Search

The well-documented difference in pentobarbital withdrawal severity between DBA/2J and C57BL/6J mice offers the opportunity to study how differences between allelic variants influence pentobarbital withdrawal via their additive and/or dominance effects and to identify modifier loci that also influence the trait via gene-gene interactions (a form of epistasis). Previous work in our laboratory identified seven provisional quantitative trait loci (QTLs) for pentobarbital withdrawal using BXD recombinant inbred strains. To date, only one of these QTLs has been confirmed, Pbw1. We hypothesized that other loci that act epistatically may also influence genetic variance in pentobarbital withdrawal severity. Using Epistat, a program developed to carry out full-genome searches for epistasis, we identified six provisional epistatic interactions (p < .002) between the provisional QTLs and modifier loci elsewhere in the genome. Verification testing of these interactions using 404 B6D2F2 mice provided supporting evidence that a QTL on chromosome 11 contributes to genetic variance in pentobarbital withdrawal, but only in the presence of a modifier allele on distal chromosome 1 (p = .0004). This modifier is in the same genomic vicinity as loci detected for a variety of withdrawal and seizure phenotypes.

Quantitative trait locus (QTL) mapping has allowed dramatic progress toward the detection and chromosome mapping of minor and major gene loci involved in murine responses to alcohol and other drugs of abuse. Here we focus on the identification of QTLs for one particular trait relevant to addiction, drug withdrawal following acute or chronic drug administration. To date, five significant QTLs (p < 5 x 10(-5)) and six suggestive QTLs (p < 0.001) have been mapped to specific murine chromosomes for alcohol and pentobarbital withdrawal, indicating the presence of a relevant gene or genes at each location. Overlapping QTLs for alcohol withdrawal and pentobarbital withdrawal are identified on murine chromosomes 1, 4, and 11, and may detect the influence of common genes. For many QTLs, candidate genes with relevant neurobiological function lie within the mapped region. Notably, several QTLs for alcohol and pentobarbital withdrawal are in proximity to genes that directly or indirectly affect GABAA receptor-mediated transmission, which has been implicated in some of the actions of alcohol and other drugs. These include a cluster of GABAA receptor genes and genes encoding the enzymes steroid 5 alpha-reductase-1 (involved in biosynthesis of the neuroactive steroid allopregnanolone) and glutamic acid decarboxylase-1 (involved in GABA biosynthesis). This paper will discuss data that examines the involvement of GABAergic genes in withdrawal and other drug responses, including genetic variation in gene sequence, expression and function.

Quantitative genetics and quantitative trait locus (QTL) mapping have undergone a revolution in the last decade. Progress in the next decade promises to be at least as rapid, and strategies for fine-mapping QTLs and identifying underlying genes will be radically revised. In this Commentary we address several key issues: first, we revisit a perennial challenge--how to identify individual genes and allelic variants underlying QTLs. We compare current practice and procedures in QTL analysis with novel methods and resources that are just now being introduced. We argue that there is no one standard of proof for showing QTL = gene; rather, evidence from several sources must be carefully assembled until there is only one reasonable conclusion. Second, we compare QTL analysis with whole-genome mutagenesis in mice and point out some of the strengths and weakness of both of these phenotype-driven methods. Finally, we explore the advantages and disadvantages of naturally occurring vs mutagen-induced polymorphisms. We argue that these two complementary genetic methods have much to offer in efforts to highlight genes and pathways most likely to influence the susceptibility and progression of common diseases in human populations.

Chronic ethanol treatment is known to alter gene expression and function of gamma-aminobutyric acid type-A (GABA(A)) receptors. Here we focus on the beta(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 beta(2) subunit mRNA and peptide content following chronic ethanol administration. In humans, polymorphism at the beta(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 beta(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 beta(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 beta(2) subunit expression.

