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RSA 2008
Transcriptional analyses of alcohol consumption
Susan E. Bergeson1, Yuri A. Blednov2 and Amanda J. Roberts3
Texas Tech University Health Science Center1, University of Texas at Austin2, and The Scripps Research Institute3Genetic and environmental factors are known to contribute to preference for alcohol drinking, including high initial and increasing levels of consumption. INIA collaborators have utilized several standardized models for alcohol consumption and measured alcohol drinking and other traits; and present here, genetical genomic and transcriptomic analyses. Gene expression was measured using a cDNA hybridization platform in brain following: 1) a repeated abstinence paradigm of two-bottle choice drinking in several hybrid and inbred strains of mice, 2) DID – “drinking-in-the-dark” in FVB.B6 hybrid mice, and 3) WID – “withdrawal-induced-drinking” in C57BL6J mice. Resource sharing of the results is accomplished through the Alcohol Research Integrator database located at URL: http://aridb.ttuhsc.edu Further analysis of the expression using several over-representational analyses for chromosomal location, transcription factor binding site usage and biological pathways narrow the focus on several “alcohol-responsive” candidate genes isolated for further study. These findings from several mouse models may allow a better understanding of the molecular consequences in brain of high alcohol consumption. The authors thank NIAAA for INIA funding: 5U01AA013475, 5U01AA013520 and 5U01AA013523.
MICE LACKING THE GABAA ALPHA2 SUBUNIT:PARADIGM-DEPENDENT REGULATION OF ALCOHOL CONSUMPTION
Blednov Y.A. and Harris R.A.
University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
.
The GABAA alpha2 subunit is a candidate gene for human alcoholism (Soyka et ak., 2008) and we asked if this subunit regulates alcohol consumption in mice. There are several distinct measure alcohol consumption in mice (see Rhodes et al., 2005 for rev.). Mice with continuous access to alcohol and water do not generally drink sufficient alcohol to achieve intoxication. However, some tests with limited access to alcohol promote sufficient consumption that blood ethanol concentration reach levels that produce effects on physiology and/or behavior. We asked ‘Will gene deletion produce the same effect on different tests of alcohol intake?’ Wild type and alpha2 subunit GABAA receptor null mice were used for 4 different tests for alcohol intake: 1) Standard 24-hrs two-bottle choice (2BC); 2) An ethanol acceptance method (5% ethanol) using scheduled, restricted fluid access (SHAC) (Finn et al., 2005); 3) Consumption of ethanol with limited access and one bottle (no water bottle) (DID-1B) (Rhodes et al., 2005); 4) A test similar to #3 except using two bottles (choice between ethanol and water) (DID-2B). Null mutant mice of both sexes consumed less alcohol with lower preference in 24-hrs 2BC. No differences between wild type and alpha2 GABAAR knockout mice in DID-1B test were found. In SHAC, only female null mice showed reduced consumption of alcohol but they also consumed less water after the alcohol sessions. In the DID-2B test, male null mice showed higher ethanol intake with higher preference for alcohol than wild type at all concentrations tested (???% ethanol), whereas female null mice demonstrated stronger adaptation to drinking the more concentrated alcohol solutions (??? % ethanol). Thus, the effect of mutation on ethanol intake strongly depends on choice of experimental procedure chosen for measurement of ethanol intake. These results demonstrate that ethanol intake measured in different tests is driven by different mechanisms and using only one test for alcohol consumption may fail to detect effects of mutations. Supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (AA U01 13520 -INIA Project; AA06399).
The effects of ethanol consumption during peri-adolescence or adulthood on subsequent ethanol-induced motor impairment in alcohol-preferring (P) rats
J.B. Cook, A.L. Bracken, Z.A. Rodd, J.E. Toalston, L. Lumeng, J.M. Murphy, W.J. McBride & R.L. Bell
Depts. Psych, Med & Biochem, Indiana University School of Medicine, and Dept. Psychology, Purdue School of Science, IUPUI, Indianapolis, IN 46202.The objectives of this study were to examine the effects of ethanol (E) drinking on subsequent motor impairment induced by moderate E doses in female P rats. Peri-adolescent P rats (n = 5-11) were given free-choice access 15 and 30% E in their home cages starting on post-natal day (PND) 30, whereas adult P rats (n = 3-9) started E drinking on PND 90. There were 3 groups: (a) a naïve control group had water (W) only; (b) Intermittent E (IE) rats had 24-hr access for an initial 7 days followed by 5 cycles of 3 days of E deprivation and 3 days of E re-exposure, with the last re-exposure extended to 7 days, for a total of 41 days; and (c) Continuous E (CE) rats had 24-hr access 7 days a week for 41 days. At the end of the 41 days, both the IE and CE groups were drinking ~10 g/kg/day of E. Motor impairment was assessed using an oscillating bar task. Rats received 5 days of training (the 7th through 11th days after the pretreatment phase), in which they were required to stay on an oscillating bar for 120 sec to avoid an electric shock. After training, E-induced motor impairment was assessed once a day for 5 consecutive days. On test days, animals were injected IP with E (0.0, 1.0 or 1.5 g/kg) and tested at 15-min intervals until they reached criterion (120 sec at 40 os/min). At the 1.0 g/kg dose, adult, but not peri-adolescent, E pretreated rats developed tolerance to the motor impairing effects of E, [51 ± 10, 47 ± 15, 42 ± 5 (min to recover, mean ± SEM), on the 1st test day vs, 42 ± 9, 20 ± 3, 20 ± 3, on the 5th test day, for the W, IE and CE groups respectively]. At the 1.5 g/kg dose, adult E pretreated rats developed tolerance quicker than W pretreated rats [122 ± 20, 120 ± 38, 97 ± 14, on the 1st test day vs, 126 ± 22, 65 ± 20, 77 ± 5, on the 2nd test day, for the W, IE and CE groups respectively]. At the 1.5 g/kg dose, peri-adolescent CE rats were significantly less impaired by E than the IE and W rats [86 ± 20, 75 ± 20, 51 ± 3, on the 1st test day for the W, IE and CE groups, respectively]. Overall, these results suggest that continuous and intermittent E drinking during adulthood readily produces the development of tolerance to low to moderate doses of E. In contrast, peri-adolescent E drinking under continuous access (but not intermittent access) produces tolerance that persists into adulthood. (AA07462, AA07611, AA10256, AA13522 an INIA project).
Selective up-regulation of Homer2/glutamate receptor-mediated signaling within the nucleus accumbens by binge alcohol drinking in C57BL/6J mice.
D.K. Cozzoli, A.J. Rahn, K.K. Szumlinski
Dept. Psychology and the Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA, 93106-9660.
