Withdrawal Seizure-Resistant Mice Research Articles

Affective Behavior in Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant Mice during Long-Term Alcohol Abstinence.

While the acute alcohol withdrawal syndrome has been well characterized both in human clinical studies and in experimental animals, much less is known regarding long-term affective disturbances that can sometimes persist during protracted abstinence. Nevertheless, since relapse often occurs long after acute detoxification and may be predicted by persistent affective disruption, a better understanding of the long-term behavioral consequences of prior alcohol dependence may lead to improved strategies for relapse prevention. Male and female Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice from the second selection replicate (WSP-2, WSR-2) were exposed to a 10-day chronic-intermittent ethanol vapor protocol (CIE) or plain air and then tested repeatedly on the sucrose preference test (SPT), marble burying test (MBT), and the light-dark box test (LDT) over 7weeks of (forced) abstinence. While WSP and WSR mice differed significantly on tests of anxiety-like behavior (LDT, MBT), we found little evidence for long-term affective disruption following CIE in either line. The major exception was in the LDT, in that WSP but not WSR mice displayed longer latencies to enter the light compartment following CIE relative to air-controls. Selective breeding for acute withdrawal severity has resulted in differences in anxiety-like behavior between WSP and WSR mice. In contrast, however, genes contributing to the severity of acute withdrawal convulsions appear to have little overlap with those predisposing to affective disruption during long-term abstinence.

Ethanol drinking in Withdrawal Seizure-Prone and -Resistant selected mouse lines

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.

Escalation of intake under intermittent ethanol access in diverse mouse genotypes

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.

Importance of genetic background for risk of relapse shown in altered prefrontal cortex gene expression during abstinence following chronic alcohol intoxication

Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.

Elevated testosterone in females reveals a robust sex difference in altered androgen levels during chronic alcohol withdrawal

The endocrine disruption associated with alcohol (ethanol) abuse in both males and females is widely recognized. Ethanol intoxication and withdrawal in males results in significant reductions in androgen levels. Less is known about female alcoholics, and because the changes in testosterone concentrations remain controversial, we systematically characterized changes in sex steroids after chronic ethanol exposure and withdrawal in both sexes. Testosterone and 17β-estradiol concentrations were determined during chronic high intoxication, over a withdrawal time course, and following a period of abstinence using a genetic model of withdrawal vulnerability, the Withdrawal Seizure-Resistant (WSR) and -Prone (WSP) selected lines. In males, testosterone concentrations were significantly lower in intoxicated WSP mice after chronic ethanol exposure, and were dramatically and transiently reduced during the withdrawal period in both WSR and WSP lines. In contrast, testosterone levels were increased in intoxicated WSP females and in both WSR and WSP mice during withdrawal. Chronic ethanol exposure disrupted normal estrous cycling in WSP mice, associated with hyperandrogenemia while intoxicated. In abstinence, elevated testosterone was observed in both sexes but only in WSR mice. Estrogen levels were modestly reduced during withdrawal in both WSR and WSP lines, predominantly in males. These findings identify a mechanism based on altered androgen signaling that likely contributes to sex-specific responses during withdrawal. However, only WSR mice showed similar elevations in androgen long after withdrawal in both sexes, suggesting that genotype is an important determinant of steroid responses after abstinence. Increased androgen signaling in females as a consequence of chronic ethanol exposure may play an important and relatively uncharacterized role in sexually dimorphic responses to alcohol abuse.

Sex differences in acute ethanol withdrawal severity after adrenalectomy and gonadectomy in Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice

Recent findings suggest that the ability of ethanol (EtOH) to increase the levels of neurosteroids with potent γ-aminobutyric acid (GABA)ergic properties can influence measures of EtOH sensitivity. Earlier studies determined that removal of the adrenals and gonads diminished the steroidogenic effect of EtOH and significantly increased acute EtOH withdrawal severity in two inbred mouse strains that differed in withdrawal severity, suggesting the contribution of anticonvulsant GABAergic steroids to acute withdrawal in intact animals. Thus, the goal of the present study was to investigate the consequence of steroid removal on acute EtOH withdrawal through excision of the adrenals and gonads, in another genetic animal model of EtOH withdrawal differences, the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines. Male and female WSP and WSR mice underwent surgical removal of the adrenals and gonads or no organ removal (SHAM). One to 2 weeks later, baseline handling-induced convulsions (HICs) were assessed, mice were given a 4 g/kg dose of EtOH, and HICs were measured hourly for 12 h and then at 24 h. The combination surgery significantly increased EtOH withdrawal in WSP and WSR female mice, as measured by area under the curve (AUC) and peak HIC scores. The AUC was significantly positively correlated with plasma corticosterone levels and significantly negatively correlated with progesterone levels. In contrast, surgical status did not alter withdrawal severity in male WSP and WSR mice. Overall, the increase in acute EtOH withdrawal severity in female WSP and WSR mice after adrenalectomy and gonadectomy corroborate our recent evidence that withdrawal from a high dose of EtOH can be modulated by anticonvulsant steroids produced in the periphery.

Decreased anticonvulsant efficacy of allopregnanolone during ethanol withdrawal in female Withdrawal Seizure-Prone vs. Withdrawal Seizure-Resistant mice

The GABAergic neurosteroid allopregnanolone (ALLO) has been repeatedly shown to have an increased anticonvulsant effect during ethanol withdrawal in rats and in C57BL/6J mice. In contrast, the seizure prone DBA/2J inbred strain and the Withdrawal Seizure-Prone (WSP) selected line exhibited decreased sensitivity to ALLO’s anticonvulsant effect during ethanol withdrawal, with no change in sensitivity in the Withdrawal Seizure-Resistant (WSR) line. To date, only male mice have been tested. Thus, the present study examined ALLO sensitivity during ethanol withdrawal in female WSP and WSR mice, since females display less severe physical symptoms of withdrawal and have higher circulating ALLO levels than males. Female WSP and WSR mice were exposed to ethanol vapor or air for 72 h. During peak ethanol withdrawal, separate groups of mice were injected with vehicle or ALLO (0, 3.2, 10, or 17 mg/kg, i.p.) prior to the timed tail vein infusion of pentylenetetrazol (PTZ). ALLO injection significantly increased the threshold dose for onset to PTZ-induced convulsions, indicating an anticonvulsant effect, in female WSP and WSR mice. During ethanol withdrawal, sensitivity to ALLO’s anticonvulsant effect was slightly increased in female WSR mice but was significantly decreased in female WSP mice. This line difference in sensitivity to ALLO during ethanol withdrawal in female mice was similar to that in the male mice. Notably, all seizure prone genotypes tested to date displayed tolerance to the anticonvulsant effect of ALLO during ethanol withdrawal, suggesting that decreased sensitivity of GABA A receptors to ALLO may contribute to the increased ethanol withdrawal phenotype.

Anticonvulsive effects of κ‐Opioid receptor modulation in an animal model of ethanol withdrawal

Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.

Effects of Finasteride on Chronic and Acute Ethanol Withdrawal Severity in the WSP and WSR Selected Lines

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of gamma-aminobutyric acidA (GABAA) receptors that can modulate ethanol (EtOH) withdrawal. The 5alpha-reductase inhibitor finasteride blocks the formation of ALLO from progesterone and was recently found to reduce certain effects of EtOH. Using the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines, in the present studies we examined the effect of finasteride on acute and chronic EtOH withdrawal severity. In the first two studies, male WSP and WSR mice were exposed to 72-hr EtOH vapor or air and received four injections of finasteride (50 mg/kg intraperitoneal (IP) or vehicle 24 hr before and each day of the vapor exposure. After removal from the inhalation chamber, mice were scored for handling-induced convulsions (HICs) hourly for 12 hr and then again at 24 hr (study 1) or were tested on the elevated plus maze at 24 hr after removal from the inhalation chamber (study 2). In the third experiment, mice were pretreated with finasteride or vehicle 24 hr before an acute dose of EtOH (4 g/kg ip) or saline and then were tested for HICs as in the chronic study. In both chronic EtOH studies, finasteride pretreatment reduced EtOH withdrawal severity, measured by HICs, and anxiety-related behavior, but only in the WSP selected line. However, finasteride pretreatment also significantly decreased blood EtOH concentration on the initiation of withdrawal in both chronic EtOH studies in WSP and WSR mice. In contrast, pretreatment with finasteride slightly enhanced acute EtOH withdrawal severity in WSP mice, whereas there was no effect of finasteride or EtOH injection on HICs in WSR mice. Collectively, these findings indicate that the WSP line is more sensitive than the WSR line to the modulatory effects of finasteride in terms of both chronic and acute EtOH withdrawal severity. The differential effect of finasteride on acute versus chronic EtOH withdrawal severity may result from an indirect effect of finasteride on EtOH pharmacokinetics in the chronic paradigm.