Background: Behavioral genomics has made dramatic progress toward mapping quantitative trait loci (QTLs) that contain genes responsible for phenotypic differences in a variety of behavioral responses to alcohol (ethanol). We previously identified a QTL on mouse Chromosome 11 that affects genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, this QTL (Alcw3) accounts for 12% of the genetic variability in withdrawal liability. Candidate genes within this QTL encode the gamma-aminobutyric acid type A (GABA A) receptor gamma2, alpha1, alpha6, and beta2 subunits. We recently identified a coding sequence polymorphism between the B6 and D2 strains for the GABA A receptor gamma2 subunit gene (Gabrg2). In this study, we expand our analysis to a panel of BXD strains derived from the B6 and D2 progenitor strains. These BXD strains provide 26 fixed recombinant genotypes that can be used to examine genetic correlations, for example, between a phenotype of interest and allelic variation in a candidate gene., Methods: Gabrg2 was cloned and sequenced from the 26 BXD recombinant inbred strains. We analyzed genetic correlations between allelic variation in Gabrg2 and alcohol phenotypes previously measured in the BXD strain means., Results: Allelic variation in Gabrg2 is correlated genetically with predisposition to acute alcohol withdrawal and may underlie the Alcw3 locus. In addition, Gabrg2 is associated with ethanol-conditioned taste aversion, ethanol-induced motor incoordination, and ethanol-induced hypothermia. A trend is observed for chronic ethanol withdrawal, ethanol-induced loss of righting reflex, and tolerance to ethanol-induced hypothermia and ataxia., Conclusions: Functionally relevant variation in Gabrg2, or a closely linked gene, is correlated genetically with some, but not all, behavioral responses to alcohol. The alcohol-related phenotypes associated with Gabrg2 generally may be characterized as debilitating or motivationally negative.

New methods for identifying chromosomal regions containing genes that affect murine responses to alcohol and drugs have been used to identify many provisional quantitative trait loci (QTLs) since 1991. By 1998, 24 QTLs had been definitively mapped (P<5x10(-5)) to specific murine chromosomes, which indicates the presence of a relevant gene or genes at each location. The syntenic (homologous) region of the human genome for these genes is often known. For many mapped QTLs, candidate genes with relevant neurobiological function lie within the mapped region. Data that implicate candidate genes for specific responses include studies of knockout animals. Current strategies for gene identification include the use of congenic strains containing QTL regions introduced from another strain. There is increasing emphasis on gene-gene and gene-environment interactions in such studies.

Quantitative trait locus (QTL) mapping efforts in alcohol (ethanol) research are beginning to generate promising data that may ultimately lead to the identification of genes influencing alcohol addiction. Rodents have been extensively utilized to study ethanol's rewarding and aversive effects, and to demonstrate the existence of genetic influences on traits such as free-choice ethanol-consumption, ethanol-conditioned place preference and ethanol-conditioned taste aversion. The purpose of the current investigation was to verify or eliminate from further consideration putative QTLs for free-choice ethanol consumption originally identified in BXD Recombinant Inbred (RI) strains and other informative genetic crosses. B6D2F2 mice were utilized in a verification testing strategy to evaluate the viability of putative ethanol consumption QTLs. When data were combined from BXD RI, B6D2F2 and short-term selected line (STSL) mapping studies, verification was obtained for two QTLs, one on Chromosome (Chr) 9 (proximal-mid) and another on Chr 2 (distal), and suggestive verification was obtained for QTLs on Chrs 2 (proximal), 3, 4, 7, and 15. In addition, the possible genetic association of ethanol consumption with conditioned place preference was evaluated. Genetic correlations were estimated from BXD RI strain means, and QTL maps for these traits were compared to evaluate the possibility of a genetic association. The correlational analysis yielded a trend (r = 0.34, p = 0.09), but no statistically significant results. However, comparisons of QTL mapping results between phenotypes suggested some possible genetic overlap for these traits, both putative measures of ethanol reward. These data suggest that the determinants of these two measures are genetically diverse, but may share some common genetic elements.

