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.

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.

The focus of this review is on progress achieved in identifying specific genes conferring risk for anxiety disorders through the use of genetic animal models. We discuss gene-finding studies as well as those manipulating a candidate gene. Both human and animal studies thus far support the genetic complexity of anxiety. Clinical manifestations of these diseases are likely related to multiple genes. While different anxiety disorders and anxiety-related traits all appear to be genetically influenced, it has been difficult to ascertain genetic influences in common. Mouse studies have provisionally mapped several loci harboring genes that affect anxiety-related behavior. The growing array of mutant mice is providing valuable information about how genes and environment interact to affect anxious behavior via multiple neuropharmacological pathways. Classical genetic methods such as artificial selection of rodents for high or low anxiety are being employed. Expression array technologies have as yet not been employed, but can be expected to implicate novel candidates and neurobiological pathways.

Both the nucleus accumbens (NAcc) and central amygdala (CeA) are thought to play roles in tolerance to, and dependence on, abused drugs. Although our past studies in rat brain slices suggested a role for NMDA receptors (NMDARs) in NAcc neurons in the effects of acute and chronic opiate treatment, the cellular and molecular mechanisms remained unclear. Therefore, we examined the effects of morphine dependence on electrophysiological properties of NMDARs in freshly isolated NAcc neurons and on expression of mRNA coding for NR2A-C subunits using single-cell RT-PCR. Chronic morphine did not alter the affinity for NMDAR agonists glutamate, homoquinolinate, or NMDA, but decreased the affinity of the coagonist glycine. Chronic morphine altered the NMDAR inhibition by two NMDAR antagonists, 7-Cl-kynurenate and ifenprodil, but not that by d-APV or Mg2+. Chronic morphine accelerated the NMDA current desensitization rate in NAcc neurons. In single-cell RT-PCR, chronic morphine predominantly reduced the number of neurons expressing multiple NR2 subunits. Ethanol also alters NMDARs. We found that low ethanol concentrations (IC50 = 13 mM) inhibited NMDA currents and NMDA-EPSPs in most NAcc neurons in a slice preparation. NAcc neurons from ethanol-dependent rats showed enhanced NMDA sensitivity. In CeA neurons, acute ethanol decreased (by 10-25%) non-NMDA- and NMDA-EPSPs in most neurons. In CeA neurons from ethanol-dependent rats, acute ethanol decreased the non-NMDA-EPSPs to the same extent as in naïve rats, but inhibited (by 30-40%) NMDA-EPSPs significantly more than in controls, suggesting sensitization to ethanol. Preliminary studies with microdialysis and real-time PCR analysis support this idea: local ethanol administration in vivo had no effect on glutamate release, but chronic ethanol nearly tripled the expression of NR2B subunits (the most ethanol sensitive) in CeA. These combined findings suggest that changes in glutamatergic transmission in NAcc and CeA may underlie the neuroadaptions that lead to opiate and ethanol dependence.

Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in the spinal cord and brainstem. To better define the function of this receptor in vivo, we constructed a point mutation that impairs receptor function in the α1-subunit and compared these knock-in mice to oscillator (spdot) mice lacking functional GlyR α1-subunits. Mutation of the serine residue at amino acid 267 to glutamine (α1S267Q) results in a GlyR with normal glycine potency but decreased maximal currents, as shown by electrophysiological recordings using Xenopus oocytes. In addition, single-channel recordings using human embryonic kidney 293 cells indicated profoundly altered properties of the mutated GlyR. We produced knock-in mice bearing the GlyR α1 S267Q mutation to assess the in vivo consequences of selectively decreasing GlyR efficacy. Chloride uptake into brain synaptoneurosomes from knock-in mice revealed decreased responses to maximally effective glycine concentrations, although wild-type levels of GlyR expression were observed using 3H-strychnine binding and immunoblotting. A profound increase in the acoustic startle response was observed in knock-in mice as well as a “limb clenching” phenotype. In contrast, no changes in coordination or pain perception were observed using the rotarod or hot-plate tests, and there was no change in GABAA-receptor-mediated chloride uptake. Homozygous S267Q knock-in mice, like homozygous spdot mice, exhibited seizures and died within 3 weeks of birth. In heterozygous spdot mice, both decreased 3H-strychnine binding and chloride flux were observed; however, neither enhanced acoustic startle responses nor limb clenching were seen. These data demonstrate that a dominant-negative point mutation in GlyR disrupting normal function can produce a more dramatic phenotype than the corresponding recessive null mutation, and provides a new animal model to evaluate GlyR function in vivo.

Two-dimensional gel electrophoresis (2-DE) was used to separate protein samples solubilized from the nucleus accumbens and hippocampus of alcohol-naïve, adult, male inbred alcohol-preferring (iP) and alcohol-nonpreferring (iNP) rats. Several protein spots were excised from the gel, destained, digested with trypsin, and analyzed by mass spectrometry. In the hippocampus, 1629 protein spots were matched to the reference pattern, and in the nucleus accumbens, 1390 protein spots were matched. Approximately 70 proteins were identified in both regions. In the hippocampus, only 8 of the 1629 matched protein spots differed in abundance between the iP and iNP rats. In the nucleus accumbens, 32 of the 1390 matched protein spots differed in abundance between the iP and iNP rats. In the hippocampus, the abundances of all 8 proteins were higher in the iNP than iP rat. In the nucleus accumbens, the abundances of 31 of 32 proteins were higher in the iNP than iP rat. In the hippocampus, only 2 of the 8 proteins that differed could be identified, whereas in the nucleus accumbens 21 of the 32 proteins that differed were identified. Higher abundances of cellular retinoic acid-binding protein 1 and a calmodulin-dependent protein kinase (both of which are involved in cellular signaling pathways) were found in both regions of the iNP than iP rat. In the nucleus accumbens, additional differences in the abundances of proteins involved in (i) metabolism (e.g., calpain, parkin, glucokinase, apolipoprotein E, sorbitol dehydrogenase), (ii) cyto-skeletal and intracellular protein transport (e.g., β-actin), (iii) molecular chaperoning (e.g., grp 78, hsc70, hsc 60, grp75, prohibitin), (iv) cellular signaling pathways (e.g., protein kinase C-binding protein), (v) synaptic function (e.g., complexin I, γ-enolase, syndapin IIbb), (vi) reduction of oxidative stress (thioredoxin peroxidase), and (vii) growth and differentiation (hippocampal cholinergic neurostimulating peptide) were found. The results of this study indicate that selective breeding for disparate alcohol drinking behaviors produced innate alterations in the expression of several proteins that could influence neuronal function within the nucleus accumbens and hippocampus.

Background The power of microarray analysis can be realized only if data is systematically archived and linked to biological annotations as well as analysis algorithms. Description The Longhorn Array Database (LAD) is a MIAME compliant microarray database that operates on PostgreSQL and Linux. It is a fully open source version of the Stanford Microarray Database (SMD), one of the largest microarray databases. LAD is available at Conclusions Our development of LAD provides a simple, free, open, reliable and proven solution for storage and analysis of two-color microarray data.