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RSA 2003 Abstracts

Indiana University - Purdue University, Indianapolis

ANALYSIS OF GENE EXPRESSION OF ALCOHOL PREFERRING P AND NON-PREFERRING NP RATS IN FIVE BRAIN REGIONS

H. J. Edenberg, W.-M. Liu, W. N. Strother, J.N. McClintick, M. Stephens, R. Jerome, C. Nicholson, E. R. Edenberg, R. Goldberg, L. Lumeng, T.-K. Li, W. J. McBride

Indiana University School of Medicine, Indianapolis, IN 46202

Differences in preference for drinking alcohol are in part under genetic control, as demonstrated by the selectively bred P and NP rats. We hypothesized that the difference in preference may be a result of differences in gene expression in various regions of the brain. Therefore, we used the Affymetrix rat genome microarray RGU34A to study the differences in gene expression of adult male inbred alcohol preferring (iP) and non-preferring (iNP) rats in five brain regions: nucleus accumbens, striatum, anterior cortex, amygdala, and hippocampus. Because differences in gene expression in the brain are usually small, we examined 6 individual rats from each group. We used both t-test and rank sum test on the discrimination scores and signals for better sensitivity at low target concentrations. We then applied singular value decomposition on genes that significantly differed between iP and iNP in at least one region. The first four eigengenes (also known as the left singular vectors) can explain 98.4% of the total variance. The first eigengene represented the direction of the largest variance for both groups, and the projections on the second eigengene clearly differentiate the expression of iP and iNP rats. The third and fourth eigengenes provide a projection plane showing the different expression patterns in different brain regions. The genes that primarily characterize these eigengenes include aldehyde dehydrogenase, aldehyde reductase, catalase, chemokine ligand, and a potassium channel regulator. [AA07611, AA13521 [INIA], Indiana Genomics Initiative]Category: 1g Molecular Genetics and Biology/Microarrays/genomics

CHRONIC ALCOHOL DRINKING BY ALCOHOL-PREFERRING (P) RATS INCREASES THE SENSITIVITY OF THE POSTERIOR VTA TO THE REINFORCING EFFECTS OF ETHANOL

Z.A. Rodd; R.L. Bell; Y. Zhang; C. Hsu; T.-K. Li; L. Lumeng; J.M. Murphy; W.J. McBride

Departmentsof Psychiatry and Medicine Indiana University School of Medicine, Indianapolis, IN 46202

Previously our laboratory reported that P rats readily self-administered 75-200 mg % ethanol (E) directly into the posterior VTA (pVTA). The objective of the present study was to determine the effects of 8 weeks of 24-hr free-choice drinking of 15% E on the self-infusion of E directly into the pVTA. Rats were implanted with guide cannulae aimed at the posterior VTA. Subjects were tested in standard 2-lever (active and inactive) operant chambers. Naïve and E drinking P rats were assigned to one of two groups (n=5-8/group) that self-administered (FR1 schedule) 50 or 75 mg % E for the initial 4 sessions (acquisition), aCSF for sessions 5 and 6 (extinction), and the original infusate for session 7 (reinstatement). The group drinking E prior to surgery self-infused both the 50 and 75 mg % E and discriminated between the active and inactive lever throughout all 4 acquisition sessions. The E-naïve group self-infused only the 75 mg % E during sessions 2-4. Moreover, responses on the active lever and reinforcements for 75 mg % E were 2-fold higher in sessions 2-4 of the E drinking group (120 + 10 responses/session; 44 + 3 infusions/session) than the naïve group (55 + 3 responses/session; 22 + 4 infusions /session). Both groups extinguished responding on the active lever when aCSF was substituted for 75 mg % E in sessions 5 and 6, and reinstated responding on the active lever when E was restored in session 7. These preliminary data suggest that chronic alcohol drinking by P rats increased the sensitivity of the pVTA to the reinforcing effects of E. (AA07611, AA12262, AA11261, AA10717).