The ultimate goal of quantitative trait locus (QTL) mapping is to identify the genes affecting complex traits. Using animal models, we recently identified QTLs on mouse Chromosomes (Chrs) 1, 4, and 11 affecting genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, the locus identified on Chr 11 ( approximately 20 cM) accounted for 12% of the genetic variability in withdrawal liability. Candidate genes in proximity to this QTL encode the gamma2, alpha1, and alpha6 subunits of GABAA receptors. The present studies identify a polymorphism between the B6 and D2 strains in the gamma2 subunit gene, Gabrg2, and expand genotypic analysis to their BXD recombinant inbred strains. This polymorphism predicts a difference in amino acid sequence (Ala-11 vs. Thr-11) within the extracellular amino-terminal region of the gamma2 subunit. Analysis using BXD strain means for acute alcohol withdrawal severity suggests that the gamma2 subunit polymorphism is genetically correlated with alcohol withdrawal severity. This is the first report that QTL mapping for an alcohol-related trait has successfully led to the identification of a polymorphic candidate gene product that is genetically associated with the trait.

Alcohol dependence (alcoholism) is accompanied by evidence of tolerance, withdrawal (physiological dependence), or compulsive behavior related to alcohol use. Studies of strain and individual differences using animal models for acute physiological dependence liability are useful means to identify potential genetic determinants of liability in humans. Behavioral and quantitative trait analyses were conducted using animal models for high risk versus resistance to acute physiological dependence. Using a two-step genetic mapping strategy, loci on mouse chromosomes 1, 4, and 11 were mapped that contain genes that influence alcohol withdrawal severity. In the aggregate, these three risk markers accounted for 68% of the genetic variability in alcohol withdrawal. Candidate genes in proximity to the chromosome 11 locus include genes encoding the alpha1, alpha6, and gamma2 subunits of type-A receptors for the inhibitory neurotransmitter, GABA. In addition, suggestive linkage is indicated for two loci on mouse chromosome 2, one near Gad1 encoding glutamic acid decarboxylase, and the other near the El2 locus which influences the seizure phenotype in the neurological mutant strain El. The present analyses detect and map some of the loci that increase risk to develop physiological dependence and may facilitate identification of genes related to the development of alcoholism. Syntenic conservation between human and mouse chromosomes suggests that human homologs of genes that increase risk for physiological dependence may localize to 1q21-q32, 2q24-q37/11p13, 9p21-p23/1p32-p22.1, and 5q32-q35.

Recent studies implicate the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in many neurochemical actions of ethanol and a variety of behavioral responses to acute and chronic ethanol treatment. However, the molecular mechanisms responsible for genetic differences in initial neurochemical or behavioral sensitivity to ethanol, and adaptation following chronic or repeated ethanol administration, remain to be elucidated. Pharmacogenetic research will increasingly move toward mapping, cloning, identification, and functional analysis of the genes underlying the actions of ethanol. The approaches discussed here permit molecular analysis of both known and previously unknown genes regulating behavioral sensitivity to ethanol. The synthesis of molecular methods and behavioral genetics offers immediate hope for delineating the role of the GABA(A) receptor complex, and other determinants of GABAergic neurotransmission, in determining genetic variation in behavioral responses to ethanol.

The successful generation and functional expression of a series of recombinant chimeric transporters, in which distinct functional properties of NET and DAT are exchanged, have allowed the assignment of a number of important functional properties of MPP+ and antidepressant-sensitive catecholamine transporters to specific domains within their primary structure. These studies are the first comprehensive structure-function analysis of members of the rapidly growing superfamily of Na+/Cl- carriers using chimeric transporters. This represents the first step in identifying the specific structural or regulatory determinants that differentiate NET and DAT. An appreciation of the potentially distinct sites for substrate recognition, translocation, and transport inhibition of NET and DAT may facilitate the development of more selective drugs for the treatment of stimulant addiction, human depression, and other affective disorders.

