Our previous work indicated a role for fyn-kinase in mediating several ethanol- and GABAA agonist-mediated behaviors. In the present work we investigate behavioral sensitivity to ethanol and several GABAA compounds in mice that over-express fyn-kinase in forebrain to further characterize the role of this non-receptor tyrosine kinase in the mediation of ethanol sensitivity. Transgenic mice over-expressing fyn-kinase were tested for sensitivity to ethanol-induced loss of righting reflex and ethanol preference drinking using a two-bottle choice drinking paradigm. Loss of righting reflex induced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; GABAA agonist) and etomidate (GABAA positive allosteric modulator) were also assessed. Fyn over-expressing mice exhibited shorter durations of ethanol-induced loss of righting reflex in the absence of differences in the rate of blood ethanol clearance, and exhibited reduced ethanol preference drinking. The genotypes did not differ in initial sensitivity to ethanol-induced loss of righting reflex suggesting development of greater acute tolerance to this ethanol action. Fyn over-expressing and wild-type mice also did not differ in sensitivity to loss of righting reflex induced by THIP and etomidate. The present results suggest regional specificity for fyn-kinase in the modulation of ethanol and GABAergic behavioral sensitivity. Fyn-kinase over-expression in forebrain structures modulates ethanol's hypnotic actions, as well as ethanol preference and consumption. Moreover, fyn over-expression in forebrain does not alter hypnotic sensitivity to THIP or etomidate, supporting data from fyn null mutant mice suggesting that cerebellar structures mediate the hypnotic actions of these GABAergic compounds.

Animal models designed to examine different facets of alcohol-related behaviors have been developed to study genetic and neurobiological factors underlying alcoholism and alcohol abuse. One goal has been to develop valid, congruent, complementary animal models of alcohol craving and relapse, with the ultimate objective of assessing the effectiveness of pharmacological agents with these models. Animal models of alcohol craving include drug-induced responding (drug reinstatement), cue-induced responding, Pavlovian Spontaneous Recovery (PSR), and appetitive/consummatory responding. A primary experimental approach to study alcohol relapse has been through expression of the Alcohol Deprivation Effect (ADE) following a single deprivation or multiple deprivations. To date, five selectively bred lines of rats have been developed to study alcohol-drinking behavior. These are the ALKO/Alcohol (AA), alcohol-preferring (P), high alcohol-drinking (HAD-1 and HAD-2 replicates), and the Sardinian alcohol-preferring (sP) lines of rats. Findings thus far indicate that only the P line of rats meets all the criteria established for a valid animal model of alcoholism, with progress having been made in characterizing the AA, HAD and sP lines of rats. The focus of the current review will be to analyze the various models of alcohol craving, emphasizing the use of the Indiana University selected rat lines (P and HADs). Overall, the findings indicate substantial progress has been made in developing animal models of alcohol abuse, relapse and craving using these selectively bred rat lines, as well as outbred rats.

This article represents the proceedings of a symposium at the 2002 RSA Meeting in San Francisco, California, organized and co-chaired by L. Judson Chandler and Richard A. Morrisett. The presentations were (1) PKA regulates chronic ethanol-induced synaptic targeting of NMDA receptors, by L. Judson Chandler; (2) Long-lasting potentiation of GABAergic synapses in dopamine neurons after a single in vivo ethanol exposure, by Antonello Bonci; (3) The DARPP-32 cascade and regulation of the ethanol sensitivity of NMDA receptors in the nucleus accumbens, by Richard A. Morrisett; (4) and The cAMP/PKA signal transduction pathway modulates ethanol consumption and sedative effects of ethanol, by Gary S. Wand.