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.

Withdrawal from alcohol in withdrawal seizure-prone and -resistant mice: evidence for enkephalin resistance

Methionine enkephalin (Met-enkephalin) functions as an endogenous anticonvulsant. Peptide transport system-1 (PTS-1) is an important regulator of Met-enkephalin levels in brain and transports the peptide from brain to blood. In outbred mice, alcohol dependence is associated with decreased PTS-1 activity and increased levels of Met-enkephalin. In contrast, alcohol withdrawal is associated with recovery of PTS-1 activity, decreased levels of Met-enkephalin, and seizures. In this study, we examined the PTS-1/Met-enkephalin system in two replicates of withdrawal seizure-resistant (WSR) and withdrawal seizure-prone (WSP) mouse lines. We measured levels of preproenkephalin (PPE) mRNA and Met-enkephalin peptide in brain and the activity of PTS-1 during alcohol-naive, -dependent, and -withdrawal states. In alcohol-naive animals, Met-enkephalin levels were higher in WSP than in WSR mice. In alcohol-withdrawal animals, Met-enkephalin levels remained elevated in WSP mice, whereas they increased in WSR mice. Peptide levels were unrelated to levels of PPE mRNA or activity of PTS-1. Factorial analysis showed that proneness to seizures was genetically linked to Met-enkephalin levels in alcohol-naive, -dependent, and -withdrawing mice but not to mRNA levels or PTS-1 activity. Overall, these results may be explained by resistance to enkephalin in WSP mice and suggest that the dysregulation of the PTS-1/Met-enkephalin system contributes to susceptibility to seizures in WSP mice.

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.

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.

Acute neuroactive steroid withdrawal in withdrawal seizure-prone and withdrawal seizure-resistant mice

Allopregnanolone (3α-hydroxy-5α-pregnan-20-one) is an endogenously derived metabolite of progesterone, and a potent positive modulator of γ-aminobutyric acid A (GABA A) receptors. A withdrawal syndrome, characterized by central nervous system (CNS) hyperexcitability, has been demonstrated following abrupt discontinuation of high progesterone levels in rats, which was due in part to altered levels of allopregnanolone. The purpose of the present study was to determine if a single administration of pregnanolone or allopregnanolone could produce an acute withdrawal response in mice selected for susceptibility (Withdrawal Seizure-Prone, WSP) or resistance (Withdrawal Seizure-Resistant, WSR) to ethanol withdrawal convulsions. WSP mice administered 75 mg/kg pregnanolone showed a significant increase in handling-induced convulsion (HIC) scores over a 25-h testing period. In contrast, HIC scores in WSR mice were negligible after acute administration of 25, 50, 75, or 100 mg/kg pregnanolone. WSP mice also showed a similar increase in HIC after withdrawal from 75 mg/kg allopregnanolone. This effect was evident at both the 10-h and 25-h overall withdrawal severity assessment. These results demonstrate that neuroactive steroids can elicit an acute withdrawal response similar to that of other positive modulators of GABA A receptors in WSP mice, supporting the notion that a common set of genes underlie acute and chronic withdrawal severity from multiple agents with depressant effects on the central nervous system.

Elevated prodynorphin expression associated with ethanol withdrawal convulsions

The hypothesis that κ-opioid system activity may in part mediate convulsions exhibited during ethanol withdrawal was tested by exposing Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice to chronic ethanol. Whole brain was harvested for RNA isolation and prodynorphin mRNA steady-state levels in whole brain were examined using Northern blot analysis. The data revealed significantly increased levels of prodynorphin mRNA expression in mice susceptible to ethanol withdrawal convulsions after withdrawal, with no corresponding increase in prodynorphin steady-state levels in mice resistant to ethanol withdrawal convulsions. These findings were not due to basal differences in prodynorphin expression between the WSP and WSR mice. To verify that the differences observed were not due to an ethanol-induced global alteration in gene transcription, mRNA levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were measured. Glyceraldehyde-3-phosphate dehydrogenase expression was unchanged following both chronic exposure to ethanol and chronic exposure followed by withdrawal. These results extend our understanding of prodynorphin’s role in generalized seizure activity to include ethanol withdrawal-induced convulsions. Our findings suggest that prodynorphin expression is modulated during ethanol withdrawal convulsions, or alternatively, prodynorphin may mediate the severity of ethanol withdrawal convulsions.

