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RSA 2005 Abstracts
UNIVERSITY OF TEXAS AT AUSTIN
ALCOHOL DRINKING INDUCED ALTERATIONS IN DOPAMINE D2 RECEPTOR EXPRESSION IN CHOLINERGIC INTERNEURONS OF THE PREFRONTAL CORTEX, DORSAL STRIATUM AND NUCLEUS ACCUMBENS OF ALCOHOL-PREFERRING (P) RATS
M.C. Camp, A.A. Alcantara
The University of Texas at Austin, Austin, Texas 78712
The prefrontal cortex (PFC), dorsal striatum and nucleus accumbens (NuAcc) are among the key brain areas implicated in the development and maintenance of alcohol abuse and dependence. Previous findings in our lab have revealed the involvement of cholinergic interneurons, as measured by Cdk5 immunoreactivity, in these brain areas of P rats following 1 month of alcohol drinking. Neuroadaptive changes in dopamine D2 receptor levels have previously been reported to occur following alcohol drinking and withdrawal. Evidence that overexpression of D2 attenuates alcohol drinking (Thanos et al., 2004) further implicates D2 receptors as putative targets for pharmacotherapies for the treatment of alcohol abuse and dependence. Alcohol driven changes in D2 expression in specific cell types, however, have not been previously examined. The present study investigated neuroadaptive changes in D2 receptor expression in cholinergic neurons of the PFC, dorsal striatum and NuAcc of P rats following 1 month of voluntary alcohol drinking, using an unlimited access two-bottle choice (10% alcohol versus water) drinking paradigm.
Findings from the present study revealed an 86% decrease in the number of cholinergic neurons that expressed D2 receptors in the prelimbic region of the PFC, a 34% decrease in the core NuAcc and 46% increase in the ventromedial striatum as compared to controls. Our results provide evidence that alcohol induced alterations in D2 receptors in cholinergic neurons of the PFC, striatum and NuAcc may contribute to the myriad of neuroadaptive changes in the brain that contribute to the development of alcohol abuse and dependence. Identifying alcohol driven neuroplasticity in specific brain areas and cellular microcircuits should contribute to the development of site-specific targeted pharmacotherapies and behavioral prevention and treatment programs for alcohol abuse and alcoholism.
This research was supported by grants from the National Institute of Alcohol Abuse and Alcoholism, NIH (AA13497-04-INIA Project; 5 T32 AA07471-18- Alcohol Training Grant), Jones Fellowship, and Waggoner Center for Alcohol and Addiction Research.
THE GENETIC DETERMINANTS OF ALOHOL PREFERENCE: BRAIN GENE EXPRESSION ANALYSIS IN DIFFERENTIALLY ALCOHOL PREFERRING C57BL/6J, FVB/NJ AND F1 PROGENY
M. K. Mulligan; Y.A. Blednov; J.C. Crabbe; B. Tabakoff; S.V. Bhave; P.S. Levin; J.A. Owen; S.E. Bergeson.
University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
Differences in alcohol preference and consumption between inbred strains of mice have been well characterized. However, although C57BL/6J (B6) mice are the most utilized murine choice for alcohol drinking studies, under most experimental conditions they fall short of meeting general requirements for a “good” model of alcoholism. Recently, a two-bottle choice “screen” of F1 offspring from reciprocal crosses of B6 and several other inbred strains revealed that FVBxB6 and B6xFVB hybrid mice consume more, and show a stronger preference for alcohol than their B6 progenitor strain (Blednov et. al., unpublished data). [FVB/NJ mice show a strong preference for water over alcohol in the two-bottle choice standard paradigm.] To determine whether differences in drinking were related to inherent differences in alcohol metabolism or induced changes that result in metabolic tolerance, several tests were completed. Measurement of initial blood ethanol concentration and clearance following alcohol consumption, gavage or i.p. injection revealed no significant differences in metabolism between B6 and F1 hybrid mice, regardless of previous drinking history. The novel F1 mouse model creates a new opportunity to study high levels of alcohol self-administration. Whole brain microarray analysis was performed using naïve female mice from each parental strain and reciprocal F1 cross (n=5/group) in order to identify molecular mechanisms underlying the F1 high alcohol preference phenotype. Candidate genes were prioritized by level of significance and similarity of gene expression differences to the pattern of alcohol consumption for each genotype. Supported by NIAAA grants; U01AA013475 – INIA Project, K01AA103403, and R21AA103182, the Waggoner Center for Alcohol and Addiction Research, and a Bruce Jones Fellowship (MKM).