Levels of neuropeptide Y (NPY) mRNA expression in discrete brain regions of alcohol preferring (P) rats and alcohol nonpreferring (NP) rats were examined using in situ hybridization. NPY mRNA expression was significantly lower in the central nucleus of amygdala (CeA) of P rats than NP rats, whereas no differences were found in the medial or basolateral amygdaloid nuclei. This study suggests that reduced NPY gene expression in the CeA may contribute to differences in alcohol preference and other behavioral differences observed between P and NP rats.

The pharmacological profile of allopregnanolone, a neuroactive steroid that is a potent positive modulator of gamma-aminobutyric acidA (GABAA) receptors, is similar to that of ethanol. Recent findings indicate that acute injection of ethanol increased endogenous allopregnanolone to pharmacologically relevant concentrations in male rats. However, there are no comparable data in mice, nor has the effect of ethanol drinking on endogenous allopregnanolone levels been investigated. Therefore, the present studies measured the effect of ethanol drinking and injection on allopregnanolone levels in male and female C57BL/6 mice. One group was given 17 days of 2-h limited access to a 10% v/v ethanol solution in a preference-drinking paradigm, while another group had access to water only. The ethanol dose consumed in 2 h exceeded 2 g/kg. Then, separate groups of mice were injected with either 2 g/kg ethanol or saline. Mice were killed 30 min after the 2-h drinking session or injection. Blood ethanol concentration was significantly higher in the ethanol-injected versus ethanol-drinking groups, even though the dose was similar. Consumption of ethanol significantly increased brain allopregnanolone levels in male but not female mice, compared with animals drinking water, but did not alter plasma corticosterone levels. In contrast, injection of ethanol did not significantly alter brain allopregnanolone levels in male or female mice and only significantly increased plasma corticosterone levels in the male mice, when compared with saline-injected animals. The sex differences in the effect of ethanol administration on endogenous allopregnanolone levels suggest that the hormonal milieu may impact ethanol's effect on GABAergic neurosteroids. Importantly, these data are the first to report the effect of ethanol drinking on allopregnanolone levels and indicate that ethanol consumption and ethanol injection can produce physiologically relevant allopregnanolone levels in male mice. These results have important implications for studies investigating the potential role of endogenous allopregnanolone levels in modulating susceptibility to ethanol abuse.

Dopamine D1 receptors (D1DRs) mediate a major component of dopaminergic neurotransmission, and alterations in their synaptic and subcellular distribution may underlie a variety of neurological diseases. In order to monitor D1DR localization in real time, we subcloned a sindbis virus containing an enhanced-GFP coding region inserted at the C-terminal region of a dopamine D1 receptor (eGFP-D1DR). Two-photon excitation of expressed eGFP-D1DRs was monitored in a variety of viable neural preparations. Infection of primary cultured rat ventral striatal neurons, verified for neuronal phenotype using patch clamp electrophysiology, was induced by the simple addition of the virus to media. Parasagittal slice cultures, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), were infected by manual injection below the glia surface. NAc-containing parasagittal slices prepared from mice in which the virus was administered via stereotaxic injection in vivo also displayed robust eGFP-D1DR expression. Expression of functional D1DRs following infection in baby hamster kidney (BHK) cells was monitored by DA-stimulated cAMP production that was also blocked by a selective D1 antagonist. Taken together, these findings provide the first demonstration of the functional expression and real-time imaging of eGFP-D1DRs, and indicate that sindbis virus is an effective method for D1 receptor expression in a variety of native neuronal preparations.