Reuptake systems for monoamines are the initial sites of action for a wide range of therapeutic antidepressants and drugs of abuse, such as cocaine. To delineate structural domains of the dopamine and norepinephrine transporters that contribute to differential interaction with reuptake inhibitors with antidepressant or reinforcing properties, a series of recombinant transporter chimeras were generated and transiently expressed in HeLa cells. The inhibition constants (Ki values) for cocaine and a variety of selective transport inhibitors were determined for each chimera. Analyses of functional chimeras delineate a segment spanning transmembrane domains 5-7 of the norepinephrine transporter of primary importance for high affinity binding of tricyclic and nontricyclic antidepressants (e.g., Ki < 20nM desipramine or nisoxetine). In contrast, all chimeras containing dopamine transporter sequences from this region resemble the dopamine transporter, which demonstrates higher affinity for psychomotor stimulants compared with antidepressants (e.g., Ki = 391 +/- 39 nM cocaine compared with 9365 +/- 1260 nM desipramine). A region including transmembrane domains 1-3 of the norepinephrine transporter also contributes to the interaction of desipramine and nisoxetine, whereas the analogous region of the dopamine transporter influences the affinity for piperazine derivatives (e.g., GBR12909 and LR1111) that are selective for the dopamine transporter. These analyses provide a framework for identifying the precise structural determinants of monoamine transporters involved in selective interactions with antidepressant and psychomotor stimulant reuptake inhibitors.

Most responses to alcohol in both humans and animals are heritable, and this genetic sensitivity to ethanol is determined by multiple genes. However, the number of genes, their identities, and just how they determine susceptibility to the actions of alcohol are unknown. Herein, we describe a multistage strategy for mapping quantitative trait loci (QTLs) using recombinant inbred strains and F2 mice. Precise mapping of the chromosome positions of these QTLs should increase our understanding of the genetic causes for individual differences in behavioral sensitivity to alcohol by (1) identifying genomic markers associated with sensitivity to alcohol, (2) allowing the genes specifying behavior to be cloned by position, and (3) elucidating "candidate" genes demonstrating linkage to markers associated with behavioral responses to alcohol. Syntenic conservation between the mouse and human genomes should facilitate the eventual mapping and cloning of human homologs of these QTLs. Ultimately, cloning of these genes may allow the development of gene therapies or other therapeutic interventions for management or prevention of alcoholism and alcohol abuse.

A quantitative trait locus (QTL) was detected and mapped to proximal chromosome 10 near the markers Mpmv5 and D10Mit51 with a strong influence on morphine-induced analgesia in the BXD recombinant inbred (RI) strains and in an F2 cross (B6D2F2) between the BXD progenitor strains, C57BL/6 and DBA/2. A LOD score of 3.9 (p < .00002) was seen for analgesia using the hot plate assay. Naloxone Bmax was also associated with this chromosome region in BXD RI mice. The mu opioid receptor gene (Oprm) has recently been mapped to this same chromosome region. The observation that several morphine-related traits and naloxone Bmax appear to be partly determined by this presumed single locus is consistent with the hypothesis that the mu opioid receptor gene, or one of its modulators, is the basis for the QTL.

The dopamine (DA) and norephinephrine (NE) transporters demonstrate important differences in their selectivity for catecholamines and the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+), yet their primary sequences and predicted topology are strikingly similar. To delineate discrete structural domains contributing to pharmacologic and kinetic differences between the DA and NE transporters, a series of recombinant chimeras was generated by a restriction site-independent method and expressed in mammalian cells. Functional analyses of the chimeras delineate two discrete regions spanning the first through the third transmembrane domains (TM1-3) and TM10-11 that contribute to differences in their apparent affinities for DA, NE, and MPP+. These studies also suggest that TM2-3 of the DA transporter have a role in selectively increasing the rate of DA uptake as compared with NE. TM4-8 of the DA transporter may influence the relative rate with which MPP+ is taken up into cells and could contribute to its selective toxicity in neurons expressing the DA transporter. These structure-function studies using chimeras of members of the superfamily of Na(+)- and Cl(-)-dependent transporters provide a framework for identifying the specific structural or regulatory determinants contributing to substrate recognition and translocation by the DA and NE transporters.