GENETIC AND BEHAVIORAL ANALYSIS OF DISPARATE ALCOHOL PREFERENCES IN TWO CLOSELY RELATED INBRED STRAINS OF C57BL/6 MICE
M.K. Mulligan; S.L. Boehm, II; I. Ponomarev; P.S. Levin; S.E. Bergeson.
University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
C57BL/6 (B6) mice have been widely used as a research model for the investigation of alcohol related traits and behaviors. Here we report that two inbred strains of B6 mice, separated by a span of approximately 50 years at two different breeding facilities, Charles River Laboratories (C57BL/6NCrl) and Jackson Laboratories (C57BL/6J), show a statistically significant difference in alcohol preference. Female C57BL/6J mice show a higher preference for 3-15% alcohol solutions than do female C57BL/6NCrl mice. Preference was determined by continuous two-bottle choice access to either water or alcohol solutions. In order to determine if these strains differed on alcohol-related behaviors other than preference, Loss of Righting Reflex (LORR), initial sensitivity (IS) and Acute Functional Tolerance (AFT) to ethanol-induced hypnosis, saccharin and quinine preference, and alcohol metabolism were measured between strains. LORR, IS and AFT were measured after an I.P. injection of 3.8 g/kg 20% alcohol (w/v), n=10). No difference was observed between either B6 strain for LORR, IS, AFT, alcohol metabolism, saccharin or quinine preference. The results of this study indicate that the two strains examined differ mainly in their preference for alcohol, not as a consequence of taste discrimination, and show very little difference in other alcohol related traits. The strain differences present a unique opportunity to determine the genes underlying the observed difference in alcohol preference. Microarray analysis was used to determine genetic (strain) differences in brain gene expression that may be causal to the observed differences in alcohol preference. Preliminary results reveal that there are significant differences in brain gene expression between the C57BL/6J and C57BL/6NCrl strains. Supported by NIAAA grants; [U01AA013475 –INIA Project, K01AA103403, and R21AA103182], the Waggoner Center for Alcohol and Addiction Research, and a Bruce Jones Fellowship (MKM).
HYBRID C57BL/6J x FVB/NJ MICE DRINK MORE ALCOHOL THAN C57BL/6J INBRED STRAIN
Y.A. Blednov1; P. Metten2; D.A. Finn2; J.S. Rhodes2; S.E. Bergeson
1; R. A. Harris1; J.C. Crabbe2 1 Waggoner Center for Alcohol and Addictions Research, University of Texas, Austin, Texas, 78712;
2 Portland Alcohol Research Center, Veteran Affairs Medical Center, and Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon, 97239From several recent strain surveys (28 strains: Bachmanov et al, personal communication; 22 strains: Finn et al., unpublished), and from data in >100 other published studies of 24-hr, two-bottle ethanol preference in C57BL/6 (B6) mice, it is known that male B6 mice self-administer about 10-14 g/kg/day and that female B6 mice self-administer about 12-18 g/kg/day (choice between 10% ethanol and water). No strain has been found to consume more ethanol than B6. In one of our laboratories (Texas), we noted a markedly greater intake of ethanol in an F1 hybrid of B6 and FVB/NJ (FVB) mice. To confirm and extend this finding, we repeated the study at another site (Portland) and also tested B6FVBF1 mice in limited access drinking procedures that produce high levels of alcohol intake (Finn et al., in press; Rhodes et al., 2005). At both sites, we found that B6FVBF1 female mice self-administered high levels of ethanol during two-bottle preference tests (up to 30-35 g/kg/day). The F1 hybrids accepted much more concentrated ethanol solutions than the B6 strain and drank solutions containing 20-25% ethanol with preferences > 0.5. In the limited access tests, ethanol consumption the F1 hybrids was equivalent to that in B6 mice. These data show that this new genetic model has some significant advantages, when compared to existing inbred strains, and which can be useful for alcohol research. Supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (AA U01 13520; AA UO1 13478; AA U24 13519 - INIA Projects).