EFFECTS OF CHRONIC ETHANOL CONSUMPTION ON THE DOPAMINE TRANSPORTER AND DOPAMINE CONTENT IN THE EXTENDED AMYGDALA OF HIGH ALCOHOL DRINKING (HAD-2) RATS

M.R. Carroll; R.L. Bell; J.R. Simon

Indiana University School of Medicine, Institute of Psychiatric Research, Indianapolis, IN. 46202

The present study was undertaken to examine the effects of ethanol (E) consumption on several dopaminergic parameters within the extended amygdala (AMYG). It was hypothesized that chronic E consumption by HAD-2 rats would alter dopamine (DA) uptake, dopamine transporter (DAT) binding, and/or tissue DA content. HAD-2 rats were divided into 3 groups: rats had continuous access to E for 56-days, binge-like access to E for the 56-days or served as water controls. Following E treatment, rats were sacrificed and the extended AMYG was dissected from a fresh, unfrozen, 1.6 mm coronal section. Samples were immediately homogenized in 0.32 M sucrose, and aliquots taken for the determination of DA content and DA uptake. The remainder of each homogenate was frozen for subsequent DAT binding. DA content was determined by HPLC analysis of acid extracts of the homogenates. DA uptake was determined following a 2-min incubation of homogenates at 30oC in the presence of 7 nM 3H-DA, and DAT binding was assessed following a 90-min incubation at 4oC in the presence of 2 nM 3H-WIN 35,428. Post hoc analyses revealed significant differences in tissue content of DA between continuous access rats and both binge-like access and water control rats. Analyses of DA uptake and binding revealed no significant differences. These findings suggest that chronic E consumption by HAD-2 rats results in an increase in DA content within the extended AMYG that does not appear to be mediated by alterations in DA uptake. The observed changes in tissue DA content may, however, be the result of alterations in DA release and/or DA synthesis. (AA13522, INIA)

GENE EXPRESION IN CNS REGIONS FOLLOWING SELF-ADMINISTRATION OF ETHANOL INTO THE POSTERIOR VTA BY INBRED ALCOHOL-PREFERRING (iP) RATS

Z. A. Rodd, W.J. McBride, R. L. Bell; W. N. Strother; H. Aloor; J.N. McClintick; M. Stephens; R. Jerome; W.-M. Liu; L. Lumeng; H. J. Edenberg

Indiana University School of Medicine, Indianapolis, IN 46202

Previously our laboratory reported that the alcohol-preferring (P) rat would self-administer ethanol directly into the posterior ventral tegmental area (pVTA). The objective of the current study was to examine the effects of ethanol self-administration into the pVTA on gene expression in the nucleus accumbens (NA), medial prefrontal cortex, amygdala, caudate putamen and hippocampus. Male iP rats (strain 5-C) were stereotaxically implanted with a guide cannula aimed at the right pVTA. Subjects were tested in standard 2-lever operant chambers (active and inactive). Rats were randomly assigned to one of two groups (n=5/group) that self-administered (FR1 schedule) either aCSF or 150 mg% ethanol for 4 sessions. Rats self-administered ethanol at a 5-fold higher rate than aCSF (31 + 2 and 6 + 1 infusions/session, respectively). Animals were killed immediately after the 4-hr test session, and the brain regions dissected, RNA extracted and purified for microarray analysis. Affymetrix Rat Genome RGU34A GeneChips were used to analyze gene expression differences between the animals that self-administered ethanol and those that administered aCSF. After filtering out genes not reliably detected in at least one of the groups, t-tests were applied to detect expression differences. In the NA, 104 genes changed out of the 4102 detected. A very small number of genes (21-30) changed in the other 4 brain regions. The higher number of changes observed in the NA likely reflect its high dopamine innervation from the VTA, and the involvement of these mesolimbic structures in mediating the rewarding effects of ethanol. [Supported by AA07611, AA13521 (INIA), AA13496 (INIA), Indiana Genomics Initiative.]