Behavioral and pharmacological responses of selectively bred and inbred rodent lines have been analyzed to elucidate many features of drug sensitivity and the adverse effects of drugs, the underlying mechanisms of drug tolerance and dependence, and the motivational states underlying drug reward and aversion. Genetic mapping of quantitative trait loci (QTLs) has been used to identify provisional chromosomal locations of genes influencing such pharmacological responses. Recent advances in transgenic technology, representational difference analysis, and other molecular methods now make feasible the positional cloning of QTLs that influence sensitivity to drugs of abuse. This marks a new period of synthesis in pharmacogenetic research, in which networks of drug-related behaviors, their underlying pharmacological, physiological, and biochemical mechanisms, and particular genomic regions of interest are being identified.

A recently-developed method of gene mapping is reviewed. Several responses to EtOH were studied with the purpose of identifying genes with modest effects (Quantitative Trait Loci, or QTLs). As an example, results from a study of acute ethanol withdrawal severity are discussed. Mice from inbred strains C57BL/6J and DBA/2J, and 19 of their Recombinant Inbred (BXD RI) strains, were given 4 g/kg EtOH and their acute withdrawal severity assessed with the handling-induced convulsion (HIC). HIC scores varied markedly among strains. Comparison of the pattern of strain means for withdrawal with a database comprising genotype of each BXD RI strain for almost 800 mapped polymorphic genetic markers revealed associations with several potential QTLs appearing on several mouse chromosomes. To verify the presence of a gene affecting withdrawal, we then withdrawal-tested individual F2 mice bred from the F1 cross of the parental C57 and DBA strains. These mice were then genotyped for several polymorphic markers close to a putative QTL on chromosome 2. Possession of the DBA allele in severely withdrawing F2 animals was significantly associated with one such marker, D2Mit9, confirming the presence of a gene nearby affecting withdrawal. As a further test, mice of the replicated Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) lines, selected for severity of EtOH withdrawal HIC, were also genotyped. Alleles at the D2Mit9 locus assorted disproportionately (and consistently) between the two pairs of WSP and WSR lines, while alleles at other loci did not. Thus, three tests consistently suggest the influence of a gene, tentatively termed Aw1, 37 cM from the centromere on chromosome 2, that appears to control as much as 40% of the genetic variance in withdrawal. The provisional locus is located very near to two candidate genes. Gad1 codes for the synthesis of glutamic acid decarboxylase, the rate-limiting enzyme for synthesis of GABA. A cluster of genes (Scn1, Scn2, Scn3) code for voltage-sensitive sodium channel proteins. These genes are plausible candidates for affecting withdrawal HIC.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.

To determine whether genetic differences in development of ethanol dependence are related to changes in gamma-aminobutyric acidA (GABAA) receptor function, we measured 36Cl- uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Muscimol-stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]- [1,4]benzodiazepine-3-carboxylate) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was not different for ethanol-naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15-4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15-4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.

We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits.

Chronic ethanol treatment is known to alter the function of the gamma-aminobutyric acidA (GABAA) benzodiazepine receptor complex. To determine if genetic differences in development of ethanol dependence are related to expression of GABAA receptor subunits, we measured whole brain levels of mRNA for the alpha 1, alpha 3, alpha 6, gamma 2s, gamma 2L, and gamma 3 receptor subunits in withdrawal seizure-prone and -resistant (WSP and WSR, respectively) mice fed an ethanol-containing liquid diet or a control diet. Brain poly(A)+ RNA was converted to cDNA and amplified by the polymerase chain reaction using primers conserved among GABAA receptor subunits. Quantification was carried out by densitometric analysis of Southern blots generated using subunit-specific probes. Chronic ethanol treatment decreased the content of alpha 1 mRNA in WSP but not WSR mice and decreased the content of alpha 6 mRNA in WSR but not WSP mice. The content of gamma 3 mRNA was increased by chronic ethanol in both lines. In untreated mice, the WSP line had lower levels of alpha 3 and alpha 6 mRNA than the WSR line. Thus, a decrease in the content of alpha 1 mRNA is most clearly linked with development of withdrawal signs, although the amounts of alpha 6 and alpha 3 may also be important in the genetic differences between WSP and WSR mice. In contrast, levels of mRNA for gamma 2S and gamma 2L subunits do not appear to be altered in ethanol dependence.