Glial Differences Between Naïve Withdrawal Seizure‐Prone and ‐Resistant Mice

Withdrawal seizure-prone (WSP) and withdrawal seizure-resistant (WSR) mice were bred in replicate (i.e., WSP-1 and WSP-2) to exhibit differences in handling-induced convulsion severity during ethanol withdrawal. We examined the role of the glutamatergic system in susceptibility to ethanol-withdrawal convulsions in naive mice by measuring the density of immunolabeling for several glutamate transporters and the glutamate-metabolizing enzyme, glutamine synthetase. The density of glial fibrillary acidic protein immunolabeling (a marker of glial structure) and cytochrome oxidase activity (a marker of neuronal activity) were also characterized in naive mice. We observed a significantly greater density of immunolabeling for the glial transporter, glutamate/aspartate transporter, in CA1 subfield of the hippocampus (CA1) of naive WSP-2 mice as compared to WSR-2 mice. No other significant differences were observed. However, as compared to WSR mice, naive WSP mice exhibited a trend toward (a) greater immunolabeling for the glial glutamate transporter, glutamate transporter-1, in CA3, (b) greater immunolabeling for glial-specific glutamate-metabolizing enzyme, glutamine synthetase, in CA1 (replicate-2 only), and (c) less immunolabeling for the glial structural protein, glial fibrillary acidic protein, in all brain regions tested. In contrast, no trends or significant differences in the labeling density for the neuronal transporter, excitatory amino acid carrier 1, or the neuronal activity marker, cytochrome oxidase, were observed between the selected lines. These data suggest that the glutamatergic system and glia may play a pivotal role in the increased susceptibility to handling-induced convulsions observed in WSP mice.

Glutamate uptake in mice bred for ethanol withdrawal severity.

Withdrawal seizure-prone and withdrawal seizure-resistant mice were selectively bred to exhibit differences in handling-induced convulsion severity during ethanol withdrawal. The glutamatergic system has been implicated in seizure activity as well as ethanol withdrawal symptoms. This study assessed L-[3H]glutamate uptake into hippocampal synaptosomes prepared from withdrawal seizure-prone and- resistant mice. Glutamate uptake was characterized following repeated handling-induced convulsions, during acute intoxication, and during peak withdrawal following chronic ethanol exposure. Hippocampal synaptosomal L-[3H]glutamate uptake did not differ between convulsion- and ethanol-naive withdrawal seizure-prone and- resistant mice. Furthermore, exposure to convulsions or to a hypnotic dose of ethanol (4 g/kg) did not alter L-[3H]glutamate uptake. However, withdrawal from 72 h of ethanol exposure significantly increased L-[3H]glutamate uptake in both mouse lines as compared to their respective ethanol-naive controls. These data suggest that glutamate uptake is influenced by chronic ethanol exposure similarly in both withdrawal seizure-prone and- resistant mice. The observed increases in glutamate uptake during withdrawal may be associated with compensatory mechanisms triggered by chronic intoxication and are independent of the selected differences for withdrawal severity.

