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RSA 2007 Abstracts
Indiana University , Indianapolis
CROSSED HAPs: CHARACTERIZATION OF EXCESSIVE ALCOHOL DRINKING MODELS IN MICE SELECTED FROM TWO HIGH ALCOHOL PREFERRING LINES OF MICE: N. J. Grahame. Indiana University Purdue University School of Science, Indianapolis, IN 46202.
Selective breeding for high alcohol drinking can yield animal models of alcoholism. Previously, 2 lines of High Alcohol Preferring (HAP) mice were derived by selectively breeding for high 24-h free-choice 10% alcohol consumption over 4 weeks. However, inbreeding can limit the number of high drinking alleles present in any single line. The purpose of the present study was to maximize the number of these alleles, and presumably drinking behavior, in a single population of mice by crossing the two HAP lines and selectively breeding from the result. Crossed HAPs (cHAPs) were derived from a reciprocal cross of HAP1 and HAP2 mice and selected for the same phenotype as both parent lines over 5 generations. We tested 30 cHAP and 30 mice from one of the parent lines, HAP1, for limited access alcohol drinking. Some mice had simultaneous free-choice water access (the LAC procedure) while others didn’t (the Drinking in the Dark, or DID procedure). In 11 weeks, mice were tested for LAC and DID when 5, 10, 15, or 20% concentrations of ethanol were presented for 30, 60, or 120 min a day, starting 1-2 h after onset of dark. We hypothesized that cHAPs would drink more than HAP1 mice during 24-h and limited access. As expected, 4 generations of selective breeding of cHAPs was successful, yielding 24-h intakes exceeding either parent line: 19+g/kg/day in males and 20+g/kg/day in females. However, on the 5th generation, intakes in cHAPs no longer exceeded HAP1s. During 30-min access, cHAP S4 mice drank more ethanol than HAP1 S31 mice. Highest intake was seen with 15% ethanol, when cHAP mice drank 2.05 g/kg (+/- 0.091) and HAP1 mice drank 1.68 (+/- 0.078) g/kg ethanol in 30 min. At no time did ethanol intake differ between LAC and DID, showing that ethanol drinking in these mice is unaffected by concurrent water availability, and suggesting that HAP and cHAP mice are drinking alcohol solutions for their pharmacological effect. These data show for the first time that crossing two high-drinking selected lines can concentrate high drinking alleles in a single population. The resulting cHAP line may be a useful tool in characterizing excessive alcohol drinking behavior. AA13483 to NJG and AA07611 to David Crabb.
Differences in Expression of Genes Involved in Neuronal Function and Development in the VTA of P and NP Rats: M. W. Kimpel; D. Ji; J. N. McClintick; H. J. Edenberg; W. J. McBride. Depts Psych, Biochem & Mol Biol, Medical & Mol Genetics, Ctr Med Genomics, Inst Psych Res, Indiana Univ Sch Med, Indianapolis, IN 46202-4887.
The objective of this study was to determine if there are innate differences between selectively bred P and NP rats (n = 4 per group) in gene expression in the ventral tegmental area (VTA), a region involved in regulating alcohol drinking and the reinforcing effects of ethanol. The VTA was dissected using a micro-punch technique and microarrays were run using Affymetrix Rat Genome 230 2.0 arrays. Preprocessing was performed using RMA. Filtering of probe sets was performed using the following criteria: 1.) Affymetrix absent/present filter (threshold of 0.5 present within group) and, 2.) inter-quartile range (IQR fell within the upper 75% of the IQR distribution of all probe sets). Only probe sets assigned to named genes were selected for further statistical analysis. Of the 31,099 probe sets in the initial dataset, 6,177 passed all filters. Linear Modeling for Microarray Analysis (LIMMA) with moderated t-statistics was used to calculate p-values, which were adjusted for false discovery rate (FDR). There were 805 probe sets, representing 736 unique genes, differentially expressed between the strains (FDR<0.20). Analysis for over-representation of Gene Ontology categories amongst the significant genes revealed 81 significant categories (p < 0.05), including synaptic transmission (40 genes, p < 0.000) and dendrite development (6 genes, p < 0.003). In the synaptic transmission category, 6 genes demonstrated lower expression in the P vs. NP, whereas 34 demonstrated higher expression. Those with lower expression included: GABA-A receptor subunit gamma 3; ionotropic glutamate receptor 4; AMPA1 (alpha 1) glutamate receptor; and metabotropic glutamate receptors 2 and 8. Those with higher expression included: dopamine D1A receptor; GABA-A receptor alpha 1 subunit; kainate glutamate receptor 2; glutamine synthase; neuregulin 1; neuronal pentraxin 1; and neuropeptide Y receptor Y5. Additional differences included genes for pre- and post-synaptic proteins, transcription factors, and genes involved in neuro-development. The many differences in gene expression between the VTA of the P and NP rat lines may contribute to the divergent alcohol drinking phenotypes of these rats. (AA07611, AA13521 [INIA], AA16652 [INIA], Indiana Genomics Initiative [INGEN®] of Indiana University).