Recent studies have identified two alternatively spliced forms of the GABA(A) receptor gamma(2) subunit that differ by the presence (gamma(2L)) or absence (gamma(2S)) of an eight-amino acid segment. This insert in the gamma(2L) isoform exists in the proposed cytoplasmic loop region, between M3 and M4, and contains a consensus sequence for phosphorylation by protein kinase C. To examine the regional distribution of this novel receptor subunit in the brain, gamma(2L) subunit mRNA was detected using both in situ hybridization histochemistry and and PCR amplification methods. Hybridization histochemistry with a gamma(2L), subunit-specific oligonucleotide probe revealed that the gamma(2L), subunit mRNA is widely distributed throughout the mouse brain. The highest levels of expression are found in the cerebral cortex, hippocampus, olfactory lobe, and cerebellum. The presence of the gamma(2L), subunit in these regions was confirmed using PCR. Additionally, PCR experiments detected yes subunit mRNA in the cerebral cortex and hippocampus but not in the cerebellum. To examine the functional properties of the gamma(2) subunit isoforms, gamma(2S) and gamma(2L), subunit mRNAs were coexpressed with alpha(1)beta(1) subunit mRNAs in Xenopus oocytes. These experiments indicate that the gamma(2L) and gamma(2S) subunit variants exhibit similar pharmacological properties, including the ability of both isoforms to confer diazepam sensitivity to the receptor complex. In addition, potentiation of GABA responses by pentobarbital in oocytes expressing either subunit isoform is similar. These data indicate that the presence or absence of the additional eight amino acids in the gamma(2) subunit isoforms does not appear to alter the response of the GABA(A) receptor complex to either benzodiazepines and barbiturates at the level of protein phosphorylation present in the oocyte.

Chronic exposure to ethanol is associated with the development of tolerance to the acute effects of ethanol and a withdrawal syndrome characterized by anxiety and seizure susceptibility. In the present study we examined the ability of flumazenil (Ro15-1788), a benzodiazepine receptor antagonist, to reverse neuronal and behavioral manifestations of ethanol tolerance and dependence. A single injection of flumazenil (10 mg/kg, 14 hr before withdrawal) to mice administered a liquid diet containing ethanol for 10 days, reduced seizure severity during withdrawal from ethanol. Acute tolerance to ethanol-induced hypothermia was not sensitive to flumazenil treatment, but tolerance and diazepam-induced cross-tolerance to the ataxic effects of ethanol were reversed by a single injection of flumazenil given 2 to 26 hr before evaluation of tolerance. At a biochemical level, the ability of benzodiazepine inverse agonists (e.g., Ro15-4513) to reduce the activity of gamma-aminobutyric acid (GABA) receptor-operated chloride channels may represent a neuronal manifestation of ethanol dependence (Buck and Harris, 1990). Flumazenil treatment of ethanol-dependent mice 14 hr before isolation of brain membrane vesicles partially reversed the augmentation of Ro15-4513 inhibition of muscimol-stimulated 36Cl- uptake in vitro. These results demonstrate that brief occupation of benzodiazepine receptors by an antagonist may reset the cellular mechanisms responsible for the development of ethanol tolerance and dependence, and support the hypothesis that increased sensitivity to benzodiazepine inverse agonists is involved in the development of ethanol dependence.

Cellular responses of neuronal tissue to chronic ethanol exposure are reviewed. Evidence for adaptive responses to the acute actions of ethanol is available for five systems: GABA-activated chloride channels, voltage-sensitive calcium channels, NMDA-activated cation channels, receptors coupled through stimulatory guanine nucleotide binding proteins, and membrane lipid order. We suggest that at least some of these adaptive responses occur because of ethanol actions at the level of gene expression.

