Ethanol Withdrawal Severity Research Articles

Alteration of kappa-opioid receptor system expression in distinct brain regions of a genetic model of enhanced ethanol withdrawal severity

Abrupt withdrawal from chronic alcohol exposure can produce convulsions that are likely due to ethanol (EtOH) neuroadaptations. While significant efforts have focused on elucidating dependence mechanisms, the alterations contributing to EtOH withdrawal severity are less well characterized. The present studies examined the kappa-opioid receptor (KOP-R) system in Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice, selected lines that display severe and mild convulsions upon removal from chronic EtOH exposure. Previous data demonstrated significant increases in whole brain prodynorphin ( Pdyn) mRNA in WSP mice only during EtOH withdrawal. No significant effects of EtOH exposure or withdrawal were observed in WSR mice. The present study characterized Pdyn mRNA and the KOP-R in WSP and WSR mice during EtOH withdrawal using in situ hybridization (ISH) and KOP-R autoradiography. Analyses were performed in brain regions that express Pdyn mRNA and/or KOP-R and that might participate in seizure circuitry: the piriform cortex, olfactory tubercle, nucleus accumbens, caudate–putamen, claustrum, dorsal endopiriform nucleus, and cingulate cortex. ISH analyses confirmed previous findings; EtOH withdrawal increased Pdyn mRNA in multiple brain regions of WSP mice, but not WSR. Basal KOP-R binding was higher in WSR mice than in WSP mice, suggesting an anti-convulsant role for receptor activation. Finally, increased KOP-R density was present during EtOH withdrawal in WSP mice. These data suggest that differences in the KOP-R system among the lines might contribute to their selected difference in EtOH withdrawal severity.

The Syntaxin Binding Protein 1 Gene (Stxbp1 ) Is a Candidate for an Ethanol Preference Drinking Locus on Mouse Chromosome 2

We previously mapped a quantitative trait locus (QTL) for ethanol preference drinking to mouse chromosome 2 (mapped with high confidence, LOD = 15.5, p = 3 x 10(-16)). The specific gene(s) in the QTL interval responsible for phenotypic variation in ethanol preference drinking has not been identified. In the current study, we investigated the association of the syntaxin binding protein 1 gene (Stxbp1) with ethanol preference drinking and other ethanol traits using a panel of B6 x D2 (BXD) recombinant inbred (RI) strains derived from the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains. Confirmation analyses for ethanol consumption and withdrawal were performed using a large B6D2 F2 cross, short-term selected lines derived from the B6 and D2 progenitor strains, and standard inbred strains. BXD RI strain analysis detected provisional associations between Stxbp1 molecular variants and ethanol consumption, as well as severity of acute ethanol withdrawal, ethanol-conditioned taste aversion, and ethanol-induced hypothermia. Confirmation analyses using three independent genetic models supported the involvement of Stxbp1 in ethanol preference drinking but not in ethanol withdrawal. Stxbp1 encodes a Sec1/Munc18-type protein essential for vesicular neurotransmitter release. The present study provides supporting evidence for the involvement of Stxbp1 in ethanol preference drinking.

Alcohol Effects on Central Nervous System Gene Expression in Genetic Animal Models

This article summarizes the proceedings of a symposium presented at the 2004 annual meeting of the Research Society on Alcoholism in Vancouver, British Columbia, Canada. The organizers and chairs were William J. McBride and Michael F. Miles. The presentations were (1) Molecular Triangulation on Gene Expression Patterns in Behavioral Responses to Acute Ethanol, by Robnet T. Kerns; (2) Gene Expression in Limbic Regions After Ethanol Self-Infusion Into the Posterior Ventral Tegmental Area, by Zachary A. Rodd; (3) Microarray Analysis of CNS Limbic Regions of Inbred Alcohol-Preferring and -Nonpreferring rats and Effects of Alcohol Drinking, by Wendy N. Strother and Howard J. Edenberg; and (4) Microarray Analysis of Mouse Lines Selected for Chronic Ethanol Withdrawal Severity: The Convergence of Basal, Ethanol Regulated, and Proximity to Ethanol Quantitative Trait Loci to Identify Candidate Genes, by Joel G. Hashimoto and Kristine M. Wiren.