PRIOR EXPERIENCE WITH DIFFERENT TASTES INCREASES ALCOHOL PREFERENCE IN C57Bl/6 MICE
H. Alva;Y.A. Blednov.
University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
Significant correlation between consumption of sweet substances and alcohol has been reported for different rodent models (Sinclair et al., 1992; Stewart et al., 1994; Belknap et al, 1993; Kampov-Polevoy et al., 1999; Dess et al., 1998) and we found that deletion one of the genes responsible for perception of sweet and bitter tastes (alpha-gustducin) results in markedly decreased alcohol preference (Blednov et al., 2004). However, there are no studies asking whether prior exposure to different tastes alters subsequent perception of alcohol in laboratory mice? Three groups of naïve C57Bl/6 (B6) females had free access to 3 different tastes: sweet (saccharin 0.066%), umami (monosodium glutamate 0.XX mM) and bitter (quinine, 0.0X mM). Order of their presentation was different for each group. Each taste was presented as two-bottle choice paradigm for 4 days with two days break with water between each taste. A control group always had access to two bottles of water. One week after presentation of the last taste mice all groups were offered ascending concentrations of ethanol (3, 6, 9, 12, 15 and 18%) in two-bottle choice procedure. Independent of order of presentation all 3 groups of ‘taste experienced’ mice showed preference for sweet and umami substances and avoidance for quinine. However, all groups of mice demonstrated increased preference for moderate concentrations of alcohol (3 and 6%) compared with the taste-naïve control group. Repeated presentation of alcohol after one week of abstinence produced a decrease of alcohol preference in the control group. However, taste experience created highly variable responses to the repeated presentation of alcohol. These data show that response to alcohol can be modified by prior exposure to different tastes and suggest that the taste deprived environment of laboratory mice may be one factor which suppresses acceptance of alcohol by these mice. Supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (AA U01 13520 -INIA Project).
INCREASED ETHANOL CONSUMPTION IN MICE LACKING FATTY ACID AMIDE HYDROLASE: IMPLICATIONS FOR ENDOCANNABINOIDS
D. Walker1, Y.A. Blednov1, B.F. Cravatt2, R.A. Harris1.
1 University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712;
2 The Scripps Research Institute, La Jolla, California 92037.
Fatty acid amide hydrolase (FAAH) is a key membrane protein for FAA metabolism. One member of the large class of FAAs which is metabolized by a FAAH is anandamide, an important endocannabinoid (Cravatt et al., 1996). The role of endocannabinoid signaling in regulation of preference to ethanol was demonstrated recently (Wang et al., 2003; Hungund et al., 2003). A missense mutation in human FAAH was associated with problem drug/alcohol use (Sipe et al., 2002).To determine if FAAH regulates ethanol consumption, we studied mutant mice with deletion of the FAAH gene (Cravatt et al., 2001). Null mutant mice showed significantly higher preference for alcohol and consumed more alcohol in a two-bottle choice test as compared with wild-type littermates. There was no significant difference in consumption of sweet or bitter solutions. To determine the specificity of FAAH for ethanol intake, we studied additional ethanol-related behaviors. There were no differences between null mutant and wild type mice in ethanol-induced (3.8 g/kg) loss of righting reflex, severity of ethanol-induced acute withdrawal (4.0 g/kg), motor stimulant effect of alcohol (0.75, 1.5, 2.0 and 2.5 g/kg), conditioned taste aversion to alcohol (1.5 and 2.5 g/kg) and conditioned place preference. These data show that FAAH regulates ethanol consumption in mice and suggest that endocannabinoids or other FAAs selectively promote consumption without affecting other actions of alcohol. Supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (AA U01 13520 -INIA Project; AA06399).