Converging behavioral, neurochemical and immunoblotting data support a necessary and active role for Homer2-mGluR signaling within the nucleus accumbens in mediating alcohol intake in continuous alcohol access paradigms. The role for Homer2 and its associated glutamate receptors in regulating excessive, binge drinking is under-characterized. To this end, the present study employed immunoblotting to assess the effects of short-term withdrawal from intermittent bouts of binge alcohol drinking using the Scheduled High Alcohol Consumption (SHAC) paradigm upon the expression of Homer proteins and their associated glutamate receptors and kinases within mesolimbic structures implicated in alcohol reward. C57BL/6J (B6) mice were presented with 5% alcohol for 30 min, every 3 days, for a total of 6 bottle presentations (mean BEL attained=109 mg%). Twenty-four hours later, animals were sacrificed for immunoblotting for Homer1b/c, Homer2a/b, mGluR1a/5, NR2a/b, as well as total and phosphorylated PI3K and PKCe, within the entire nucleus accumbens, dorsal striatum, prefrontal cortex and hippocampus. Compared to water controls, SHAC mice exhibited an increase in the total protein expression of Homer2, NR2a, NR2b, as well as the ratio of phosphorylated to total PI3K and PKCe levels within the nucleus accumbens, but did not differ with respect to any of the proteins within any other brain regions examined. These results extend earlier data for continuous alcohol access in B6 mice and provide further evidence to support the over-arching hypothesis that alcohol-induced increases in Homer2/glutamate receptor-mediated signaling within the nucleus accumbens is an important neuroadaptation underlying excessive alcohol intake. This work was supported by NIH grant AA016650 (INIA West) to KKS.
CRF1 receptor antagonists block the ethanol-induced release of GABA in central amygdala in vitro and in vivo.
Cruz M.T., Madamba S., Stouffer, D., Siggins G.R., Parsons L.H., and Roberto M.
Corticotropin-releasing factor (CRF) in the central nucleus of the amygdala (CeA) plays an important role in regulating ethanol consumption and in the anxiogenic response to ethanol withdrawal. We previously demonstrated that acute ethanol augmented GABA release in rat and mouse CeA in vitro. This ethanol effect is mediated by presynaptic CRF1 receptors (CRF1Rs). In CeA from chronic ethanol-treated (CET) rats, the basal GABA release was elevated and acute ethanol further enhanced GABA release, suggesting lack of tolerance. Here we have examined the effects of CRF and acute and chronic ethanol on the GABAergic system in rat CeA in vitro using a CeA slice preparation and in vivo using brain microdialysis. We found that 200 nM CRF significantly enhanced (to 140%) evoked GABAA receptor-mediated inhibitory postsynaptic current (IPSC) amplitudes in CeA of naïve rats. CRF also decreased paired-pulse facilitation (PPF) of IPSCs in rat CeA. The selective CRF1R antagonists NIH-3 (10 mM; aka LWH-63), Antalarmin (10 mM) and R121919 (1 mM) decreased basal IPSC amplitudes and prevented their increase by ethanol. The three antagonists increased (by 20%) PPF of IPSCs, suggesting a decreased GABA release that opposes the ethanol effect. In CeA of CET rats, the ability of CRF to increase IPSCs was enhanced (to 165%) compared to naïve CeA. In CeA slices from CET rats, the inhibition of basal IPSCs by the CRF1R antagonists was greater than in CeA of control rats. Interestingly, in naive rats local administration of R121919 alone did not alter the dialysate levels of GABA, but did block the ability of ethanol to increase CeA GABA levels. In CET rats, local administration of R121919 alone reduced dialysate GABA levels by almost 50%. Co-administration of the CRF1 antagonist with ethanol prevented the ethanol-induced increase in dialysate GABA. These electrophysiological and microdialysis findings suggest an important ethanol-CRF interaction with GABAergic transmission in the CeA that plays a significant role in the development and maintenance of ethanol dependence. Supported by grants from NIH (AA015566, AA013517, AA06420)
Ethanol and CRF: which is driving GABA release in the amygdala
M. T. Cruz, M. Bajo, R. G. Siggins, R. Messing and M. Roberto
The Scripps Research Institute and Alcohol Research Center, Committee on the Neurobiology of Addictive Disorders, 10550 N. Torrey Pines, La Jolla, CA 92037, USA.Ethanol and corticotrophin releasing factor (CRF) acting at CRF1 receptors in the central nucleus of the amygdala (CeA) regulate GABA release. Amygdalar CRF mediates anxiety associated with stress and drug dependence, and plays an important role in regulating voluntary ethanol intake. Here we examined signaling pathways downstream of the CRF1 receptor in the CeA that mediate GABAergic signaling and anxiety. We will provide electrophysiological data indicating that the PKCe pathway is involved in the ethanol and CRF sensitivity of GABAergic terminals in CeA. We found that CeA neurons from mice with a null mutation for protein kinase C epsilon (PKCe-/-) exhibit reduced anxiety-like behavior and show increased GABAergic tone due to enhanced GABA release when compared with wild type (PKCe+/+) neurons. CRF enhanced GABAergic transmission in PKCe+/+ CeA by increasing presynaptic release of GABA. This effect was absent in the PKCe-/- CeA. Ethanol also stimulated presynaptic GABA release in PKCe+/+ CeA neurons and this effect was also absent in the PKCe-/- CeA. A PKCe-specific peptide inhibitor blocked both CRF- and ethanol-induced GABA release in the CeA of PKCe+/+ mice, confirming findings in the PKCe-/- CeA. These results identify a PKCe signaling pathway in the CeA that is activated by CRF1 receptor stimulation, regulates neurotransmitter release at GABAergic terminals, and may contribute to increased anxiety-like behavior.
LESIONS OF THE AMYGDALA, BUT NOT THE NUCLEUS ACCUMBENS, DECREASE THE MAINTENANCE OF ETHANOL SELF-ADMINISTRATION IN ALCOHOL-PREFERING (P) RATS.
R Dhaher; ZA Rodd; CP Whalen; B Getachew; S Hauser; WJ McBride; JM Murphy; RL Bell.
Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IndianaThe central extended amygdala is a limbic region proposed to play a key role in drug addiction and alcohol dependence, and is mainly composed of the central nucleus of the amygdala (CeA), the lateral bed nucleus of the stria terminalis (LBNST), and the medial portion of the shell of the nucleus accumbens (NAc shell). The aim of this study was to determine if the CeA and NAc shell are involved in the maintenance of ethanol (E) self-administration in an operant access procedure in the P rat. Female P rats were given experience with 2-3 months of 1-hr limited access E self-administration. The rats were given a choice to lever press for a 10%, 20% or 30% (v/v) E with water freely available. Following this experience, rats were separated into groups that received bilateral ibotenic acid lesions of either the CeA or NAc shell, and a group that received sham surgery. One day following surgery, rats were returned to the operant chambers with concurrent access to the 3 E solutions; responses for the E solutions were measured over a one-month period. Responses on the 3 levers for the 10, 20 and 30% E solutions were 53.5 ± 11.6 for 10% E, 52.6 ± 20.1 for 20% E, and 88.7 ± 9.7 for 30% E before surgery and were not altered by sham operation. Post sham surgery responses were 37.9 ± 6.2 for 10% E, 64.3 ± 16.0 for 20% E, and 104.2 ± 12.1 for 30% E. Ibotenic acid lesions of the CeA reduced responding for E at the 10 % concentration by 63% (p < 0.01).. Whereas the CeA lesioned group showed a 30% and 35% decrease in responding at the 20% and 30% E concentrations, these decreases were not significant. In contrast to the CeA lesions, ibotenic acid lesions of the NAc shell did not alter responding on the E lever for any of the E concentrations. Overall, the results suggest that the CeA is necessary for maintaining instrumental self-administration of E, whereas the NAc shell may not be required for this behavior. In addition, these results contribute toward elucidating neural circuitries involved in E self-administration. (Supported in part by AA07611, AA07462, AA10721, AA10717, AA13522)
OPTIMIZING PASSIVE INFUSION PARAMETERS TO PRODUCE ETHANOL SELF-INFUSION IN C57BL/6J AND DBA/2J MICE.
T.L. Fidler; M.S. Powers; J.J. Ramirez; C.L. Cunningham.
Oregon Health & Science University, Portland, OR 97239.We have previously shown that ethanol experience determined level of ethanol selfinfusion in rats (Sprague Dawley, HAD and LAD) and in mice (C57BL/6 (B6), DBA/2J (D2), FVB/NJ and B6xFVB F1) when licks on a Kool-Aid tube were paired with intragastric (IG) infusions of ethanol. In the current experiments, we manipulated daily ethanol dose (experiment 1) or the number of ethanol infusions (experiment 2) during the passive infusion phase and assessed subsequent ethanol self-infusion in B6 and D2 mice. In Experiment 1, groups of mice with surgically implanted IG catheters initially received 3 passive infusions per day of 0 (water), 2, 3 or 4 g/kg of ethanol per infusion. In Experiment 2, the passively infused ethanol dose started at 0 (water) or 9 g/kg/day but the dose was distributed over 3, 6 or 9 infusions/day. In each experiment, passive infusion was followed by self-infusion phases in which animals had access (23 h/day) to one or two Kool-Aid solutions presented in drinking tubes (without ethanol). During the first 2 days of self-infusion, only one flavor was available (No-Choice) and licks produced IG ethanol infusions (20% v/v). During the next 5 days (Choice), a second flavor was also available and licks on this flavor were paired with infusions of water. In Experiment 1, mice in the B6 4 group self-infused more ethanol than those in the 0 or 2 groups. Intake by the B6 3 and 4 groups did not differ. Within D2s, mice in the 0 group self-infused less ethanol than mice in any of the other groups, which did not differ. In Experiment 2, there was no difference among B6 groups in ethanol intake. However, D2 mice in the 3 infusion group self-infused more ethanol than mice in any other D2 group. Although B6 mice generally self-infused more ethanol than D2 mice, there was no strain difference in the ethanol intakes of the 3-infusion groups. Distribution of ethanol intake may be even more important than total daily intake. Consistent with our earlier work, D2 mice in both experiments administered more of their ethanol in larger bouts than B6 mice. Across a range of parameters D2 mice gulp while B6 mice sip. Future work will examine IG ethanol intake in additional inbred mouse strains. Supported by INIA AA013479.
Strain differences in basal and alcohol-induced changes in members of the Homer/mGluR/NMDA signaling complex and Neurochemical sensitization.
Scott P. Goulding, Ilona Obara, Zuzana Kapasova, Karen K. Szumlinski
Department of Psychology, University of California at Santa Barbara, Santa Barbara, CA, 93106-9660Behavioural pharmacological and genetics data indicate a critical role for mesolimbic glutamate transmission in regulating various aspects of alcohol reward in both humans and laboratory animals. Decades of evidence reveals marked strain differences in alcohol intake and alcohol-induced motor sensitization between C57BL/6J (B6) and DBA2/J (D2) inbred mice but the underlying mechanisms for this divergence is not known. Thus, the present study assessed for strain differences in alcohol-induced changes in NAC glutamate levels and related these changes to the total protein expression of Homers, glutamate receptors and kinases activated upon Group1 mGluR stimulation. Adult, male B6 and D2 mice were injected repeatedly with alcohol (8 X 2 g/kg, every other day) and then in vivo microdialysis was employed to assay for (1) strain differences in the neurochemical responses to acute and repeated alcohol and (2) basal and alcohol-induced changes in NAC glutamate content. A second study employed immunoblotting to examine for strain differences in basal and alcohol-induced changes in Homer1/2, mGluR1/5, NR2a/b, PI3K and PKC epsilon within the NAC shell. While B6 and D2 mice did not differ with respect to their NAC glutamate response to an acute 2 g/kg alcohol injection, repeated alcohol injections (8 X 2 g/kg) induced robust glutamate sensitization within the NAC of B6 mice only. Alcohol-naïve B6 and D2 mice did not differ regarding NAC glutamate content nor probe recovery. However, repeated alcohol administration induced an elevation in NAC basal glutamate content only in B6 mice. Alcohol-naïve B6 mice exhibited an approximately 30-50% higher protein expression of Homer2, mGluR1 and mGluR5, as well as reduced levels of total PI3K and PKC epsilon within the NACshell. While strain differences were not observed for the alcohol-induced increase in Homer2 and mGluR1/5, only B6 mice exhibited an increase in NR2b levels following repeated alcohol treatment. Collectively, these data implicate alcohol-induced glutamate plasticity within the NAC in mediating vulnerability to high alcohol consumption and implicate genetic variance in members of the Homer/mGluR/NMDA signalling complex in this regard. This work was supported by NIH grants AA015351 and AA016650 (INIA-West) to KKS.
EXCESSIVE ALCOHOL CONSUMPTION FOLLOWING ALCOHOL INJECTIONS IS RELATED TO LOCMOTOR SENSITIZATION IN SELECTIVELY BRED HIGH ALCOHOL PREFERRING MICE.