Estrogen protects against brain lipid peroxidation in ethanol-withdrawn rats

This study examined whether 17β-estradiol (E2) administration protects against ethanol withdrawal (EW)-associated oxidative insults by assessing oxidative markers thiobarbituric-acid-reacting-substances (TBARS). Ovariectomized rats implanted with E2 (EW/E2) or oil pellets (EW/Oil) received chronic ethanol (7.5% wt./vol., 5 weeks) or control dextrin diet (Dextrin/Oil). At 24 or 48 h of EW, rats were tested for overt EW signs and the cerebellum, hippocampus, and cortex were prepared for TBARS assessment in the presence and absence of FeCl 3. For control experiments, we assessed E2 effects on blood ethanol concentrations and TBARS levels during ethanol exposure prior to EW. The EW/Oil group showed enhanced endogenous- and FeCl 3-stimulated membrane TBARS levels in the cerebellum and hippocampus in a manner inhibited by E2 treatment. There was a relationship between the severity of EW and elevation of TBARS levels, particularly in the cerebellum. The enhanced TBARS levels at 24 h of EW appeared to diminish at 48 h in the hippocampus, but persisted in the cerebellum. E2 treatment did not alter blood ethanol concentrations and ethanol exposure alone did not enhance TBARS levels. These data suggest that EW rather than ethanol enhances brain lipid peroxidation that is transient and brain-region specific. Estrogens protect against the brain lipid peroxidation in a manner independent of blood ethanol concentrations (Supported by NIH/NIAAA 013864).

Serotonin 5-HT2 receptors and alcohol: reward, withdrawal and discrimination.

This article represents the proceedings of a symposium at the 2003 Research Society on Alcoholism meeting in Fort Lauderdale, Florida. The organizer was Karl J. Buck, and the chairperson was Mark S. Brodie. The presentations were (1) The Multiple PDZ Domain Protein May Mediate Genetic Differences in Ethanol Withdrawal Severity Via Interaction With 5-HT2 Receptors, by Matthew T. Reilly and Kari J. Buck; (2) The Ionic Mechanism of Serotonin Potentiation of Ethanol Excitation of Ventral Tegmental Area Neurons, by Mark S. Brodie; and (3) 5-HT(2C) Receptor Agonists in the Discriminative Stimulus Effects of Ethanol, by Laura M. Rogers, Ken Szeliga, and Kathleen Grant.

Interaction of the nicotinic cholinergic system with ethanol withdrawal.

The observation that alcohol and nicotine are commonly abused together suggests that the two drugs have common sites of action. In vitro studies indicate that nicotinic acetylcholine receptor (nAChR) function is enhanced by ethanol. Furthermore, some ethanol-related behaviors are associated with a region of mouse chromosome 2 that contains the gene encoding the alpha4 subunit of the nAChR (Chrna4). We have identified a polymorphism in Chrna4 that results in an alanine (A) or threonine (T) residue at position 529 in the second intracellular loop of the protein. Nicotinic receptors expressing the A variant have greater responses to nicotine and ethanol than receptors with the T variant when measured in vitro, but the possible effects of the polymorphism on the severity of ethanol withdrawal have not been assessed. The handling-induced convulsion (HIC) assay is an established method for studying drug withdrawal in vivo. We monitored the HIC responses of mice for 8 h after an injection of ethanol (4 g/kg). A survey of 16 mouse strains, as well as previously published data, indicated an association of the A/T polymorphism with ethanol withdrawal. This association was also found in wild-type animals from an F2 intercross of the A/J (A529-genotype) strain with C57BL/6J (T529-genotype) mice that also lack expression of the beta2 nAChR subunit. Beta2 -/- animals, which do not express alpha4beta2 nAChRs in the brain, exhibited significantly lower HIC responses and no effect of the polymorphism. These results suggest that the nicotinic cholinergic system and the A/T polymorphism modulate ethanol withdrawal.