Development of procedures yielding substantial blood alcohol concentrations during voluntary access to an alcohol solution in mice is necessary to further characterize genetic and neurobiologic mechanisms underlying alcohol self-administration. Although, in experimental situations, some populations of mice readily drink an alcohol solution, results from previous studies have not typically revealed high blood alcohol concentrations after voluntary access, probably because of the high alcohol metabolism rate in mice. Toward development of a murine drinking model, 36 selectively bred high-alcohol-preferring mice of both sexes were subjected to a 30-min scheduled-access procedure by using saccharin fading to gradually introduce an alcohol solution. Mice had ad libitum access to food and water 24 h a day. The alcohol solution was available 1 h after the start of the dark part of the cycle for 30 min per day, 5 days per week. After complete removal of saccharin from the drinking tubes, mice consistently drank 1.4 g/kg of a 10% [volume/volume (vol./vol.)] alcohol solution in 30 min. Analysis of tail blood samples, taken immediately after the end of the 30-min access period, indicated blood alcohol concentrations were tightly correlated with alcohol intakes (range, 6-130 mg/dl; average, nearly 60 mg/dl). A concentration-response function of 10%, 12%, 15%, 18%, and 21% (vol./vol.) alcohol solutions indicated an inverted U-shaped relation between alcohol intake and alcohol concentration, with peak intake of greater than 1.75 g/kg per 30 min when a 15% alcohol solution was available. No sex differences were seen. These findings indicate the utility of this procedure in obtaining pharmacologically relevant blood alcohol concentrations after voluntary oral self-administration of an alcohol solution in mice.

Ethanol (alcohol) withdrawal-induced convulsions are a key index of physical dependence on ethanol and a clinically important consequence of alcohol abuse in humans. In rodent models, severity of withdrawal is strongly influenced by genotype. For example, many studies have reported marked differences in withdrawal severity between the WSR (Withdrawal Seizure Resistant) and WSP (Withdrawal Seizure Prone) mouse strains selectively bred for over 25 generations to differ in chronic withdrawal severity. Therefore, we used an F2 intercross between the inbred WSP and WSR strains for a genome-wide search for quantitative trait loci (QTLs), which are chromosomal sites containing genes influencing the magnitude of withdrawal. We also used the recently developed HW, RHW (high withdrawal) and LW, RLW (low withdrawal) lines selectively bred for the same trait and in the same manner as the WSP, WSR lines. QTL analysis was then used to dissect the continuous trait distribution of withdrawal severity into component loci, and to map them to broad chromosomal regions by using the Pseudomarker 0.9 and Map Manager QT29b programs. This genome-wide search identified five significant QTLs influencing chronic withdrawal severity on Chromosomes (Chrs) 1 (proximal), 4 (mid), 8 (mid), 11 (proximal), and 14 (mid), plus significant interactions (epistasis) between loci on Chr 11 with 13, 4 with 8, and 8 with 14.

The familial occurrence of alcoholism has been known for many years. Many twin, adoption, and family studies now concur that this familial pattern is to a great extent conferred by genes transmitted to biological offspring (1,2). Approximately 50–60% of individual differences in risk for alcoholism is genetic, and this proportion is approximately equal in men and women (2). Thus, it is an easy task to predict that a close biological relative of an alcoholic is at higher risk for alcoholism. However, risk is not inherited alleles at specific risk-promoting or -protective genes are inherited. To date, there are only two specific genes known to confer substantial protection against alcoholism, variants at the ALDH2*2 and ADH2*2 metabolic enzymes. The variant alleles lead to the accumulation of alcohol’ s metabolite, acetaldehyde, when susceptible individuals drink alcohol. This toxic compound produces nausea, flushing, dizziness, and other unpleasant effects, and slow alcohol metabolizers avoid excessive drinking (3). Therefore, progress from assigning risk statistically to ascertaining whether specific individuals possess risk-promoting or -protective alleles will require the identification of the specific genes underlying risk.