Differences in Expression of Genes involved in Neuronal Function and Development in the Nucleus Accumbens of Inbred HAD and LAD Rats: M. W. Kimpel; W. N. Strother; J. N. McClintick; H. J. Edenberg; W. J. McBride. Depts Psych, Biochem & Mol Biol, Medical & Mol Genetics, Ctr Med Genomics, Inst Psych Res, Indiana Univ Sch Med, Indianapolis, IN 46202-4887.
The objective of this study was to determine if there are innate differences between alcohol-naive male iHAD and iLAD rats (n=6 per group) in gene expression in the nucleus accumbens (ACB), a region involved in regulating alcohol drinking. The ACB was dissected using standard techniques and microarrays were run using Affymetrix Rat Genome 230 2.0 arrays. Preprocessing was performed using RMA. Filtering of probe sets was performed using the following criteria: 1.) Affymetrix absent/present filter (threshold of 0.5 present within group) and, 2.) inter-quartile range (IQR fell within the upper 75% of the IQR distribution). Probe sets assigned to named genes were selected for further analysis, 6,186 met all criteria. Linear Modeling for Microarray Analysis was used to calculate p-values. There were 708 probe sets, representing 627 unique genes, differentially expressed between the strains (FDR<0.20). Analysis for over-representation of Gene Ontology categories revealed 60 significant categories (p<0.05), including synaptic transmission (35 genes, p<0.001), positive regulation of apoptosis (25 genes, p<0.000), regulation of development (23 genes, p<0.004), regulation of neurotransmitter levels (15 genes, p<0.002), axonogenesis (13 genes, p<0.049) and regulation of neurogenesis (6 genes, p<0.024). In the synaptic transmission category, 23 genes demonstrated lower expression levels in the iHAD vs. iLAD rats, whereas 12 genes showed higher expression levels. Genes with lower expression included: serotonin-3a receptor; adenosine A2a receptor; cocaine and amphetamine regulated transcript (CART); GABA-A receptor subunit gamma 3; and metabotropic glutamate receptor 2. Genes with higher expression included: aldehyde dehydrogenase family 5 subfamily A1; catechol-O-methyltransferase; and the alpha 7 subunit of the nicotinic cholinergic receptor. Additional differences included genes for pre- and post-synaptic proteins, transcription factors, calcium channels, potassium channels, and genes involved in neuronal development. The many differences in gene expression in the ACB of the iHAD and iLAD rat strains may contribute to their divergent alcohol drinking behaviors. (AA07611, AA13521 [INIA], AA16652 [INIA], Indiana Genomics Initiative [INGEN®] of Indiana University).
HIGH ALCOHOL PREFERRING MICE ARE MORE IMPULSIVE THAN LOW ALCOHOL PREFERRING MICE AS MEASURED BY A DELAY DISCOUNTING TASK: B. G. Oberlin; A. N. Henderson; N. J. Grahame. Indiana University Schools of Science and Medicine, Indianapolis, IN 46202.