Decreased alcohol self-administration and increased alcohol sensitivity and withdrawal in CB1 receptor knockout mice

Recent advances in the understanding of the neurobiological basis of alcohol dependence suggest that the endocannabinoid system may play a key role in the reinforcing effects of ethanol. In the present study, disruption of CB1 receptors in mice generated on a CD1 background decreased both ethanol consumption and preference. This decreased ethanol self-administration was associated with increased sensitivity to the acute intoxicating effects of ethanol. Mutant mice were more sensitive to the hypothermic and sedative/hypnotic effects of acute ethanol administration (1.5–4.0 g/kg), although plasma ethanol concentrations did not differ from those of controls. Moreover, wild-type mice exhibited normal locomotor activation caused by 1.0–2.5 g/kg injection of ethanol, whereas mutant mice displayed sedation in response to the injection of the same ethanol doses. The severity of alcohol withdrawal-induced convulsions was also increased in CB1 −/− mice. Our results suggest that CB1 receptors participate in the regulation of ethanol drinking and demonstrate that their disruption lead to increased ethanol sensitivity and withdrawal severity.

Evaluation of the glutamate decarboxylase genes Gad1 and Gad2 as candidate genes for acute ethanol withdrawal severity in mice

Previous studies in crosses between the C57BL/6J (B6) and the DBA/2J (D2) mice have implicated a role of the genes encoding for the 67- and 65-kDa isoforms of the glutamate decarboxylase (Gad1 and Gad2) in the manifestation and severity of multiple ethanol-related traits such as acute ethanol withdrawal severity [Buck, K.J., Metten, P., Belknap, J.K., Crabbe, J.C., 1997. Quantitative trait loci involved in genetic predisposition to acute alcohol withdrawal in mice. J. Neurosci. 17, 3946–3955], ethanol preference [Phillips, T.J., Belknap, J.K., Buck, K.J., Cunningham, C.L., 1998. Genes on mouse chromosomes 2 and 9 determine variation in ethanol consumption. Mamm. Genome 9, 936–941] and ethanol-induced locomotion [Demarest, K., McCaughran Jr., J., Mahjubi, E., Cipp, L., Hitzemann, R., 1999. Identification of an acute ethanol response quantitative trait locus on mouse chromosome 2. J. Neurosci. 19, 549–561]. Strain-specific sequencing experiments as well as gene expression studies in drug-naı̈ve and ethanol-treated D2 and B6 mice were carried out. The Gad1 sequence was similar, the Gad2 cDNA carried only a silent polymorphism (1017 G>C) between both strains. In addition, no significant GAD65 or GAD67 expression differences were detected in either drug-naı̈ve or acute ethanol withdrawn animals by Western blot experiments. Therefore, these results do not support the hypothesis of an involvement of Gad1 or Gad2 in the pathophysiology of acute ethanol withdrawal severity and the other ethanol related traits.

The Relationship Between Hippocampal Acetylcholine Release and Cholinergic Convulsant Sensitivity in Withdrawal Seizure–Prone and Withdrawal Seizure–Resistant Selected Mouse Lines

Background The septo‐hippocampal cholinergic pathway has been implicated in epileptogenesis, and genetic factors influence the response to cholinergic agents, but limited data are available on cholinergic involvement in alcohol withdrawal severity. Thus, the relationship between cholinergic activity and responsiveness and alcohol withdrawal was investigated in a genetic animal model of ethanol withdrawal severity.Methods Cholinergic convulsant sensitivity was examined in alcohol‐naïve Withdrawal Seizure—Prone (WSP) and—Resistant (WSR) mice. Animals were administered nicotine, carbachol, or neostigmine via timed tail vein infusion, and the latencies to onset of tremor and clonus were recorded and converted to threshold dose. We also used microdialysis to measure basal and potassium‐stimulated acetylcholine (ACh) release in the CA1 region of the hippocampus. Potassium was applied by reverse dialysis twice, separated by 75 min. Hippocampal ACh also was measured during testing for handling‐induced convulsions.Results Sensitivity to several convulsion endpoints induced by nicotine, carbachol, and neostigmine were significantly greater in WSR versus WSP mice. In microdialysis experiments, the lines did not differ in basal release of ACh, and 50 mM KCl increased ACh output in both lines of mice. However, the increase in release of ACh produced by the first application of KCl was 2‐fold higher in WSP versus WSR mice. When hippocampal ACh was measured during testing for handling‐induced convulsions, extracellular ACh was significantly elevated (192%) in WSP mice, but was nonsignificantly elevated (59%) in WSR mice.Conclusions These results suggest that differences in cholinergic activity and postsynaptic sensitivity to cholinergic convulsants may be associated with ethanol withdrawal severity and implicate cholinergic mechanisms in alcohol withdrawal. Specifically, WSP mice may have lower sensitivity to cholinergic convulsants compared with WSR because of postsynaptic receptor desensitization brought on by higher activity of cholinergic neurons.