N.J. Grahame and R.E. Bristow, IUPUI, Indianapolis, IN 46202Animal models of excessive alcohol intake have used oral (Alcohol Deprivation Effect) or vapor (Withdrawal Induced Drinking) alcohol exposure to increase voluntary alcohol consumption. While disparate behavioral models agree that alcohol exposure causes increases in alcohol drinking, they disagree on the types of neuroplasticity that are thought to mediate this increase. In selectively bred High Alcohol Preferring mice, repeated intermittent exposure to injected ethanol (2-3.5 g/kg) causes the sensitization to the locomotor stimulating effects of ethanol. In this study, we examine whether such injections also increase voluntary alcohol drinking. We also examined the recruitment time and durability of changes in drinking following alcohol exposure. Mice received 10 days free-choice access to 10% ethanol for 30 min daily during the dark part of the cycle, with the last 2 days of access in the locomotor testing apparatus. After baseline drinking, mice were given 2 rounds of injections of either saline (n = 15) or alcohol (3.5 g/kg, n = 28) given every 2 days over an 8-day period in the apparatus, but with the drinking tubes removed. After each round of 4 injections and a locomotor sensitization test, they were again given access to ethanol for 5 days in the apparatus, starting either 2 or 8 days after injections. Both alcohol intake and locomotor activity were measured throughout. After 4 injections, alcohol mice showed locomotor sensitization, and drinking increased from about 1 g/kg to 1.8 g/kg. The increase in drinking took at least 3 days to emerge and lasted up to 8 days. Normalization of drinking occurred more rapidly when mice had alcohol access. After two rounds of alcohol injections, greater locomotor sensitization was observed, and drinking increased from about 1.3 g/kg to 2.2 g/kg, lasting 2-11 days after alcohol injections. Results are broadly consistent with other studies showing that alcohol exposure increases drinking. However, while physical withdrawal peaks 8-12 h after ethanol exposure and tends to dissipate thereafter, we did not observe increased drinking until at least 3 days after initial alcohol injections. Like locomotor sensitization, elevated drinking persisted. These data suggest that locomotor sensitization, rather than alcohol withdrawal, is involved in the increased drinking observed after intermittent ethanol injections in these mice. AA13483 to NJG
DETECTION OF RECIPROCAL QTL FOR ETHANOL WITHDRAWAL AND CONSUMPTION.
Hitzemann R, Edmunds S, Malmanger B, Wu W, Darakjian P, McWeeney S and Belknap J
Departments of Behavioral Neuroscience and Public Health and Preventative Medicine, Oregon Health & Science University, Portland OR and Research Service, Veterans Affairs Medical Center, Portland ORSeveral studies have shown that for crosses derived from the C57BL/6J (B6) and DBA/2J (D2) mouse strains, there is an inverse relationship between ethanol withdrawal and ethanol consumption/preference (see Metten et al. 1998). Whether or not this relationship is true for other genotypes e.g. heterogeneous stock (HS) animals, is less clear. In the current study, short-term selective breeding from HS animals was used to create High and Low lines for acute ethanol withdrawal and ethanol consumption (2 bottle choice, continuous access). The HS stock was formed from the B6, D2, BALB/cJ and LP strains; this HS stock (HS4) is maintained as 48 families. The selection for both phenotypes began with 192 animals at G19 (2 males and females from each family). The selection for withdrawal was stopped at S5 and for consumption at S4. For both selections there was a marked divergence of the High and Low lines. Animals selected for acute withdrawal did not differ in ethanol consumption. However, the animals selected for ethanol consumption did differ for acute withdrawal and in the expected direction. Forty-eight to 56 animals from each line were genotyped using a 768 SNP panel. Six reciprocal QTLs (LOD > 11) were detected on chromosomes 1 (2), 3, 6, 11, 16 and 17. Haplotype analysis was used to narrow the most proximal chr 1 QTL to an interval between 170 and 173 Mbp (NCBI build 37). Three candidate genes in this relatively gene poor region are Atf6, Nos1ap and Kis (Umhk1). Nos1ap is cis-regulated (HS4 striatum database – Affymetrix 430 2.0 array). Kis is a member of a large gene network that contains several GABA and glutamate receptors and accessory proteins; in addition, the Kis network contains 7 of the 15 genes designated by the INIA-West consortium as being high priority for regulating ethanol consumption. Supported in part by AA 010760, AA 11034 and AA 13484.
GENETIC DETERMINANTS OF ALCOHOL CONSUMPTION BY MICE
P.L. Hoffman, L. Saba, S.V. Bhave, K. Kechris, W. Hu, Y. Blednov, D. Finn, N. Grahame, B. Tabakoff
University of Colorado School of MedicineThere have been a large number of studies supporting the influence of genetic factors on susceptibility to alcohol dependence. Although quantitative aspects of alcohol consumption do not currently enter into the definition of alcohol dependence in humans, it has been considered that higher alcohol consumption triggers the neuroadaptive phenomena that lead to dependence on alcohol. Therefore, the quantity of alcohol consumed may be regarded as a predisposing factor for the development of alcohol dependence. We have taken an unbiased approach to identifying genes that may influence the quantity of alcohol consumed by performing a meta-analysis correlating brain gene expression with levels of alcohol intake by selected lines of inbred strains of mice. Our filtering procedures, including QTL analyses, generated a list of eight candidate genes with highly heritable expression levels. These genes explained a significant amount of the variance in alcohol consumption. Using the Allen Brain Atlas for gene expression, we noted localization of the expression of the candidate genes in the olfactory and limbic areas of brain and the orbitofrontal cortex. Informatics techniques suggested the role of the candidate genes in neuronal migration, differentiation and synaptic remodeling. Breeding studies were used to evaluate the ability of a subset of the genes to predict the levels of alcohol consumption. Our results emphasize the importance of olfactory cues, learning and memory processes in the genetic influence on alcohol consumption by mice.
