoholism in females. One haplotype was in a genomic area in proximity to ADCY2, but actually within a lincRNA gene, 2 haplotypes were within ADCY5, and 1 haplotype was within the coding region of ADCY8. Three of the 4 haplotypes contributed independently to Type III alcoholism and together generated a positive predictive value of 72% and a negative predictive value of 78% for distinguishing women with a Lesch Type III diagnosis versus women designated as Type I or II alcoholics. CONCLUSIONS: Polymorphisms in ADCY8 and ADCY5 and within a lincRNA are associated with an alcohol-dependent phenotype in females, which is distinguished by comorbid signs of depression. Each of these genetic locations can rationally contribute to the polygenic etiology of the alcoholism/depression phenotype, and the use of these genetic markers may aid in choosing appropriate and beneficial treatment strategies.

Alcohol is widely consumed; however, excessive use creates serious physical, psychological and social problems and contributes to the pathogenesis of many diseases. Alcohol use disorders (that is, alcohol dependence and alcohol abuse) are maladaptive patterns of excessive drinking that lead to serious problems. Abundant evidence indicates that alcohol dependence (alcoholism) is a complex genetic disease, with variations in a large number of genes affecting a person's risk of alcoholism. Some of these genes have been identified, including two genes involved in the metabolism of alcohol (ADH1B and ALDH2) that have the strongest known affects on the risk of alcoholism. Studies continue to reveal other genes in which variants affect the risk of alcoholism or related traits, including GABRA2, CHRM2, KCNJ6 and AUTS2. As more variants are analysed and studies are combined for meta-analysis to achieve increased sample sizes, an improved picture of the many genes and pathways that affect the risk of alcoholism will be possible.

The neural circuitry that processes natural rewards converges with that engaged by addictive drugs. Because of this common neurocircuitry, drugs of abuse have been able to engage the hedonic mechanisms normally associated with the processing of natural rewards. Ghrelin is an orexigenic peptide that stimulates food intake by activating GHS-R1A receptors in the hypothalamus. However, ghrelin also activates GHS-R1A receptors on extrahypothalamic targets that mediate alcohol reward. The central nucleus of the amygdala (CeA) has a critical role in regulating ethanol consumption and the response to ethanol withdrawal. We previously demonstrated that rat CeA GABAergic transmission is enhanced by acute and chronic ethanol treatment. Here, we used quantitative RT-PCR (qRT-PCR) to detect Ghsr mRNA in the CeA and performed electrophysiological recordings to measure ghrelin effects on GABA transmission in this brain region. Furthermore, we examined whether acute or chronic ethanol treatment would alter these electrophysiological effects. Our qRT-PCR studies show the presence of Ghsr mRNA in the CeA. In naive animals, superfusion of ghrelin increased the amplitude of evoked inhibitory postsynaptic potentials (IPSPs) and the frequency of miniature inhibitory postsynaptic currents (mIPSCs). Coapplication of ethanol further increased the ghrelin-induced enhancement of IPSP amplitude, but to a lesser extent than ethanol alone. When applied alone, ethanol significantly increased IPSP amplitude, but this effect was attenuated by the application of ghrelin. In neurons from chronic ethanol-treated (CET) animals, the magnitude of ghrelin-induced increases in IPSP amplitude was not significantly different from that in naive animals, but the ethanol-induced increase in amplitude was abolished. Superfusion of the GHS-R1A antagonists D-Lys3-GHRP-6 and JMV 3002 decreased evoked IPSP and mIPSC frequency, revealing tonic ghrelin activity in the CeA. D-Lys3-GHRP-6 and JMV 3002 also blocked ghrelin-induced increases in GABAergic responses. Furthermore, D-Lys3-GHRP-6 did not affect ethanol-induced increases in IPSP amplitude. These studies implicate a potential role for the ghrelin system in regulating GABAergic transmission and a complex interaction with ethanol at CeA GABAergic synapses.

A crucial issue in treating opiate addiction, a chronic relapsing disorder, is to maintain a drug-free abstinent state. Prolonged abstinence associates with mood disorders, strongly contributing to relapse. In particular, substance use disorders occurring during adolescence predispose to depression later in adulthood. Using our established mouse model of opiate abstinence, we characterized emotional consequences into adulthood of morphine exposure during adolescence. Our results indicate that morphine treatment in adolescent mice has no effect on anxiety-like behaviours in adult mice, after abstinence. In contrast, morphine treatment during adolescence increases behavioural despair in adult mice. We also show that morphine exposure strain-dependently enhances sociability in adult mice. Additional research will be required to understand where and how morphine acts during brain maturation to affect emotional and social behaviours into adulthood.