Mapping murine loci for physical dependence on ethanol.

Alcoholism is associated with withdrawal (physical dependence), tolerance, or a maladaptive pattern of alcohol (ethanol) use. The well-documented difference in susceptibility to withdrawal after chronic ethanol exposure between the C57BL/6J and DBA/2J mouse strains provides an excellent starting point for dissecting genetic influences involved in physical dependence on ethanol. A quantitative trait locus (QTL) identifies the genomic location of a gene (or genes) affecting a trait of interest. A genome-wide QTL mapping study was carried out to dissect the multifactorial nature of withdrawal after chronic ethanol exposure using 400 B6D2F2 mice. To induce physical dependence, we used a standard paradigm in which mice were exposed to ethanol vapor for 72 h. The mice were then tested hourly for handling-induced convulsions (HICs) for 10 h and at hours 24 and 25. Ethanol withdrawal severity was first computed as the area under the 25-h HIC curve. Separate regression residuals were then calculated that corrected for individual differences in blood ethanol concentration at the time of withdrawal and baseline HIC severity (i.e. before ethanol exposure). Statistical mapping yielded significant evidence ( P<0.00005) for QTLs on chromosomes 19 and distal 1 that account for 45% of the genetic variance in ethanol withdrawal severity. The F2 results also provide supporting evidence for a sex-limited QTL on chromosome 13, and QTLs on chromosomes 4 and proximal 1, which may account for an additional 38% of the genetic variance. The distal chromosome 1 QTL is a locus of major effect, accounting for 26% of the genetic variance. Experiments using two congenic strains more precisely mapped this QTL. The QTLs map near candidate genes involved in neurosteroid biosynthesis and signal transduction. Syntenic homology between human and mouse chromosomes suggests that genes related to physical dependence on ethanol may localize to human chromosome regions 10q23-q26, 1q21-q43, 2q11-q32, 5p15/5q14-q21, and 9p24-p22.

Ethanol-induced conditioned taste aversion in 15 inbred mouse strains.

This study used a genetic correlational strategy to characterize the neurobiological basis of ethanol's (0, 2, or 4 g/kg) aversive effects as indexed by conditioned taste aversion. Substantial strain differences in taste aversion and hypothermia were observed, but the genetic correlation between these phenotypes was not significant. However, significant genetic correlations were observed between taste aversion and ethanol-related behaviors measured in previous studies, including home-cage ethanol preference (r = .68) and ethanol withdrawal severity (r = -.69). Strains showing stronger taste aversion tended to show lower ethanol preference and higher withdrawal severity. This pattern of findings is consistent with previous studies suggesting a commonality in neurobiological mechanisms underlying these phenotypes. These results do not support the hypothesis that ethanol-induced taste aversion is mediated by the drug's rewarding properties.

Expression profiling identifies strain-specific changes associated with ethanol withdrawal in mice.

Mice that exhibit characteristics of physical dependence following ethanol exposure serve as useful models of alcoholism in humans. The DBA/2J and C57BL/6J inbred strains differ in their behavioral response to ethanol withdrawal. Alterations in gene expression are believed to underlie neuroadaptation to ethanol dependence and tolerance. Therefore, the differences in ethanol withdrawal severity observed between the DBA/2J and C57BL/6J strains may be related to differential regulation of gene expression. We have used cDNA microarrays to determine the gene expression profile in the hippocampus of DBA/2J and C57BL/6J mice during withdrawal after chronic and acute ethanol exposure. Of the 7634 genes surveyed, approximately 2% were consistently differentially expressed by at least 1.4-fold in DBA/2J mice during chronic ethanol withdrawal. Less than 1% of the genes showed altered expression in C57BL/6J mice under the same conditions, or in DBA/2J mice during acute ethanol withdrawal. Strain- and treatment-specific patterns of altered expression were observed for multiple genes associated with the Janus kinase/signal transducers and activators of transcription and the mitogen activated protein kinase pathways. Genes associated with both pathways are regulated in DBA/2J mice during chronic ethanol withdrawal, and to a lesser extent during acute ethanol withdrawal. Only those genes associated with the mitogen-activated protein kinase (MAPK) pathway exhibited changes in expression in C57BL/6J mice during ethanol withdrawal. Furthermore, genes associated with retinoic acid-mediated signaling show differential expression exclusively in C57BL/6J mice. These findings represent significant differences in cellular adaptation to ethanol between the DBA/2J and C57BL/6J strains.