Enhancement of the activation of GABAA receptors is a common feature of many sedative and hypnotic drugs, and it is probable that the GABAA receptor complex is a molecular target for these drugs in the mammalian central nervous system. We set out to elucidate the role of the two predominant (alpha1 and beta2) subunits of GABAA receptor in sedative drug action by studying mice lacking these two subunits. Both alpha1 (-/-) and beta2 (-/-) null mutant mice showed markedly decreased sleep time induced by nonselective benzodiazepine, flurazepam, and GABAA agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol. The sleep time induced by the beta-selective drug etomidate was decreased only in beta2 (-/-) knockout mice. In contrast, alpha1 (-/-) mice were more resistant to the alpha1-selective drug zolpidem than beta2 (-/-) or wild-type animals. Knockout mice of both strains were similar to wild-type mice in their responses to pentobarbital. The duration of loss of the righting reflex produced by ethanol was decreased in male mice for both null alleles compared with wild-type mice, but there were no differences in ethanol-induced sleep time in mutant females. Deletion of either the alpha1 or beta2 subunits reduced the muscimol-stimulated 36Cl36 influx in cortical microsacs suggesting that these mutant mice have reduced number of functional brain GABAA receptors. Our results show that removal of either alpha1 or beta2 subunits of GABAA receptors produce strong and selective decreases in hypnotic effects of different drugs. Overall, these data confirm the crucial role of the GABAA receptor in mechanisms mediating sedative/hypnotic effects.

BACKGROUND: An earlier study showed that deletion of the fyn-kinase gene enhanced sensitivity to ethanol's sedative hypnotic effects and suggested that this was associated with diminished fyn-kinase phosphorylation of NMDA receptors. The authors of that study speculated that this resulted in an inability of the null mutants to develop acute tolerance to ethanol, leading to the longer ethanol-induced sleep times. However, in vivo acute tolerance to ethanol was not examined directly. METHODS: To address the role of fyn-kinase in mediating acute tolerance, as well as sensitivity to several other behavioral effects of ethanol, we studied an independently generated population of fyn null mutant and wild-type mice. RESULTS: Homozygous mutants exhibited longer ethanol sleep times that could not be attributed to differences in initial sensitivity, and impaired acute tolerance to the motor incoordinating effects of ethanol as measured by using the stationary dowel, but not the rotarod. Fyn-kinase null mutants were more sensitive to the anxiolytic effects of ethanol when tested using the elevated plus maze, and males displayed a lower preference for ethanol in a two-bottle choice paradigm. Finally, mutant and wild-type mice did not differ in sensitivity to the hypothermic effects of ethanol. The genotypes also did not differ in blood-ethanol clearance, eliminating a metabolic explanation for these behavioral differences. CONCLUSIONS: These results show that fyn-kinase modulates acute tolerance to ethanol and suggest a role for fyn in mediating ethanol's anxiolytic and reinforcing properties.

Identifying and characterizing brain regions regulating alcohol consumption is beneficial for understanding the mechanisms of alcoholism. To this aim, we first identified brain regions changing in expression of the inducible transcription factor c-Fos in the alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) mice after ethanol consumption. Drinking a 5% ethanol/10% sucrose solution in a 30 min limited access procedure led to induction of c-Fos immunoreactivity in urocortin (Ucn)-positive cells of the Edinger-Westphal nucleus (EW), suppression of c-Fos immunoreactivity in the dorsal portion of the lateral septum (LS) of both strains of mice, and strain-specific suppression in the intermediate portion of the LS and the CA3 hippocampal region. Because the EW sends Ucn projections to the LS, and B6 and D2 mice differ dramatically in EW Ucn expression, we further analyzed the Ucn EW–LS pathway using several genetic approaches. We find that D2 mice have higher numbers of Ucn-immunoreactive processes than B6 mice in the LS and that consumption of ethanol/sucrose in the F2 offspring of a B6D2 intercross positively correlates with Ucn immunoreactivity in the EW and negatively correlates with Ucn immunoreactivity in the LS. In agreement with these findings, we find that alcohol-avoiding male B6.D2Alcp1 line 2.2 congenic mice have lower Ucn immunoreactivity in the EW than male B6.B6 mice. Finally, we also find that HAP mice, selectively bred for high alcohol preference, have higher Ucn immunoreactivity in EW, than LAP mice, selectively bred for low alcohol preference. Taken together, these studies provide substantial evidence for involvement of the EW–LS Ucn pathway in alcohol consumption.

