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RSA 2004 Abstracts

University of Colorado Health Sciences Center

ROLE OF BRAIN ADENYLYL CYCLASE TYPE 7 IN ANXIOLYTIC EFFECT OF ETHANOL AND GENE EXPRESSION

W. Hu; S.V. Bhave; M. Yoshimura; P.L. Hoffman; B. Tabakoff

University of Colorado Health Sciences Center, Denver, CO 80262

Many behaviors, including anxiety, are involved in the development of alcoholism. The cAMP/PKA signaling cascade has been linked to the etiology of anxiety, depression and alcoholism. Since adenylyl cyclase Type VII (AC7) was found to be most sensitive to the effect of ethanol, we have developed transgenic mice (TG) expressing the human AC7 gene in brain, and knockout mice (HET) which lack one copy of endogenous mouse AC7 gene, for the study of the role of cAMP/PKA signaling in anxiety, depression and alcoholism. We have evaluated the role of AC7 in the anxiolytic effect of ethanol in male and female TG and HET mice using the elevated plus maze. After saline treatment, male TG mice were less anxious (p < 0.05) than WT mice. Ethanol (1.25 g/Kg) significantly increased open arm entry and time (decrease anxiety) in both WT and TG mice, but the effect of ethanol was more profound in TG. In contrast, the anxiolytic effect of ethanol (1.25 g/kg) was not evident in male HET mice. In female mice, there was no significant difference among WT, HET, and TG mice either in basal ?anxiety? level, or in the anxiolytic effect of ethanol (1.25 g/kg). Oligonucleotide microarrays were used to evaluate the patterns of gene expression in both genders of WT and genetically modified mice. The expression of the AC7 transgene, and the AC7 heterozygous knockout, produced a significantly different spectrum of gene expression differences in the brain of male and female mice. Supported in part by NIAAA (INIA) and the Banbury Foundation.

EXPRESSION OF ETHANOL METABOLIZING SYSTEMS IN BRAINS OF INBRED MOUSE STRAINS DIFFERING IN THEIR BEHAVIORAL RESPONSES TO ETHANOL

S.V. Bhave; P.L. Hoffman; R.A. Deitrich; B. Tabakoff

University of Colorado Health Sciences Center, Denver, CO 80262.

Inbred mouse strains, such as C57BL/6J and DBA/2J, have been widely used in understanding various behavioral effects of ethanol. These strains differ significantly in terms of alcohol preference, and a number of ?candidate genes? expressed in brain have been proposed to underlie the differences in ethanol preference. It has also been suggested that differences in ethanol metabolizing systems in these two strains of mice may play a role in preference for ethanol. Acetaldehyde, one of the metabolites of ethanol, has been suggested to play either an aversive or reinforcing role in ethanol preference. Steady-state acetaldehyde levels in the brain are a reflection of in situ acetaldehyde formation, delivery from the circulation and catabolism. Acetaldehyde has been proposed to be produced in the brain by alcohol dehydrogenase (ADH isoenzymes) and catalase and/or cytochrome P450 (CYP450 isoforms), and aldehyde dehydrogenases (ALDH isoenzymes) are postulated to be responsible for its removal. We have utilized oligonucleotide microarray technology to simultaneously assess expression of ethanol and acetaldehyde metabolizing enzymes including ADHs, ALDHs, catalase and cytochrome P450 isoenzymes in the whole brain of C57BL/6J and DBA/2J mice. There were no significant differences between the two strains of mice in gene expression intensity (GEI) for the majority of enzymes (ADHs, catalase and CYP450 family of genes) involved in ethanol catabolism. However, a significantly higher expression of ALDH2, an isoenzyme mainly responsible for the removal of acetaldehyde, was observed in whole brains of DBA mice compared to C57 mice. In contrast, the GEI for the ALDH7 isoenzyme was significantly higher in C57 mice compared to DBA mice. These gene expression differences may contribute to differential ethanol preference in the two strains. Supported in part by NIAAA (INIA) and the Banbury Foundation.

COMPARISONS OF GENE EXPRESSION DATA OBTAINED USING DIFFERENT MICROARRAY PLATFORMS

T. Phang; B. Soriano; J. Gaydos; S.V. Bhave; R. Lapadat; G. Zerbe; P.L. Hoffman; L. Hunter; B. Tabakoff

University of Colorado Health Sciences Center, Denver, CO 80262

Characterization of gene expression patterns using oligonucleotide based microarrays to understand the influence of genetic components and to evaluate the effects of various drugs of abuse has become popular in recent years. A wide variety of microarray platforms, based on the technologies used in manufacture, as well as the variety of the probe types, are in use at present. In spite of the popularity of these various platforms, there is a lack of standard ways of comparing the data obtained using different oligonucleotide based platforms. We have compared the patterns of gene expression in the whole brains of C57BL/6J and DBA/2J mice, strains of mice widely used to study the effects of ethanol, using three different microarray platforms. The same RNA samples were used with Affymetrix and Amersham oligo-microarray platforms and an in-house oligonucleotide array. The gene expression intensity (GEI) data were obtained from microarray images using the default settings for the given platform. The GEI values from each of these platforms were further filtered to remove genes below the detection levels. The GEI data for genes that were represented on all three platforms were further used to compare the data within and between the platforms. We have used two different approaches to analyze the genes that were represented on all three platforms, first, using the UniGene IDs for the probe sets on these microarrays and second by comparing the probe sequences for the probe sets. The data were analyzed using various statistical methods, such as correlation, and class comparison. The availability of such a knowledge base will aid comparisons of the gene expression data obtained using various platforms for large scale gene expression measurements, and will provide a basis for statistical meta-analysis. Supported in part by NIAAA (INIA) and the Banbury Foundation.