GENETIC AND “ALCOHOL DEPRIVATION-SPECIFIC” REGIONAL TRANSCRIPTOME ANALYSIS IN INBRED ALCOHOL-PREFERRING RATS

M.K. Mulligan1, J.A. Owen1, M.B. Warner1, R.L. Bell2, W.N. Strother2, W.J. McBride2 and S.E. Bergeson1

1Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology, University of Texas, Austin, TX 78712
2Depts. of Psych. and Med. Indiana Univ. School of Medicine, Indianapolis, IN 46202

Relapse is a prevalent problem in human alcoholics. Using a rat model of alcohol relapse, an mRNA differential display analysis has been initiated to test the “Two-Hit” hypothesis that both genetic and environmental factors contribute to excess alcohol intake. After chronic free-choice drinking followed by a prolonged period of alcohol deprivation, iP rats temporarily consume a greater amount of alcohol when they are next given access. This increase in ethanol intake is called the alcohol deprivation effect (ADE). Regional brain expression differences in alcohol naïve iP and inbred alcohol-non-preferring (iNP), and in iP rats that were on continuous alcohol access or were repeatedly deprived, were screened. The transcriptome from specific brain regions from these four groups was isolated and analyzed by mRNA Differential Display PCR. Total RNA was extracted from 8 brain regions including the amygdala, caudate putamen, hippocampus, hypothalamus, anterior and posterior cortex, septum and nucleus accumbens. iP/iNP genetic expression differences, iP long-term continuous alcohol drinking changes, and iP alcohol deprivation-specific expression were compared across all 8 brain regions. Preliminary results indicate that brain gene expression differences exist between iP and iNP animals, and between naive, continuous alcohol exposed, or repeated alcohol-deprived iP rats. A better understanding of the brain regions involved and the genes that contribute to either the genetic propensity toward, and/or underlying neuroadaptation should increase the likelihood of developing successful treatments for alcoholism. Supported by NIAAA INIA program grants AA13475, AA13521, AA13522 and NIAAA training grant AA07471-13.

INNATE DIFFERENCES IN PROTEIN EXPRESSION IN THE NUCLEUS ACCUMBENS AND HIPPOCAMPUS OF INBRED ALCOHOL-PREFERRING (IP) AND -NONPREFERRING (INP) RATS

Frank A. Witzmann, Junyu Li, Wendy N. Strother, William J. McBride, Lawrence Hunter, David W. Crabb, Lawrence Lumeng, Ting-Kai Li

Departments of Cell & Integrative Physiology, Medicine and Psychiatry, Institute for Psychiatric Research, Indiana Univ Sch Med, Indianapolis, IN 46202; Depart Pharmacol, University of Colorado Health Sciences Center, Denver, CO

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-non-preferring (iNP) rats (n = 5 each strain). Several protein spots were excised from the gel, de-stained, digested with typsin, and analyzed by mass spectrometry. In the hippocampus, the abundances of 8 proteins (only 2 of which were identified) were higher in the iNP than iP rat. In the nucleus accumbens, the abundances of 31 of 32 proteins (of which 21 were identified) were higher in the iNP than iP rat. 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 (a) metabolism (e.g.,calpain, parkin, glucokinase, apolipoprotein E, sorbitol dehydrogenase), (b) cyto-skeletal and intracellular protein transport (e.g., beta actin), (c) molecular chaperoning (e.g., grp 78, hsc70, hsc 60, grp75, prohibitin), (d) cellular signaling pathways (e.g., protein kinase C-binding protein), (e) synaptic function (e.g., complexin I, gamma enolase, syndapin IIbb), (f) reduction of oxidative stress (thioredoxin peroxidase), and (g) 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. (Supported by AA07611, AA13521 [INIA], AA13524 [INIA] and the Indiana Genomics Initiative).

 

 

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