The objectives of this study were to assess the effects of access to different concentrations of ethanol and sex of the animal on ethanol consumption during periadolescence [postnatal days (PNDs) 30-60] in alcohol-preferring (P) rats. On PND 28, female and male P pups were single housed in hanging stainless steel cages with ad libitum access to water and food. Beginning on PND 30, the rats were also given access to either a single concentration [15% volume/volume (vol./vol.)] or multiple concentrations [10%, 20%, and 30% (vol./vol.)] of ethanol. Differences between sex (male vs. female) and ethanol conditions (single concentration vs. multiple concentrations), for the average amount of ethanol consumed for each week (starting on PND 33) of access, were examined. Analyses of the data for ethanol drinking revealed significant (P\textless.025) main effects of week and ethanol condition, as well as a significant weekxethanol condition interaction. For the first week, both male and female P pups consumed more ethanol under the multiple-ethanol-concentration condition than under the single-ethanol-concentration condition. However, across the second through fourth weeks, this pattern was seen only in female P pups. When preference for one concentration of ethanol over the other concentrations was assessed, it was found that male P pups tended to choose the 30% concentration over the 10% and 20% concentrations, whereas female P pups did not display a preference. The findings of this study corroborate previous work indicating that periadolescent P rats readily acquire high-ethanol-drinking behavior and that, similar to adult P rats, concurrent access to multiple concentrations of ethanol further enhances ethanol intake. These findings suggest to us that innate genetically influenced mechanisms promoting high ethanol intake are present at this stage of development.

We reported that repeated alcohol deprivations prolonged the expression of an alcohol-deprivation effect (ADE) under 24-h free-choice alcohol-drinking access and that the duration of the initial deprivation period had a positive effect of prolonging the duration of the ADE. In the present study, operant techniques (including progressive ratio measures) were used to examine the effects of initial deprivation length and number of deprivation cycles on the magnitude and duration of the ADE in alcohol-preferring (P) rats to test the hypothesis that repeated deprivations can increase the reinforcing effects of ethanol (ETOH). Adult male P rats were trained in two-lever operant chambers to self-administer 15% ETOH (v/v) on a fixed-ratio 5 (FR-5) and water on a FR-1 schedule of reinforcement in daily 1-h sessions. Following 6 weeks of daily 1-h sessions, the P rats were randomly assigned to one of four groups (n=10/group): nondeprived or deprived of alcohol for 2, 5, or 8 weeks. Following this initial period, the deprived groups were given 15% ETOH again in the operant chambers for a 2-week period, following which they were deprived again for 2 weeks (all three deprived groups). Following the fourth deprivation, the rats underwent a progressive ratio test to determine the breakpoints (FR values) for the nondeprived and the deprived groups. Repeated deprivations increased both the magnitude and duration of the ADE as indicated by increased responding on the ETOH lever. However, the length of the initial deprivation had little effect on expression of the ADE except following the first deprivation, where an ADE was not observed for the 8-week group. Breakpoint values for responding on the ETOH lever for all three deprived groups were two-fold higher than the value for the nondeprived group. The results suggest that repeated cycles of alcohol deprivation and alcohol access increased the reinforcing effects of ETOH in the P rats.