Impulsivity may be an important component of human alcoholism as well as other addictive behaviors. Impulsivity can be defined by the choice of small immediate rewards over large delayed rewards as measured by a delay discounting (DD) task. To test whether genetic differences in alcohol consumption affect impulsivity, lines of mice selected for divergent free-choice alcohol consumption (High Alcohol Preferring [HAP] and Low Alcohol Preferring [LAP]) were assessed on the DD task using saccharin reward. Mice were alcohol-naïve, so line differences could be attributed to genetics. Our hypothesis was that HAP mice would be more impulsive than LAP mice. We used an adjusting amount version of the DD task which gave HAP (n = 19) and LAP (n = 17) mice concurrent choice between a fixed magnitude reward delivered after a delay and a varying reward delivered immediately. Choice was assessed at delays of 1, 2, 4, and 8 seconds to the fixed reward, with 6 sessions at each delay. At each delay, mice titrated the value of the immediate reward until it was equivalent to the fixed, delayed reward, thus giving a measure of subjective reward size. As expected, HAP mice showed greater impulsivity, as shown by a main effect of Line on mean adjusted amount averaged across all delays, and a difference between lines in the value of k. This parameter, derived from nonlinear regression to a hyperbolic discounting function, is a measure of the discounting of delayed reinforcers. These data suggest that impulsive choice behavior is genetically linked to ethanol preference, and that differences in impulsivity seen in a model of human alcoholism may be independent of ethanol exposure. To our knowledge, this is the first demonstration of differences between lines selected for divergent alcohol intake on impulsivity as measured on the DD task. Furthermore, these data corroborate human studies showing that addicts and alcoholics are more impulsive than controls, and implicate impulsivity as an endophenotype for addictive behaviors. In future studies, we will evaluate the effect of ethanol on impulsivity, as well as map relevant neuroanatomical sites. AA13483 to NJG, AA07611 to David Crabb, F31AA016430 to BGO.
CHRONIC ETHANOL DRINKING RESULTS IN LONG-TERM ALTERATIONS IN FOS B IMMUNOREACTIVITY IN LIMBIC REGIONS OF ALCOHOL-PREFERRING (P) RATS: W. N. Strother; L. Lumeng; W. J. McBride. Depts Psych, Med, Indiana Univ Sch Med, Indianapolis, IN, 46202.
Fos B induction is hypothesized to mediate neuroadaptations to chronic drug exposure and can be used as a marker to study neuronal pathways involved in long-term neuroadaptations. The objective of the present study was to determine the effects of chronic ethanol (EtOH) drinking on Fos B immunoreactivity (ir) in the limbic system of adult P rats. P rats received 24-hr free choice access to 15% EtOH (v/v) and water (n=8) or water only (n=7) for a period of 8 weeks. P rats consumed an average of 6.9 ± 0.2 g/kg/day EtOH during the final 3 weeks of access. All rats were sacrificed during the light portion of the light/dark cycle with EtOH removed 4 hrs prior. Rats were transcardially perfused with 4% paraformaldehyde and post-fixed overnight. Coronal 40 µm sections were collected in phosphate buffer using a freezing microtome. Fos B-ir (Santa Cruz @ 1:3000) was detected using a modified avidin-biotin-immunoperoxidase protocol with diaminobenzidine as the chromogen. Fos B-ir cells were counted in a standard counting frame in 3 adjacent sections per animal/per region at 10X magnification using Spot Advanced software (Diagnostic Instruments Inc). Mean cell counts were calculated as Fos B-ir cells/mm2. Significant (p < 0.05) elevations in Fos B-ir were found in the nucleus accumbens core and shell, the basolateral nucleus and central nucleus of the amygdala, the lateral septum, and ventral tegmental area of EtOH drinking P rats compared to water drinking P rats. Significant increases in Fos B-ir ranged from 33-70 % over water control levels. There were no significant differences between the groups in the medial prefrontal cortex, bed nucleus of the stria terminalis, or caudate putamen. These results demonstrate that chronic EtOH drinking resulted inchanges in transcription factor levels, suggesting that these regions have undergone neuroadaptations that may contribute to chronic EtOH addictive behaviors. (AA07611, AA10721, AA16652 [INIA]).
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