Ethanol Reward and Aversion in Mice Bred for Sensitivity to Ethanol Withdrawal

The present study examined mice selectively bred for sensitivity to ethanol withdrawal for differences in the conditioned place preference (CPP) and conditioned taste aversion (CTA) paradigms. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice and High Alcohol Withdrawal (HAW) and Low Alcohol Withdrawal (LAW) mice were selectively bred for differences in chronic and acute ethanol withdrawal, respectively. For the CPP experiment, male HAW and LAW (generation 5) mice received four pairings of ethanol (2 g/kg), with a distinctive floor stimulus. On intervening days, mice received saline paired with an alternate floor type. During the preference test, all mice received an injection of saline before 60-min access to both floor types. HAW mice showed conditioned preference for the ethanol-paired floor, whereas LAW mice did not. For the CTA experiments, male HAW, LAW, WSP, and WSR mice were adapted to a 2-hr/day water restriction regimen and subsequently received ethanol injections (0, 2, 2.5, or 4 g/kg, i.p.) immediately after 1-hr access to a NaCl-flavored solution. Dose-dependent reductions in NaCl intake reflected the development of CTA in both HAW/LAW and WSP/WSR lines. However, a smaller magnitude of CTA was observed in WSP mice relative to WSR mice after the first ethanol-NaCl pairing. WSP/WSR mice showed similar reductions of NaCl intake on subsequent conditioning trials. Overall, these data suggest that HAW mice selectively bred for high sensitivity to acute ethanol withdrawal are more sensitive to the rewarding effects of ethanol in the CPP paradigm. This outcome is consistent with a previous study showing greater CPP in WSP mice relative to WSR mice. In the CTA paradigm, sensitivity to ethanol withdrawal in the HAW/ LAW selected lines does not appear to be genetically correlated with sensitivity to the aversive properties of ethanol. However, the difference in acquisition of CTA in WSP/WSR lines suggest that some genes determining ethanol withdrawal severity may also influence initial sensitivity to ethanol's aversive effects.

Enkephalin, PPE mRNA, and PTS-1 in alcohol withdrawal seizure-prone and -resistant mice

Inbred animal strains provide an opportunity to study genetic factors in alcoholism in the absence of environmental factors. Although the concentration of methionine enkephalin (Met-enkephalin) in whole brain has been implicated in the consumption of ethanol, it has not been studied in the brains of alcohol withdrawal seizure-prone (WSP) and withdrawal seizure-resistant (WSR) mice. We compared these concentrations with the levels of preproenkephalin (PPE) mRNA and with the activity of peptide transport system-1 (PTS-1), a brain-to-blood transport system for Met-enkephalin that is affected by ethanol. The concentrations of Met-enkephalin were significantly greater in WSP mice than in WSR mice, whereas synthesis of Met-enkephalin, as reflected by PPE mRNA levels, and transport out of the brain by PTS-1 was not different. These results support a direct link between elevated concentrations of Met-enkephalin in whole brain and proneness to withdrawal-induced seizures. We suggest that the inverse relationship between the consumption of ethanol and proneness to seizures in inbred mice can be explaimed through their opposite relationships to Met-enkephalin.

Immunocytochemical analysis of glutamate and GABA in selectively bred mice

Withdrawal Seizure Prone (WSP) and Withdrawal Seizure Resistant (WSR) mice have been selectively bred for differential ethanol withdrawal handling-induced convulsions (HICs). In addition, it has been observed that WSP mice exhibit drug-naive HICs. This latter finding suggests that WSP and WSR mice differ in their susceptibility to HICs. Alterations in the glutamate and γ-aminobutyric acid (GABA) systems have been implicated in convulsive activity and have been proposed to underlie the manifestation of ethanol withdrawal symptoms. It is therefore possible that WSP and WSR mice are genetically different with respect to their glutamatergic and/or GABAergic systems. To test this hypothesis, we have analyzed WSP and WSR mice that are both drug- and HIC-naive for differences in the density of nerve terminal glutamate and GABA immunoreactivity within the CA1 subfield of the hippocampus (CA1) and layer II of the somatosensory cortex (SSC). The major finding of this study is that drug- and HIC-naive WSP mice exhibit a significantly greater density of presynaptic glutamate immunoreactivity associated with asymmetric synapses within the CA1, but not the SSC, when compared to WSR mice. The density of GABA immunoreactivity within nerve terminals associated with symmetric synapses does not differ between the selected lines in either brain region. Since prior drug exposure and HICs cannot account for the observed differences in these naive mice, the results strongly suggest that the density of nerve terminal glutamate immunoreactivity within the CA1 is a reflection of inherent genetic differences between WSP and WSR mice. Furthermore, an elevated density of presynaptic glutamate immunoreactivity may be an underlying neurochemical correlate to increased susceptibility to drug-naive and ethanol withdrawal convulsions.