Genomic Determinants of Acute Alcohol Tolerance
W. Hu; L. Saba; S. Bhave; P. Hoffman; B. Tabakoff
Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.Background: Alcohol “sensitivity” has been proposed as a predictive factor for development of alcohol dependence. Most measures of alcohol “sensitivity” in humans and animals include a component which can be ascribed to acute functional tolerance. Acute functional tolerance (AFT) to alcohol is a form of tolerance that develops within a single alcohol exposure and has a genetic component. Methods: We used microarray technology (Affymetrix), and quantitative trait locus analysis of phenotypic and gene expression data across 32 BXD RI strains, 20 inbred strains, and two replicate lines of mice selectively bred for differences in AFT (HAFT/LAFT) to identify differentially expressed candidate genes in the brain contributing to predisposition to AFT. AFT was measured in mice using the stationary dowel test. Meta-analysis was used to combine results from three types of animals. Results: Among 45021 probe sets, 5468 probe sets have a significant or suggestive eQTL (P<=0.1). 1210 probe sets have an eQTL overlapping with one of the AFT bQTLs. Fifty of these 1210 probe sets are correlated with AFT through meta-analysis of the three populations of animals (FDR <0.01). Among these 50 probe sets, 13 have above median heritability in both the 20 inbred mice (median=0.49) and the BXD mice (median=0.4). Sequence alignment on Ensembl (http://www.ensembl.org) showed that 8 probe sets align to the genes they are designed to represent and the remaining 5 probe sets do not. These 8 genes (Epb4.1l2, Wnk1, Hlf, Kcnb1, Cutl1, Glcci1, Mtch2 and A130022J15Rik) are considered candidate genes for AFT. The location of brain expression of these genes was mapped using the Allen Brain Atlas, and the transcript location and molecular pathway analysis indicated that brain structures and biochemical pathways implicated in long term potentiation and memory may also participate in the generation of acute functional tolerance. Conclusions: We identified eight candidate genes that may affect acute alcohol tolerance genetically. In a separate study, we also determined that chronic alcohol treatment affects both brain gene expression profile and AFT. Further studies are ongoing to identify the genes whose expression levels may influence AFT after chronic alcohol treatment. Candidate genes from the meta-analysis of three populations of animals and those changing after chronic alcohol treatment will be compared. (Supported by NIAAA and Banbury Foundation)
Ethanol modulation of long term depression in the nucleus accumbens: Cannabinoid-1 and nmda receptor involvement.
Z.M. Jeanes; R.A. Morrisett.
University of Texas-Austin, College of Pharmacy, Austin, TX 78712.Alterations in mesolimbic glutamatergic synaptic plasticity are believed to underlie some of the neural adaptations that contribute to the development and/or expression of drug and ethanol dependence. Our goal in this INIA-West study is to understand how changes in N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD) in the nucleus accumbens (NAc) correlate with the induction of ethanol dependence.
We used in vitro whole-cell voltage clamp techniques to study LTD at GABAergic medium spiny neurons (MSNs) of the NAc shell. Previously, we have shown NMDAR-LTD is completely abolished by acute application of 40mM ethanol (105 ± 10%, n=7) and after in vivo chronic intermittent ethanol vapor exposure (118 ± 12%, n=8) (ACER, 31:144A, 2007). Therefore, we next investigated how ethanol exerted these effects on LTD expression in the NAc shell.
One mechanism by which ethanol may modulate glutamatergic synaptic transmission is through the endocannabinoid (eCB) system. In the presence of the cannabinoid-1 receptor (CB1R) antagonist, AM-251, LTD-inducing conditioning stimuli surprisingly induced a long-term potentiation (LTP) (127 ± 12%, n=17). Neither LTD nor LTP was observed using the same conditioning stimuli in the presence of both AM-251 and the selective NMDAR antagonist, DL-APV (95 ± 14%, n=5). Co-application of 40mM ethanol with AM-251 also occluded LTD (109 ± 11%, n=6).
Our prior findings indicate that adaptations occur in the NAc after in vivo chronic intermittent ethanol exposure as evident by the occlusion of LTD. In this study, we observed that both CB1 and NMDA receptors influence the expression of LTD in the NAc.
These data suggest that a complex association exists between ethanol, NMDA receptors, and eCBs, and their combined effects on synaptic plasticity in the NAc. Because ethanol is known to interact with both the glutamatergic and endocannabinoid systems, we speculate that alterations in NAc-LTD may contribute to the adaptations that re-wire the motivational circuitry in the ethanol-dependent brain. (Supported by U01AA16651 (RAM) and R01AA15167 (RAM)).
PRIOR DISORDERED EATING INCREASES ETHANOL DRINKING IN AN OTHERWISE ALCOHOL AVOIDING MOUSE STRAIN.
D.J. Cates; H. Stern; B.L. Kieffer; A.J. Roberts.
The Scripps Research Institute, Molecular & Integrative Neurosciences Department, La Jolla, CA 92037, USAAssociations between impulse control disorders, for example certain eating disorders and alcohol abuse, have been found; however, experiments have tended to focus on the correlative nature of these with regard to genetic predispositions and relationship to comorbid psychiatric symptoms. The results of a recent study of college women suggested that disordered eating and alcohol abuse might be associated in a more causative manner. Women who dieted were more likely to abuse alcohol, independent of family history of alcohol use or depressive symptoms (Krahn et al., Eating Behaviors 6, 43-52, 2005). This has important implications in practice and supports the hypothesis that dieting can change the brain’s reward functioning. As an initial test of the hypothesis that an environmental manipulation designed to model dieting can increase alcohol drinking, we examined the effects of disrupting food availability in a mouse strain previously shown to drink little ethanol, the mu opioid receptor knockout mice (MOR-/-). Female MOR-/- and wild type (WT) mice (N = 9-10 per group) were exposed for 6 weeks to alternating 24 hr periods of food restriction and availability Monday-Friday, and then a high fat-containing lab chow was available on weekends. Control mice of each genotype received ad lib standard lab chow throughout this time. Following this manipulation, all mice were returned to ad lib lab chow for 2 weeks. At the end of this time, body weights and food intake in the “Control or C” and “Altered Diet or D” groups were equivalent. All mice were then exposed to a two bottle, free choice ethanol drinking paradigm in which they received 3, 6, 9, and 12 % ethanol (w/v) for 5 days each (24 hr exposure). As previously reported, MOR-/- mice in the C group drank less ethanol than WT-C mice overall. Very interestingly, ethanol drinking in MOR-/- mice was increased in the D group (and to a lesser, non-significant extent in WT-D mice). In fact, ethanol drinking in MOR-/--D mice reached levels seen in the more alcohol-accepting WT mice (C57BL/6). These data suggest that an environmental manipulation modeling dieting may increase alcohol acceptance. Further experiments are being designed to extend this work, but these preliminary results may have very important implications, suggesting that dieting in females actually may increase alcohol acceptance and thus the potential for abuse. Supported by INIA-West (AA013523).
