Alcoholism is a complex genetic trait; susceptibility is influenced by multiple genes of small effect. To pursue mechanistic studies, genetic animal models have been used. These models are partial, each addressing one or more of the contributing traits rather than the disease as a whole. Animal studies have modeled alcohol's rewarding effects, the development of tolerance, the pathological consequences to brain systems, and the dependence on alcohol inferred from the presence of withdrawal symptoms when the drug is removed. The classical genetic methods of inbred strain analysis and development and studies of selectively bred lines have been employed for more than 40 years. Recently, such studies have shown that a genetic tendency to experience severe withdrawal is associated with a tendency to avoid self-administration of alcohol. Also recently, attempts to identify the specific genes conferring risk or protection from alcohol's effects have been undertaken. These studies have used mapping techniques based on gene sequence polymorphisms, studies of gene expression differences, and the use of candidate gene targeting such as creation of null mutants. Studies reviewed here have mapped quantitative trait loci (QTL) for many genes affecting alcohol sensitivity, tolerance, reward, and withdrawal severity. The furthest progress in gene mapping has been made toward one withdrawal QTL on mouse chromosome 4. Using multiple congenic strains, the gene conferring increased withdrawal severity has been isolated to a region of less than 1 centiMorgan, containing fewer than 20 genes. A strong candidate gene, coding for a multiple PS095/DLG/Z0-1 (PDZ) binding domain zinc finger protein, cannot be excluded. Although many more such genes will be identified in the near future, their contribution to the mapped phenotype will be shown to be dependent on epistatic interactions with other risk genes, as well as genes in the animal's background. Progress in gene identification will also depend crucially on the precise description of the phenotypes being mapped so that their pleiotropic range of influence on the multi-behavioral phenotypic syndrome can be determined. © 2002 Wiley-Liss, Inc.

The medium spiny neurons of the nucleus accumbens receive both an excitatory glutamatergic input from forebrain and a dopaminergic input from the ventral tegmental area. This integration point may constitute a locus whereby the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors promotes drug reinforcement. Here we investigate how dopaminergic inputs alter the ethanol sensitivity of NMDA receptors in rats and mice and report that previous dopamine receptor-1 (D1) activation, culminating in dopamine and cAMP-regulated phosphoprotein-32 kD (DARPP-32) and NMDA receptor subunit-1 (NR1)-NMDA receptor phosphorylation, strongly decreases ethanol inhibition of NMDA responses. The regulation of ethanol sensitivity of NMDA receptors by D1 receptors was absent in DARPP-32 knockout mice. We propose that DARPP-32 mediated blunting of the response to ethanol subsequent to activation of ventral tegmental area dopaminergic neurons initiates molecular alterations that influence synaptic plasticity in this circuit, thereby promoting the development of ethanol reinforcement.

Previous studies have shown that ethanol enhanced [(3)H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity.

.Rationale: G-protein-coupled inwardly rectifying potassium channels (GIRKs) regulate synaptic transmission and neuronal firing rates. Co-localization of GIRK2 channels and dopamine receptors in the mesolimbic system suggests a role in regulation of motor activity. Objectives: To explore the role of GIRK channels in the regulation of motor behavior. Methods: GIRK2 null mutant mice (knockout) were used. Locomotor activity in a mildly stressful situation was conducted either in a circular open field with video tracking or in standard mouse cages equipped with infrared sensors. Drugs were injected intraperitoneally or subcutaneously. Results: GIRK2 knockout mice demonstrated a transient "hyperactive" behavioral phenotype with initially higher motor activity and slower habituation in a novel situation, increased levels of spontaneous locomotor activity during dark phase in their home cages, and impaired habituation in the open-field test. After habituation, GIRK2 knockout mice showed higher motor activity, which was inhibited by the D1 receptor antagonist SCH 23390 and was more sensitive to the activating effects of the D1 receptor partial agonist SKF 38393. In a novel environment (open-field) only the highest dose of SKF38393 used (20 mg/kg) produced significant activation, perhaps due to a ceiling effect in GIRK2 knockout mice. SCH 23390 inhibited the basal activity levels of mice of both genotypes. Conclusions: Activation of the dopamine D1 receptor in a stressful environment may be stronger in GIRK2 deficient mice, and this modified function of D1 receptors may cause the transient hyperactive behavioral phenotype of these mice.