DISTINCT PATTERNS OF GENE EXPRESSION IN HUMAN FRONTAL CORTEX OF CIRRHOTIC ALCOHOLICS
Jianwen Liua1, Joanne M. Lewohl3, R. Adron Harris1, Peter R. Dodd3, Vishwanath Iyer2, and R. Dayne Mayfield1
1 Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, TX 78712;
2Department of Psychology, University of Texas at Austin, Austin, TX 78712;
3Department of Biochemistry, University of Queensland, St Lucia 4072, Australia.Cirrhosis of the liver is a potentially life-threatening condition that is a common disease among alcoholics due to alcohol abuse. Our previous studies in prefrontal cortex have shown that cirrhotic alcoholic cases shows similar changes in the expression of G-protein-coupled receptor signaling genes compared to uncomplicated alcoholics; however, the magnitude of change appears to be greater in cirrhotic cases. Thus, it is possible that cirrhotic alcoholics represent a more severely affected population of alcoholics. To reveal the differences in gene expression on a genome-wide scale, we compared the transcription profile of frontal cortex of cirrhotic alcoholics with uncomplicated alcoholics and controls. A total of 13 controls, 14 uncomplicated alcoholics, and 8 cirrhotic alcoholics were included in the study. RNA samples from individual cases were amplified and hybridized against a common reference RNA onto 50,000-element cDNA microarrays. Genes differentially expressed in at least one of three groups were identified by one-way ANOVA analysis followed by false discovery rate corrections for multiple comparisons. Consistent with previous studies, a number of alcohol-responsive genes were identified. In addition, genes were identified that had altered expression levels only in the cirrhotic case group. These genes belong to diverse functional groups involved in synaptic transmission and protein transport. This study provides evidence that gene expression patterns in alcoholics with concomitant liver cirrhosis differ significantly from that observed in alcoholics without cirrhosis. These differences may reflect differences in the extent of alcohol abuse? Thus, increasing severity of alcoholism, as reflected by liver damage, produces additional changes in brain gene expression which may provide molecular indicators of the progression of the disease.
ETHANOL POTENTIATES [3H]DOPAMINE UPTAKE IN SK-N-SH NEUROBLASTOMA CELLS EXPRESSING THE DOPAMINE TRANSPORTER
D. G. Galindo; R.D. Mayfield
Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, TX 78712
Previous biochemical and electrophysiological studies in Xenopus oocytes have shown that ethanol increases dopamine transporter (DAT) function. This effect is consistent with an increase in the number of DAT molecules at the cell surface although it is unknown whether ethanol acts by promoting cell surface insertion of DAT or by decreasing constitutive internalization (or both) leading to an increased number of cell surface transporters. Mechanistic studies of transporter trafficking have been reported using a number of different expression systems; however, ethanol's effect on DAT function and trafficking has not been characterized in mammalian expression systems. The goal of the current study was to examine ethanol's action on DAT-mediated dopamine uptake in transiently transfected SK-N-SH human neuroblastoma cells. Cells were preincubated with ethanol (25-100 mM) for 10-120 min prior to [3H]dopamine uptake assays (100 nM; 10 min). Under these conditions, ethanol potentiated DAT-mediated uptake in a time- and concentration-dependent manner. DAT function was increased maximally (>60%) by 100 mM ethanol pretreatment. Ethanol had no effect on [3H]dopamine uptake in untransfected cells. These results in mammalian cells are consistent with those observed previously in Xenopus oocytes and suggest that this model system may be appropriate for future mechanistic studies.
This study was supported by grants from the National Institute of Alcohol Abuse and Alcoholism, NIH (U01 AA13497-INIA Project) and the Waggoner Center for Alcohol and Addiction Research.
GENE EXPRESSION PROFILE OF THE BRAIN AFTER DRINKING TO INTOXICATION IN C57BL/6J MICERhodes, J.S., Pomonarev, I., Harris, R. A., Crabbe, J. C.
University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
C57BL/6J inbred mice will self-administer alcohol to the point of reaching a pharmacologically significant blood-alcohol level when they are offered a single bottle of 20% alcohol for 4 hours in the home cage during the circadian dark phase of the light/dark cycle (Rhodes et al., 2005, Physiol Behav: 84:53). Although a majority of animals self-administer to high levels using this procedure, a small percentage of C57BL/6J animals (approximately 10 %) do not drink to significant levels and others drink at intermediate levels. Here we compared the gene expression profile of several brain regions (hippocampus, cerebellum, olfactory bulbs, frontal cortex, and amygdala) in C57BL/6J mice that self-administered high, medium, or low quantities of alcohol and compared these expression patterns with a control group exposed to only water during the 4 hour test period. This study identifies a specific pattern of gene expression changes in the brain in response to self-administration of alcohol that might contribute to the reinforcing effects of alcohol in a genetically predisposed strain of mouse.
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