ALCOHOL BINGE DRINKING ALTERS GENE EXPRESSION IN THE NUCLEUS ACCUMBENS SHELL OF ALCOHOL-PREFERRING (P) RATS
M. W. Kimpel, R. L. Bell, J. N. McClintick, H. J. Edenberg, W. J. McBride
Institute of Psychiatric Research, Depts of Psych, Biochem & Mol Biol, Medicine and Medical & Mol Genetics, Indiana University School of Medicine, Indianapolis, IN 46202The objective of this study was to determine the effects of excessive alcohol drinking by P rats on changes in gene expression in the nucleus accumbens shell (ACB-sh) of P rats. Adult male P rats were given access to 15 & 30% ethanol (EtOH) for daily one-hr sessions given 3 x during the dark cycle for a total of 8 weeks (EtOH intakes of ~2 g/kg/session); rats were killed 1, 6 & 24 hr after the last EtOH drinking session. The ACB-sh was obtained from frozen brain sections using the micro-punch technique. There were no statistically significant differences in gene expression between the control (water only) and the EtOH groups at any of the individual time points; however, an analysis of the combined data from all time points indicated a significant main effect of EtOH. In the ACB-sh, (a) there were 275 differences in named genes between the water and EtOH groups (FDR < 0.25); (b) most of the differences were small (1.1- to 1.2-fold); (c) many more genes (> 3X) had higher expression levels in the EtOH than control group; (d) 52 genes were located within rat QTLs for EtOH preference; and (e) 10 genes were within both mouse and rat QTLs for EtOH preference (including e.g., prostaglandin-endoperoxide synthase 1/COX1, neurochondrin, splicing factor proline/glutamine rich, fatty acid binding protein 3, endothelin converting enzyme 1, and neurogranin. All but COX1 were up-regulated with EtOH. GO category analysis revealed 28 significantly over-represented categories (p < 0.05) including: regulation of synaptic transmission, regulation of synaptic plasticity, and several categories relating to general metabolism. GeneSet Enrichment Analysis (GSEA) using KEGG categories demonstrated over-representation of metabolic categories such as gluconeogenesis, oxidative-phosphorylation, and other general metabolic categories; in addition, neuro-biological categories such as signaling of insulin, GnRH, and calcium, as well as long-term-potentiation were also represented. Overall, the results suggest that alcohol binge drinking produces modest changes in expression of a large number of genes within the ACB-sh, affecting multiple biological processes, the combination of which could result in marked alterations in neuronal function. Supported in part by AA07611, and INIA grants AA13521, AA13522, AA16660 and AA16652, and the Indiana Genomics Initiative (INGEN), which is supported in part by the Lilly Endowment Inc.
MUTANT ANALYSIS OF GENES ESSENTIAL FOR ALCOHOL TOLERANCE IN DROSOPHILA
Harish Krishnan, Rosie Ramazani, Jascha Pohl and Nigel Atkinson Section of Neurobiology,
The Waggoner Center for Alcohol and Addiction Research, The Un versity of Texas at AustinWe are investigating the genetic basis for alcohol and organic solvent tolerance using the fruitfly Drosophila melanogaster. Drosophila offers elegant tools for gene identification and manipulation. Tolerance is defined as a reduction in an effect of a drug caused by prior exposure to the drug. In our tolerance assay, flies that have received a prior sedating dose of ethanol recover faster from sedation than flies that are being sedated for the first time. This sedation recovery assay is thought to be analogous to the loss of righting reflex assay in mammals. We have shown that the slo gene is required for the acquisition of pharmacodynamic tolerance in flies. This gene encodes the BK-type calcium-activated potassium channel. Using a manual and a semi-automated assay for alcohol tolerance we have identified a collection of mutations that interfere with the capacity to acquire BK-channel dependent tolerance. It is well known that anesthetics and abused drugs preferentially modulate specific molecular pathways in defined regions of the brain. Using the binary GAL4/UAS system to express slo in different parts of the Drosophila nervous system we have identified brain structures in which slo induction produces alcohol resistance. Similarly, transgenic expression of a temperature sensitive dynamin gene has been used to selectively block neural signaling to ask whether the acquisition of tolerance is an emergent or a cell autonomous property of the Drosophila nervous system.
Differential effects of ethanol on protein levels in the nucleus accumbens shell of alcohol-preferring (P) and –non-preferring (NP) rats
W. J. McBride, J. A. Schultz, M. Wang, M. W. Kimpel, Z. A. Rodd
Dept of Psychiatry, Institute of Psychiatric Research, and Dept of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202The objective of this study was to determine the effects of repeated i.p. injections of ethanol (EtOH; 1 g/kg) on protein expression levels in the nucleus accumbens shell (ACB-sh) of adult male P (n = 10/group) and NP (n = 10/group) rats. Rats were injected once daily for 5 consecutive days with either saline or EtOH; 24 hr after the last injection, rats were killed and the brains extracted and frozen in isopentane in dry ice. Frozen brains were subsequently sectioned and micro-punch samples of the ACB-sh obtained; samples from each rat were individually analyzed. Proteins were extracted and subjected to trypsin digestion; resulting peptides were separated, quantified and identified with a LC-MS procedure. Over 1500 proteins were identified with a high degree (> 90%) of confidence. There were no significant differences in baseline protein expression levels between P and NP rats. In NP rats, EtOH significantly changed expression levels of almost 500 proteins. In P rats, there were approximately 170 significant changes produced by EtOH. For both the P and NP rats, most differences were in the range of 1.1- to 1.2-fold. Gene Ontology (GO) analysis indicated a number of significant biological processes (BP) and molecular function (MF) categories in which there were proteins that were differentially altered by EtOH between the P and NP lines. There were differential responses to EtOH between the P and NP rats in: (a) ‘neurotransmitter secretion’ and ‘response to hypoxia’ for the BP categories; and (b) ‘ATP binding’, ‘GTP binding’, ‘signal transducer activity’, ‘sodium-potassium-exchanging ATPase activity’, and ‘identical protein binding’ for the MF categories. In these categories, EtOH treatment increased protein levels in the NP line and decreased expression of these same proteins in the P line. Overall, the results suggest that the repeated non-contingent administration of moderate doses of EtOH produced alterations that could increase intracellular signaling and neurotransmission processes in the ACB-sh of NP rats and decrease these same processes in the ACB-sh of P rats. These differential protein changes in response to EtOH in this limbic structure might indicate alterations in neuronal function that could promote (in the P line) or deter (in the NP line) alcohol drinking. (Supported in part by AA07611, AA16652 [INIA] and INGEN®)
TARGETING GENES CONTRIBUTING TO EXCESSIVE ALCOHOL DRINKING BEHAVIOR: STUDIES WITH ALCOHOL-PREFERRING (P) AND –NON-PREFERRING (NP) RATS
W. J. McBride, M. W. Kimpel, R. L. Bell, J. A. Schultz, J. N. McClintick, Z. A. Rodd, T. Liang, W. N. Strother, H. J. Edenberg