Alcoholism is a major health problem in Western countries, yet relatively little is known about the mechanisms by which chronic alcohol abuse causes the pathologic changes associated with the disease. It is likely that chronic alcoholism affects a number of signaling cascades and transcription factors, which in turn result in distinct gene expression patterns. These patterns are difficult to detect by traditional experiments measuring a few mRNAs at a time, but are well suited to microarray analyses. We used cDNA microarrays to analyze expression of approximately 10 000 genes in the frontal and motor cortices of three groups of chronic alcoholic and matched control cases. A functional hierarchy was devised for classification of brain genes and the resulting groups were compared based on differential expression. Comparison of gene expression patterns in these brain regions revealed a selective reprogramming of gene expression in distinct functional groups. The most pronounced differences were found in myelin-related genes and genes involved in protein trafficking. Significant changes in the expression of known alcohol-responsive genes, and genes involved in calcium, cAMP, and thyroid signaling pathways were also identified. These results suggest that multiple pathways may be important for neuropathology and altered neuronal function observed in alcoholism.

In previous work, we identified genetic correlations between cAMP accumulation in the cerebellum and sensitivity to the incoordinating effects of ethanol. A genetic correlation suggests that common genes underlie the phenotypes investigated. One method for provisionally identifying genes involved in a given phenotypic measure is quantitative trait locus (QTL) analysis. Using a panel of 30 BXD recombinant inbred strains of mice and the progenitors (DBA/2J and C57BL/6J), and the dowel test for ataxia, we measured the blood ethanol concentrations at the time an animal first fell from the dowel and acute functional tolerance (AFT), and investigated cAMP signaling in the cerebellum. Cyclic AMP accumulation was measured in whole-cell preparations of cerebellar minces from individual mice under basal or stimulated conditions. We conducted a genome-wide QTL analysis of the behavioral and biochemical measures with \textgreater2000 genetic markers to identify significant associations. Western blot and comparative sequencing analysis were used to compare cAMP response element binding protein (CREB) levels and protein-coding sequence, respectively. QTL analyses correlating strain means with allelic status at genetic markers identified several significant associations (p \textless 0.01). Analysis of variance revealed an effect of strain on behavioral and biochemical measures. There was a significant genetic correlation between initial sensitivity and basal cAMP accumulation in the cerebellum. We identified 6 provisional QTLs for initial sensitivity on four chromosomes, 6 provisional QTLs for AFT on four chromosomes, and 11 provisional QTLs for cAMP signaling on nine chromosomes. Two loci were found to overlap for measures of initial sensitivity and for cAMP signaling. Given the genetic correlation between initial sensitivity and basal cAMP accumulation, we investigated candidate genes in a QTL on chromosome 1. Comparative sequence analysis was performed, and protein levels were compared between C57 and DBA mice for Creb1. No significant differences were detected in coding sequence or protein levels for CREB. These results suggest that although ethanol sensitivity and cAMP signaling are determined by multiple genes, they may share certain genetic codetermination.

The overall scheme of a microarray experiment is summarized in Figure 3. The real benefit of using microarrays in research is gaining knowledge from the abundant data provided from the series of related microarray experiments. The core laboratory has produced three cDNA arrays, a mouse 15K array, a human 13K array, and a human apoptosis array with 350 plus apoptotic elements. Recently, the 15K array is being used in building a database for gene expression in several areas of the mouse brain and for studying transgenic and knockout mice. The apoptosis array has been used by cancer researchers to further elucidate changes and interactions between genes leading to cell death and cancer. The UCHSC Gene Expression Array Core is supported by the National Institute on Aging and the National Cancer Institute (Bethesda, MD) to serve all academic users and has a special interest in providing a solid technological base for genomic researchers interested in alcohol and cancer research.

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that inhibit neurotransmission in the adult brainstem and spinal cord. GlyR function is potentiated by ethanol in vitro, and a mutant GlyR subunit α1(S267Q) is insensitive to the potentiating effects of ethanol. To test the importance of GlyR for the actions of ethanol in vivo, we constructed transgenic mice with this mutation. Under the control of synapsin I regulatory sequences, transgenic expression of S267Q mutant GlyR α1 subunits in the nervous system was demonstrated using [3H]strychnine binding and immunoblotting. These mice showed decreased sensitivity to ethanol in three behavioral tests: ethanol inhibition of strychnine seizures, motor incoordination (rotarod), and loss of righting reflex. There was no change in ethanol sensitivity in tests of acute functional tolerance or body temperature, and there was no change in ethanol metabolism. Transgene effects were pharmacologically specific for ethanol, compared with pentobarbital, flurazepam, and ketamine. These results support the idea that glycine receptors contribute to some behavioral actions of ethanol and that ethanol sensitivity can be changed in vivo by transgenic expression of a single receptor subunit.