Effects of nicotine on ethanol dependence and brain damage

Almost all alcoholics (80%–95%) smoke tobacco. When alcoholics binge, they achieve high, sustained blood alcohol levels, become physically dependent, and often suffer loss of cognition and other higher cortical functions. Nicotine could have a modulatory effect on ethanol drinking behavior and ethanol-induced brain damage through its cholinergic actions. To determine whether nicotine altered alcohol dependence, alcohol-induced brain damage, or both, a rat model of binge drinking was used to study the effects of nicotine on the alcohol withdrawal syndrome and its associated brain damage. After administration of the last dose of ethanol in a 4-day binge model, rats remained intoxicated for approximately 5 h, slowly returned to a neutral state, and entered a hyperexcited period, which peaked around 24 h and lasted a total of 60 h. Behavioral signs of withdrawal included splayed limbs, tremors, and seizures. Continuous transdermal nicotine did not alter the duration or severity of ethanol withdrawal. The 4-day binge ethanol treatment caused considerable brain damage in the perirhinal cortex, entorhinal cortex, ventral dentate gyrus, and olfactory bulb as visualized with amino cupric silver stain. Nicotine alone caused little or no brain damage and did not markedly alter binge ethanol-induced damage in cortical or hippocampal regions. In the olfactory bulb, nicotine reduced ethanol-induced brain damage. Although results of other studies seem to indicate that nicotine increases alcohol consumption, our findings indicate that nicotine does not markedly change the development of alcohol dependence or alcohol-induced cortical damage.

Ethanol-regulated gene expression of neuroendocrine specific protein in mice: brain region and genotype specificity

Neuroendocrine specific protein or reticulon 1 (NSP/RTN1) was identified as a putative ethanol-regulated gene using mRNA differential display in mice genetically selected for severe ethanol withdrawal (withdrawal seizure-prone, WSP). One transcript of RTN1 (3.0 kb) showed a statistically significant increase (13%) in relative abundance in whole brain of ethanol-treated WSP mice but not in mice selected for resistance to ethanol withdrawal convulsions (WSR). We hypothesized that ethanol-induced regulation of gene expression of mRTN1 is specific to mice predisposed to exhibit severe ethanol withdrawal and that the gene might be regulated differentially in specific brain regions. WSP and WSR selected lines and DBA/2J and C57BL/6J inbred strains of mice were exposed to ethanol vapor or air for 72 h. mRNA steady-state expression of RTN1 was assessed in hippocampus, cortex, and cerebellum. Results indicated that the pattern of ethanol-induced changes in gene expression was dependent upon transcript size, brain region, and genotype. Modest increases in the relative abundance of both transcripts of RTN1 were observed in the hippocampus and cortex of all ethanol-treated mice. Results from cerebellum showed a moderate decrease in expression of RTN1 (3.0 kb transcript) in WSP and DBA/2J mice, but not in the mice resistant to ethanol withdrawal (C57BL/6J and WSR). These results suggest a genotype-specific effect of chronic ethanol exposure on steady-state mRNA levels of RTN1 in the cerebellum. Overall, the results indicate a complex pattern of ethanol-induced regulation of the putative mouse homologue of RTN1 and suggest that specific brain regional changes may be involved in the expression of physical dependence.

Reduced ethanol withdrawal severity and altered withdrawal-induced c-fos expression in various brain regions of mice lacking protein kinase C-epsilon.