Depts of Psychiatry, Biochemistry & Molecular Biology, Medicine and Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202The objective of this study was to identify genes that could contribute to a predisposition to high alcohol drinking in P rats. In experiment 1, adult male alcohol-naïve P and NP rats were used. In experiment 2, adult male P rats were given access to 15 & 30% ethanol (EtOH) for 3 one-hr sessions during the dark cycle for a total of 8 weeks (EtOH intakes of ~2 g/kg/session), and were killed 1, 6 & 24 hr after the last EtOH drinking session. The nucleus accumbens shell (ACB-sh) was obtained from frozen brain sections using the micro-punch technique. There were 158 named genes that had different expression levels in the ACB-sh between P and NP rats. Most of the differences were between 1.3 to 1.7-fold. A Gene Ontology (GO) analysis indicated several significant categories, which included synaptic transmission, neuron and neurite development, and small GTPase mediated signal transduction. There were several genes that were in common with genes differentially expressed between inbred HAD and LAD rats and located within rat QTLs for EtOH preference, e.g., protein kinase C epsilon, purinergic receptor P2X ligand-gated ion channel 4. In the 2nd experiment, there were no differences between the control and the EtOH groups at any of the individual time points. However, an overall analysis of the combined data from all time points indicated a significant EtOH effect. There were over 380 differences in named genes between the water and EtOH groups; most of the differences were small (1.1 to 1.2-fold) and many more genes (3-fold) had higher expression levels in the EtOH group. Over 80 genes were located within rat QTLs for EtOH preference, and there were 10 genes that were within both mouse and rat QTLs for EtOH preference, e.g., neurogranin, neurochondrin, Rab14. Comparison of the results from both experiments produced a list of common genes that were different between the P and NP rats and were also altered by EtOH drinking, some of which included guanine nucleotide binding proteins, phosphatidylinositol 3-kinase catalytic alpha subunit, glutamate receptor ionotropic AMPA 1 (alpha 1) and syntaxin 5a. Overall, the results of this study suggest a combination of genes may contribute to vulnerability to alcohol drinking and maintaining excessive alcohol drinking of P rats. [Supported in part by AA07611, and INIA grants AA13521, AA13522, AA16660 and AA16652, and the Indiana Genomics Initiative (INGEN), which is supported in part by the Lilly Endowment Inc.]
ALCOHOL AS A POTENTIAL ANXIOGENIC DURING THE ADAPTATION TO CHRONIC ALCOHOLISM
M.K. McDonald, J.B. Hoek, B.A. Ogunnaike, J.S. Schwaber
Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA 19107
Department of Chemical Engineering, University of Delaware, Newark, DE, USA 19716Although alcohol is generally thought to be anxiolytic, we have recorded behavioral anomalies within a subset of our alcohol-fed animals that were indicative of increased anxiety. Male Sprague Dawley rats (Harlan, Indianapolis, IN) were fed a Lieber DeCarli liquid alcohol diet and their daily alcohol intake was recorded. We scored videotapes of the animals for anxiety-like behaviors and tested the level of anxiety in these animals on an elevated zero maze. We also found that some animals within the heightened anxiety subset died suddenly within the first 35 days on the Lieber DeCarli diet, predominantly during the second and third week on the diet, with a peak at ~18 days. Prior to death, the animals were more likely to have large variations in their daily consumption rates including episodes of voluntary withdrawal. In order to determine whether we could predict the animals susceptible to increased anxiety and/or death based on their intake patterns, we performed a principal component analysis of the alcohol intake data. This analysis uncovered changes in the intake pattern up to six days prior to death, indicating that it might be possible to identify susceptible animals before their death by careful monitoring of intake patterns.
Preliminary pathology reports indicate that the deaths were active with no identifiable cause, potentially indicative of sudden cardiac death. Although the animals’ deaths were typically preceded by a period of voluntary withdrawal, we found that involuntary withdrawal during the time of greatest susceptibility (18 days) did not lead to death in any animal tested. Therefore, we hypothesize that, for a subset of animals, alcohol is anxiogenic during the adaptation process. This increased anxiety then causes changes in alcohol intake as well as deficiencies in the regulation of cardiopulmonary homeostasis via the anatomical and functional interconnection between homeostatic and emotional regulatory regions of the brain (the nucleus tractus solitarius and central nucleus of the amygdala). Furthermore, we hypothesize that these dysregulations lead to the deaths we have observed. In our future studies, we will test these hypotheses and will also include the correlation of molecular measurements in the associated regions of the brain during this process. This work is supported by AA015601 to JSS and T32 AA007463 support of MKM.
Short-term withdrawal from intermittent alcohol access elevates Homer and glutamate receptor expression selectively in the NAC core and CeA of P rats.
I. Obara1, R.L. Bell2, C. Reyes1, L.A. Larson2, A. Ary1, W.A. Truitt2 and K.K. Szumlinski1
1Dept. Psychology and the Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA, 93106-9660.
2Dept. Psychiatry, Indiana University School of Medicine, Indianapolis, IN, 46202.Homers are constituents of multi-protein scaffolding complexes that regulate the trafficking and function of NMDA and Group1 metabotropic glutamate receptors in the brain. This study extends earlier immunoblotting data from C57BL/6J (B6) mice by examining the effects of continuous and intermittent alcohol consumption on protein expression of Homers and their associated glutamate receptors within various brain structures of selectively bred, alcohol-preferring P rats. For 6 months, P rats had 24-hr free-choice access to alcohol (15% and 30% concurrently) under continuous (7 days/week) or intermittent access (4 days/week; binge) conditions in their home cage. Rats were sacrificed 24 hrs or 4 weeks after termination of alcohol access, regions of interest were micropunched and tissue processed for immunoblotting. At the end of their drinking, the binge and continuous rats were drinking 6.9 ± 0.3 and 7.7 ± 0.4 g/kg/day, respectively. Alcohol-consuming groups did not differ from water-drinking controls vis-à-vis protein expression within the NAC shell, the dorsal striatum or the basolateral amygdala. In contrast, binge rats withdrawn for 24 hrs exhibited an approximate doubling of Homer1b/c, Homer2a/b, mGluR5 and NR2a/b levels within the NAC core and the central nucleus of the amygdala (CeA). These changes were not observed in the other alcohol groups tested. These data provide further support for the hypothesis that an increase in NAC Homer-glutamate signaling is an important neuroadaptation for excessive alcohol consumption. While our data for continuous alcohol consumption in P rats did not replicate earlier findings from B6 mice, this discrepancy might relate to species/strain differences in alcohol intake during a 24-hr period. Finally, the present data show for the first time that binge-like alcohol consumption can produce short-term increases in Homer/glutamate receptor expression within the CeA, which may increase the aversiveness of early alcohol withdrawal and consequently the negative reinforcing properties of alcohol. Taken together, these data demonstrate regionally selective increases in Homer/glutamate receptor expression during short-term withdrawal from binge-like alcohol-drinking, which may be predictive of increases in the excitability of brain regions mediating both the positive and negative reinforcing properties of this drug. This work was supported by NIH grants AA013522 (INIA West) to RLB and AA016650 (INIA West) to KKS.