NMDA RECEPTORS IN THE AMYGDALA OF ETHANOL-TREATED P RATS

P.L. Hoffman; K. Hedman; R.L. Bell; W.N. Strother; B. Tabakoff

University of Colorado Health Sciences Center, Denver, CO 80262.

Dynamic changes in the neuronal localization of NMDA receptors, including movement of receptor subunits to and from the cell surface and synaptic sites, play a key role in mechanisms of synaptic plasticity. Neuroadaptive (tolerance, dependence, ?craving?) changes in neuronal function induced by chronic ethanol exposure and withdrawal may also be mediated in part by altered levels and localization of NMDA receptors. We have measured NMDA receptor subunit proteins in homogenates and synaptic membrane fractions from amygdala of control and ethanol-treated alcohol-preferring P rats. The rats were given 6-week free-choice access to ethanol followed by ethanol deprivation and reinstatement periods, in order to produce an alcohol deprivation effect that is thought to model alcohol ?craving?. Control groups had continuous access to ethanol or to water only. NMDA receptor subunit proteins and the scaffolding protein, PSD-95, were measured after subcellular fractionation and immunoblotting. Initial results showed increases in NR1, NR2B and PSD-95 in the synaptic membrane fraction following the alcohol deprivation period. NR1 remained elevated after ethanol reinstatement, while NR2B and PSD-95 returned toward control levels. NR1 in the total homogenate was not affected by the ethanol treatment, while levels of NR2A, NR2B and PSD-95 increased following deprivation and/or reinstatement. The results suggest that ethanol deprivation, following free-choice intake, is associated with changes in the synaptic localization of NMDA receptors in the rat amygdala, potentially leading to altered NMDA receptor function associated with anxiety, reinforcement and/or craving for alcohol. Supported by NIAAA (AA14101, AA07611, AA13522, AA13521) and Banbury Fund.

TRANSPLANTATION OF STEM CELLS EXPRESSING THE HUMAN DOPAMINE TRANSPORTER INTO THE CNS IN AN EFFORT TO MODIFY ALCOHOL PREFERENCE

S.Jones, T. Grammatapoulous, M. Yoshimura, B. Hoover, E. Snyder, N. Zahniser, B. Tabakoff, M. Zawada

University of Colorado Health Sciences Center, Denver CO 80262

-To determine whether dopamine (DA) levels in the mesolimbic system modulate the preference for alcohol, we have stably transfected C17.2 neural stems cells derived from mouse with the human dopamine transporter (hDAT) gene to generate the C17.hDAT stem cell line. The C17.2 cells were stably transfected using a construct containing the hDAT gene driven by a chicken b-actin promoter and a cytomegalovirus enhancer. Several clones exhibited constitutive [3H]-DA uptake with normal affinity, but varying maximal velocities. We hypothesized that reduction of the extracellular DA levels via hDAT will alter alcohol preference. To test the engraftability of these cells, the clones were injected into the ventricles of one day old C57/BL6 mouse pups. Two and three weeks after grafting, the transplanted stem cells were localized in the brains of recipients by staining with X-gal. The b-galactosidase-expressing stem cells were seen in the olfactory bulb, and in the case of some clones, widely distributed throughout the brain. Those clones showing the longest duration of survival were transplanted into nucleus accumbens of the adult mouse brain, one of the nuclei thought to affect ethanol preference. Prior to transplantation, mice were tested for ethanol preference using a two bottle choice paradigm. After 10 days of exposure, 80% of C57/BL6 mice consumed more ethanol than water. Mice exhibiting a preference for alcohol were chosen for transplantation with either C17.hDAT cells, or a mock-transfected C17.2 clone. One week after grafting, surviving C17.hDAT cells were identified in the transplants by X-gal staining, antibodies against b-galactosidase, and in some cases staining for BrdU. Expression of hDAT was detected in transplanted cells by hDAT immunocytochemistry. Two and three week survival paradigms and potential alteration of the ethanol preference are currently being tested. In addition, the C17.hDAT stem cells will be transplanted into DAT knockout mice to determine whether they are capable of normalizing DAT levels and function in the CNS. Transplantation of hDAT-expressing stem cells into brain nuclei known for their role in substance seeking should be a useful tool for exploring the role of DA in alcohol dependence. Supported by NIH AA13473.

EXPLOITING THE GENE ONTOLOGY AND OTHER KNOWLEDGE-BASES FOR ALCOHOL NEUROINFORMATICS

L. Hunter, T. Phang, G. Acquaah-Mensah, J. Eberlein, P. Ogren, A. Gabow

Center for Computational Pharmacology, Box C236 University of Colorado Health Sciences Center, Denver, CO 80262

Interpretation of high throughput molecular biology data such as gene expression arrays and shotgun proteomics is best done in the light of as much existing biological knowledge regarding the genes, gene products and interactions among them as is possible. However, such knowledge about all potentially relevant human and model organism genes arises from a wide variety of different biological subdisciplines, and is growing at a prodigious rate. Building on the Gene Ontology, integrating a wide variety of data sources and using computational techniques for knowledge representation, we have built the Biognosticopaea, a knowledge-base of molecular biology focused on the area of alcohol research. We will describe the knowledge-base, methods required for its construction, and novel analyses made possible by its existence. Supported by NIAAA INIA Consortium Grant 5 U01 AA13524-03


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