Pharmacological data suggest that delta opioid receptors modulate learning and memory processes. In the present study, we investigated whether inactivation of the delta opioid receptor modifies hippocampus (HPC)- and striatum-dependent behaviors. We first assessed HPC-dependent learning in mice lacking the receptor (Oprd1(-/-) mice) or wild-type (WT) mice treated with the delta opioid antagonist naltrindole using novel object recognition, and a dual-solution cross-maze task. Second, we subjected mutant animals to memory tests addressing striatum-dependent learning using a single-solution response cross-maze task and a motor skill-learning task. Genetic and pharmacological inactivation of delta opioid receptors reduced performance in HPC-dependent object place recognition. Place learning was also altered in Oprd1(-/-) animals, whereas striatum-dependent response and procedural learning were facilitated. Third, we investigated the expression levels for a large set of genes involved in neurotransmission in both HPC and striatum of Oprd1(-/-) mice. Gene expression was modified for several key genes that may contribute to alter hippocampal and striatal functions, and bias striatal output towards striatonigral activity. To test this hypothesis, we finally examined locomotor effects of dopamine receptor agonists. We found that Oprd1(-/-) and naltrindole-treated WT mice were more sensitive to the stimulant locomotor effect of SKF-81297 (D1/D5), supporting the hypothesis of facilitated striatonigral output. These data suggest, for the first time, that delta receptor activity tonically inhibits striatal function, and demonstrate that delta opioid receptors modulate learning and memory performance by regulating the HPC/striatum balance.

The molecular mechanisms underlying alcohol dependence involve different neurochemical systems and are brain region-dependent. Chronic Intermittent Ethanol (CIE) procedure, combined with a Two-Bottle Choice voluntary drinking paradigm, represents one of the best available animal models for alcohol dependence and relapse drinking. MicroRNAs, master regulators of the cellular transcriptome and proteome, can regulate their targets in a cooperative, combinatorial fashion, ensuring fine tuning and control over a large number of cellular functions. We analyzed cortex and midbrain microRNA expression levels using an integrative approach to combine and relate data to previous protein profiling from the same CIE-subjected samples, and examined the significance of the data in terms of relative contribution to alcohol consumption and dependence. MicroRNA levels were significantly altered in CIE-exposed dependent mice compared with their non-dependent controls. More importantly, our integrative analysis identified modules of coexpressed microRNAs that were highly correlated with CIE effects and predicted target genes encoding differentially expressed proteins. Coexpressed CIE-relevant proteins, in turn, were often negatively correlated with specific microRNA modules. Our results provide evidence that microRNA-orchestrated translational imbalances are driving the behavioral transition from alcohol consumption to dependence. This study represents the first attempt to combine ex vivo microRNA and protein expression on a global scale from the same mammalian brain samples. The integrative systems approach used here will improve our understanding of brain adaptive changes in response to drug abuse and suggests the potential therapeutic use of microRNAs as tools to prevent or compensate multiple neuroadaptations underlying addictive behavior.

GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism.

BACKGROUND: Allopregnanolone (ALLO) is a potent positive modulator of γ-aminobutyric acidA receptors (GABAA Rs) that affects ethanol (EtOH) withdrawal. Finasteride (FIN), a 5α-reductase inhibitor that blocks the formation of ALLO and other GABAergic neurosteroids, alters EtOH sensitivity. Recently, we found that Withdrawal Seizure-Prone mice from the first genetic replicate (WSP-1) exhibited behavioral tolerance to the anticonvulsant effect of intrahippocampal ALLO during EtOH withdrawal and that intrahippocampal FIN significantly increased EtOH withdrawal severity. The purpose of this study was to determine whether neurosteroid manipulations in the substantia nigra reticulata (SNR) and ventral tegmental area (VTA) produced effects during EtOH withdrawal comparable to those seen with intrahippocampal ALLO and FIN. METHODS: Male WSP-1 mice were surgically implanted with bilateral guide cannulae aimed at the SNR or VTA at 2 weeks prior to EtOH vapor or air exposure for 72 hours. Initial studies examined the anticonvulsant effect of a single ALLO infusion (0, 100, or 400 ng/side) at a time corresponding to peak withdrawal in the air- and EtOH-exposed mice. Separate studies examined the effect of 4 FIN infusions (0 or 10 μg/side/d) during the development of physical dependence on the expression of EtOH withdrawal. RESULTS: ALLO infusion exerted a potent anticonvulsant effect in EtOH-naïve mice, but a diminished anticonvulsant effect during EtOH withdrawal. Administration of FIN into the SNR exerted a delayed proconvulsant effect in EtOH-naïve mice, whereas infusion into the VTA increased EtOH withdrawal duration. CONCLUSIONS: Activation of local GABAA Rs in the SNR and VTA via ALLO infusion is sufficient to exert an anticonvulsant effect in naïve mice and to produce behavioral tolerance to the anticonvulsant effect of ALLO infusion during EtOH withdrawal. Thus, EtOH withdrawal reduced sensitivity of GABAA Rs to GABAergic neurosteroids in 2 neuroanatomical substrates within the basal ganglia in WSP-1 male mice.