Acute exposure to ethanol was found to enhance the ability of a benzodiazepine (BZ) inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), to reduce muscimol-activated 36Cl- uptake by membranes isolated from mouse cerebral cortex. Pretreatment in vivo with a hypnotic dose of ethanol (but not a subhypnotic dose), or exposure to a corresponding concentration in vitro, was effective. This increase in sensitivity of gamma-aminobutyric acid receptor-operated chloride channels to the actions of DMCM was due to an increase in both the potency and efficacy of DMCM. Sensitization to DMCM was reversible and was not observed 24 hr after a single injection of ethanol. Pretreatment with ethanol (10, 50 and 100 mM) in vitro produced sensitization to DMCM in a concentration-dependent manner, similar to that produced by in vivo exposure; this increase in sensitivity did not develop if the membranes were pretreated with ethanol at 0 degrees C. Similarly, in vitro exposure to pentobarbital (200 microM) or flunitrazepam (1 microM) enhanced the actions of the inverse agonist Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]BZ-3- carboxylate). Acute ethanol exposure did not alter low-affinity gamma-aminobutyric acidA receptor binding or muscimol action, or the ability of a BZ agonist, flunitrazepam, to augment muscimol-activated chloride flux. Ethanol exposure did not alter [3H]flumazenil (Ro15-1788) binding to central BZ receptors, its displacement by DMCM or allosteric modulation of DMCM binding by muscimol (muscimol-shift).(ABSTRACT TRUNCATED AT 250 WORDS)

Mice were made tolerant to and dependent on ethanol by administration of a liquid diet. Gamma-aminobutyric acid (GABA) receptor-dependent uptake of 36Cl- by mouse cortical microsacs was used to study the actions of benzodiazepine (BZ) agonists and inverse agonists. Chronic exposure to ethanol attenuated the ability of a BZ agonist, flunitrazepam, to augment muscimol-stimulated uptake of 36Cl- and enhanced the actions of BZ inverse agonists, Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,4]-benzodiazepine - 3-carboxylate) and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), to inhibit GABAA receptor-operated chloride channels. Augmentation of chloride flux by pentobarbital was not reduced by chronic ethanol exposure. Attenuation of flunitrazepam efficacy was transient and returned to control levels within 6 to 24 hr after withdrawal from ethanol, but increased sensitivity to Ro15-4513 was observed as long as 8 days after withdrawal. Chronic exposure to ethanol did not alter [3H]SR 95531 ([2-(3'-carbethoxy-2'propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide] binding to low-affinity GABAA receptors or muscimol stimulation of chloride flux; and did not alter [3H]Ro15-4513 or [3H]flunitrazepam binding to central BZ receptors or allosteric modulation of this binding by muscimol (i.e., muscimol-shift). These results suggest that chronic exposure to ethanol reduces coupling between BZ agonist sites and the chloride channel, and may be responsible for the development of cross-tolerance between ethanol and BZ agonists. In contrast, coupling between BZ inverse agonist sites and the chloride channel is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravenous administration of 2-[2-methoxyethoxy]-ethyl 8-[cis-2-n-octylcyclopropyl]-octanoate (A2C) was found to disorder brain membranes but did not produce intoxication or anesthesia in mice. The abilities of A2C and an anesthetic (benzyl alcohol) to inhibit [35S]t-butylbicyclophosphorothionate (TBPS) binding, and modify gamma-aminobutyric acid (GABA) receptor-mediated 36Cl- influx into brain vesicles were then compared. Both of the perturbants inhibited [35S]TBPS binding at the same concentrations at which they reduced membrane order; however, the anesthetic was nearly 4 times more effective in reducing [35S]TBPS binding than was A2C. Muscimol-stimulated 36Cl- uptake was enhanced by benzyl alcohol at a concentration which produced little or no change in membrane order. Concentrations of both A2C and benzyl alcohol which reduced membrane order inhibited muscimol-stimulated 36Cl- influx. Similarly, membrane order and muscimol-activated 36Cl- uptake were reduced in brain vesicles prepared from mice which had received A2C in vivo. The effects of anesthetics on the GABAA receptor-chloride channel complex were analyzed by a two site model of action in which a 'perturbant' site is responsible for decreased 36Cl- uptake; but a distinct 'anesthetic' site is responsible for augmentation of chloride flux and anesthesia.