Withdrawal from chronic ethanol consumption can be accompanied by motor seizures, which may be a result of altered GABA(A) receptor function. Recently, we have generated and characterized mice lacking the epsilon isoform of protein kinase C as being supersensitive to the behavioral and biochemical effects of positive GABA(A) receptor allosteric modulators, including ethanol. The aim of the present study was to determine whether protein kinase C-epsilon null mutant mice display altered seizure severity during alcohol withdrawal. In addition, we used c-fos immunohistochemistry immediately following seizure assessment to identify potential brain regions involved in any observed differences in withdrawal severity. Mice were allowed to consume an ethanol-containing or control liquid diet as the sole source of food for 14 days. During the 7-h period following removal of the diet, both ethanol-fed wild-type and protein kinase C-epsilon null mutant mice displayed an overall increase in Handling-Induced Convulsion score versus control-fed mice. However, at 6 and 7h following diet removal, the Handling-Induced Convulsion score was reduced in ethanol-fed protein kinase C-epsilon null mutant mice compared to ethanol-fed wild-type mice. Ethanol-fed protein kinase C-epsilon null mutant mice also exhibited a decrease in the number of Fos-positive cells in the lateral septum, and an increase in the number of Fos-positive cells in the dentate gyrus, mediodorsal thalamus, paraventricular nuclei of the thalamus and hypothalamus, and substantia nigra compared to ethanol-fed wild-type mice. These data demonstrate that deletion of protein kinase C-epsilon results in diminished progression of ethanol withdrawal-associated seizure severity, suggesting that selective pharmacological inhibitors of protein kinase C-epsilon may be useful in the treatment of seizures during alcohol withdrawal. These data also provide insight into potential brain regions involved in generation or suppression of ethanol withdrawal seizures.

Anxiety and Sensitivity to Ethanol and Pentobarbital in Alcohol Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant Mice

Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were selectively bred for high and low handling-induced convulsions, respectively, after chronic ethanol treatment. Withdrawal severity is one factor that may contribute to the development of alcoholism and/or substance abuse, and anxiety is another. We sought to explore whether these factors are genetically related. WSP and WSR mice of two replicate pairs of selected lines were tested for anxiety-related behaviors on the canopy stretched-attend-posture apparatus 20 min after intraperitoneal injection of ethanol (2 g/kg, 20% v/v), pentobarbital (20 mg/kg), or an equivalent volume of saline. Dependent measures of anxiety included number of stretched attend postures (SAP) and time spent in the exposed area of the apparatus. Number of line crossings, which measures overall activity, was also scored. WSP mice given saline exhibited more SAP than WSR mice given saline, which indicated greater baseline anxiety. Ethanol and pentobarbital both reduced SAP and increased time spent in the exposed area of the apparatus, which indicated that both drugs exerted an anxiolytic effect. Despite baseline differences in SAP between selected lines, both anxiolytic drugs reduced SAP to similar levels in WSP and WSR mice. These results support the hypothesis that WSP mice are more sensitive than WSR mice to the anxiety-reducing effects of ethanol and pentobarbital. Some genes that influence this difference are likely to be the same as those that influence ethanol withdrawal severity. Thus, higher basal anxiety and greater genetic sensitivity to anxiolytic drug effects may relate to a greater genetic predisposition to the development of severe alcohol withdrawal signs.

Anxiety and Sensitivity to Ethanol and Pentobarbital in Alcohol Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant Mice

Background: Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were selectively bred for high and low handling-induced convulsions, respectively, after chronic ethanol treatment. Withdrawal severity is one factor that may contribute to the development of alcoholism and/or substance abuse, and anxiety is another. We sought to explore whether these factors are genetically related. Methods: WSP and WSR mice of two replicate pairs of selected lines were tested for anxiety-related behaviors on the canopy stretched-attend-posture apparatus 20 min after intraperitoneal injection of ethanol (2 g/kg, 20% v/v), pentobarbital (20 mg/kg), or an equivalent volume of saline. Dependent measures of anxiety included number of stretched attend postures (SAP) and time spent in the exposed area of the apparatus. Number of line crossings, which measures overall activity, was also scored. Results: WSP mice given saline exhibited more SAP than WSR mice given saline, which indicated greater baseline anxiety. Ethanol and pentobarbital both reduced SAP and increased time spent in the exposed area of the apparatus, which indicated that both drugs exerted an anxiolytic effect. Despite baseline differences in SAP between selected lines, both anxiolytic drugs reduced SAP to similar levels in WSP and WSR mice. Conclusions: These results support the hypothesis that WSP mice are more sensitive than WSR mice to the anxiety-reducing effects of ethanol and pentobarbital. Some genes that influence this difference are likely to be the same as those that influence ethanol withdrawal severity. Thus, higher basal anxiety and greater genetic sensitivity to anxiolytic drug effects may relate to a greater genetic predisposition to the development of severe alcohol withdrawal signs.