BACKGROUND: Mesolimbic dopamine is thought to play a role in the reinforcing properties of ethanol, but ethanol-induced changes in extracellular dopamine in the ventral striatum have not been well characterized in mouse models. METHODS: Two experiments were used to characterize the pharmacodynamic response of ethanol in the ventral striatum in C57BL/6 mice. The first experiment determined the effect of ethanol on ventral striatal dopamine in male and female mice after intraperitoneal injection of either 2.0 g/kg ethanol or saline. The second experiment was a replication in males, except that the mice were habituated to intraperitoneal injections before the dialysis experiment. RESULTS: Distinct patterns of dopamine activity in response to ethanol were demonstrated in male and female C57BL/6 mice. A significant increase in dialysate dopamine relative to saline injection was observed in females but not in males. With habituation to intraperitoneal injection before the dialysis experiment, ethanol administration caused a significant dopamine response in males as well. A linear decline was observed in dialysate ethanol concentrations after the peak concentration was reached. Concurrent analysis of the time course of dopamine and ethanol content showed that the dopamine response declined significantly faster than the ethanol concentrations. CONCLUSIONS: The C57BL/6 mouse strain is a justifiable model system for studying the mechanisms involved in ethanol regulation of mesolimbic dopamine activity. Habituation to intraperitoneal injection should be used in male C57BL/6 mice for experiments in which the dopamine response is measured after intraperitoneal injection of a drug. The dissociation between dopamine and ethanol may indicate an acute neural adaptation to ethanol-induced dopamine response in the ventral striatum after a single ethanol injection.

We examined the interaction of ethanol with the gamma-aminobutyric acid (GABA)ergic system in neurons of slices of the rat central amygdala nucleus (CeA), a brain region thought to be critical for the reinforcing effects of ethanol. Brief superfusion of 11-66 mM ethanol significantly increased GABA type A (GABA(A)) receptor-mediated inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in most CeA neurons, with a low apparent EC(50) of 20 mM. Acute superfusion of 44 mM ethanol increased the amplitude of evoked GABA(A) IPSPs and IPSCs in 70% of CeA neurons. The ethanol enhancement of IPSPs and IPSCs occurred to a similar extent in the presence of the GABA type B (GABA(B)) receptor antagonist CGP 55845A, suggesting that this receptor is not involved in the ethanol effect on CeA neurons. Ethanol superfusion also decreased paired-pulse facilitation of evoked GABA(A) IPSPs and IPSCs and always increased the frequency and sometimes the amplitude of spontaneous miniature GABA(A) IPSCs as well as responses to local GABA application, indicating both presynaptic and postsynaptic sites of action for ethanol. Thus, the CeA is the first brain region to reveal, without conditional treatments such as GABA(B) antagonists, consistent, low-dose ethanol enhancement of GABAergic transmission at both pre- and postsynaptic sites. These findings add further support to the contention that the ethanol-GABA interaction in CeA plays an important role in the reinforcing effects of ethanol.

The goal of this chapter is to focus on recent advances in identifying specific genes and their function in mouse models of the addictions, concentrating on quantitative trait loci (QTL) mapping and the alternative strategy of randomly induced mutagenesis. This approach recognizes that individuals may differ because of differences in the expression of genes as well rather than in the structure of their coded protein. Gene expression arrays are just beginning to be used as a gene discovery tool, and the limited data available are discussed. Finally, candidate gene studies using transgenic technologies to over- or underexpress specific genes in mice are mentioned. Throughout the chapter, the author pleads the case for a behavioral genomics approach to understanding gene function, which depends on the careful analysis of phenotypic variation in its environmental context and on the rigorous mapping of specific behaviors to the psychological constructs they are intended to represent.

Mice lacking either the alpha1 or beta 2 subunit of the GABAA receptor were tested for ethanol, saccharin, or quinine consumption, ethanol-conditioned place preference, ethanol-conditioned taste aversion, ethanol-simulated motor activity, and handling-induced seizures following chronic consumption of an ethanol liquid diet. The alpha1 null mutants showed decreased ethanol and saccharin consumption, increased aversion to ethanol, and a marked stimulation of motor activity after injection of ethanol. The beta 2 null mutants showed decreased consumption of saccharin and quinine, but not ethanol. Surprisingly, neither mutant showed marked changes in handling induced seizures before or after withdrawal of ethanol. The unique effects of deletion of these two GABAA receptor subunits on ethanol responses are discussed in terms of the distinct changes in different populations of GABAA receptors.