GABA A receptor β 2 subunit mRNA content is differentially regulated in ethanol-dependent DBA/2J and C57BL/6J mice

Chronic ethanol treatment is known to alter gene expression and function of γ-aminobutyric acid type-A (GABA A) receptors. Here we focus on the β 2 subunit which is widely expressed in the mammalian brain, and plays a key role in the GABA binding site. Previous studies using rodent models of ethanol dependence show either increased or no change of β 2 subunit mRNA and peptide content following chronic ethanol administration. In humans, polymorphism at the β 2 subunit is associated with ethanol dependence in some, but not all, populations. In the present study we measured mRNA content in the cerebellum and cerebral cortex using ethanol-naive and ethanol-dependent DBA/2J and C57BL/6J mice. The DBA/2J strain displays severe ethanol withdrawal severity, while the C57BL/6J strain shows milder withdrawal reactions. RNase protection analysis demonstrated that the DBA/2J strain is more sensitive to ethanol-induced increases in β 2 subunit mRNA content in the cerebellum, showing significant increases at lower blood ethanol concentrations than C57BL/6J mice. The ethanol-induced regulation in C57BL/6J mice appears to be more complex, with decreases in β 2 subunit mRNA content at low blood ethanol concentrations, and increases at higher concentrations. These data suggest that differences between C57BL/6J and DBA/2J mice in the degree of physical dependence (withdrawal) on ethanol may be related to differential sensitivity to ethanol regulation of β 2 subunit expression.

Genetic Association Between Chronic Ethanol Withdrawal Severity and Acoustic Startle Parameters in WSP and WSR Mice

The present study examined the genetic association between chronic ethanol withdrawal severity and acoustic startle response (ASR) in replicated lines of mice selected for high (Withdrawal Seizure-Prone; WSP) and low (Withdrawal Seizure-Resistant; WSR) susceptibility to handling-induced convulsions after withdrawal from chronic exposure to ethanol. Any differences on a nonselected (correlated) trait between the opposite-selected lines is strong evidence for pleiotropic effects of the genes fixed by selection. Naive WSP and WSR mice of both replicates were placed in startle chambers and exposed to a series of white noise stimuli of different intensities. In Experiment 1, two parameters [the maximal acoustic startle response (Rmax), and the sound intensity necessary to produce 50% of the maximal startle response (dB50)] were obtained from a least-squares nonlinear regression by fitting data for each subject to a sigmoidal function that best described the relationship between sound intensity and mean ASR. Response habituation of WSP and WSR mice to a repeated acoustic stimulus of high intensity was examined in Experiment 2. When ASR amplitude was plotted versus sound intensity, the sigmoid intensity-response curves of both WSP replicates were shifted to the right relative to the responses of WSR mice, which suggested decreased startle sensitivity in the WSP animals. Statistical analysis showed that naive WSP mice were less sensitive (higher dB50) to acoustic stimulation than Seizure-Resistant animals whereas Rmax was similar for both lines. The selected lines also differed in their responses to repeated acoustic stimulation, with WSP mice demonstrating greater habituation. Results of the present study suggest some common genetic mechanisms underlying behavioral responsiveness to acoustic stimulation and severity of ethanol withdrawal.

Genetic Association Between Chronic Ethanol Withdrawal Severity and Acoustic Startle Parameters in WSP and WSR Mice

Genetic Association Between Chronic Ethanol Withdrawal Severity and Acoustic Startle Parameters in WSP and WSR Mice