Epigenetic regulation of ethanol intake: increased drinking following withdrawal from intermittent ethanol exposure is potentiated by sodium butyrate.
Oberbeck, DL, Finn, DA, Snelling, C, Hitzemann, RJ
Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, ORStudies in rodents have determined that intermittent exposure to ethanol vapor can increase subsequent ethanol self-administration. While the increase in ethanol intake after this intermittent withdrawal paradigm has been associated with alterations in gene expression, the regulation of these gene products is as yet unknown. One method of transcriptional regulation is the post-translational modifications of histone proteins within the nucleus of activated neurons; the present study was designed to examine how the inhibition of histone acetylases during intermittent withdrawal from ethanol vapor affects subsequent ethanol self-administration. Baseline ethanol intake was established in male C57BL/6J (B6) mice using a limited access 2-bottle preference paradigm, with daily 2h access to water or ethanol (15% v/v) beginning 3h into the dark phase of a 12:12 light-dark cycle. Mice were matched for baseline intake and divided into two cohorts of withdrawal experience; intermittent withdrawal or continuous withdrawal; each cohort was further subdivided into two drug treatment groups; sodium butyrate or vehicle. Mice in the intermittent withdrawal groups underwent 3 bouts of 16h exposure to ethanol vapor (adjusted to yield BECs of 1.5-2.0 mg/ml) and 8h exposure to room air (withdrawal); mice in continuous withdrawal groups were exposed to room air only. Depending on treatment group, mice received daily injections (i.p.) of ethanol (2.0g/kg) and/or pyrazole (68.1g/kg), and sodium butyrate (1.2g/kg) and/or saline vehicle. Post-withdrawal, daily ethanol intake was measured using 2h, 2-bottle preference tests as above. Following two cycles of vapor exposure and post-withdrawal testing, mice who received sodium butyrate during either intermittent or continuous withdrawal had significantly higher levels of ethanol intake on the first two days of testing compared to vehicle-injected mice; vehicle-injected mice subjected to intermittent withdrawal had significantly higher ethanol intakes than continuous withdrawal controls for at least one week. These results suggest a role for the epigenetic regulation of ethanol intake; future studies are planned which will examine putative gene targets which may be upregulated by the administration of sodium butyrate during withdrawal.
LONG-TERM ALCOHOL EXPOSURE BY VAPOR CHAMBER ALTERS MRS DETECTABLE METABOLITES IN RAT STRIATUM
N.M. Zahr; D. Mayer; S. Vinco; J. Orduna; E.V. Sullivan; A. Pfefferbaum
Neuroscience Program, SRI International, Menlo Park, CA
Stanford University School of Medicine, Stanford, CAIn vivo magnetic resonance spectroscopy (MRS) can be used in longitudinal studies to track alterations in brain metabolites during the development of alcohol dependence in animal models of human alcoholism. We used proton MRS on a clinical 3T GE human scanner equipped with a high-strength insert gradient coil to track brain metabolite changes in the striatum in response to long-term ethanol (EtOH) exposure in 10 sibling pairs of wild-type male Wistar rats. Following baseline scanning, one rat from each sibling pair was exposed to ethanol (EtOH), the other to air (Ctrl), using a rodent vapor inhalation system (La Jolla Alcohol Research Inc., La Jolla, CA); rats had free access to food and water. After 16 weeks of escalating EtOH exposure, average blood alcohol levels (BALs) were 292mg/dl (range: 124-475mg/dl); 2 EtOH rats died during this exposure period. EtOH rats lost 1.2% body weight (mean wt=606g), whereas the controls gained 13.4% (Ctrl mean wt=714g). Four of 8 EtOH rats showed neurological signs (e.g., loss of righting reflex). Eight more weeks of higher dose EtOH exposure resulted in average BALs of 445mg/dl (range: 342-499mg/dl; EtOH wt=567g, -6.8%, Ctrl wt=733g, +2.9%); all 8 EtOH rats exhibited neurological signs. Major proton metabolite signals were quantified and referenced to water. A 2-group repeated measures (3 times and 7 metabolites) analysis of variance revealed a significant group-by-metabolite-by-time interaction (p=.009). The groups did not differ at baseline, but after 16 weeks of EtOH exposure, the EtOH group had higher total choline (Cho) levels (p=.0004) than Ctrls. After 24 weeks of greater EtOH exposure, the EtOH group had higher glutamate (Glu; p=.026), Glu+glutamine (Glx; p=.007) and Cho (p=.002) than the Ctrl group. This pattern of metabolite changes suggests onset of neurotoxicity in response to EtOH and comports with findings in actively drinking alcoholic humans, in whom Cho levels were reported to be above normal in parietal gray matter. (Support: AA013521-INIA, AA005965, AA017168).
VENTRICULAR EXPANSION AFTER ALCOHOL EXPOSURE BY VAPOR CHAMBER IN WISTAR RATS
A. Pfefferbaum; N.M. Zahr; D. Mayer; S. Vinco; J. Orduna; T. Rohlfing; E.V. Sullivan
Neuroscience Program, SRI International, Menlo Park, CA
Stanford University School of Medicine, Stanford, CAChronic alcoholism is commonly marked by neuroradiological evidence for ventricular expansion and shrinkage of regional brain tissue. For an animal model to parallel human alcoholism, excessive alcohol exposure should produce widespread brain damage, manifest as tissue shrinkage and complementary ventriculomegaly. Our prior longitudinal rodent studies indicate, however, that despite achieving blood alcohol levels (BAL) of ~125 mg/dl with voluntary consumption, the alcohol-preferring P rat exhibited only modest brain dysmorphology on structural magnetic resonance imaging (MRI). To enhance BALs, we used a rodent alcohol inhalation system (La Jolla Alcohol Research Inc., La Jolla, CA) to expose sibling pairs of wild-type, male Wistar rats to high doses of vaporized alcohol; rats had free access to food and water. MRI, acquired on a 3T GE human scanner equipped with a high-strength insert gradient coil, was conducted before and after 16 weeks of vaporized alcohol exposure. Post-exposure, average BALs were 291.9mg/dl (range=124.1 to 474.6mg/dl). Alcohol rats lost 1.2% body weight (N=9; mean weight=596g), whereas the controls gained 13.4% (N=10; mean weight=714g). MRI data were quantified with atlas-based parcellation computations based on delineation of the lateral ventricles on the baseline images of one untreated, reference animal. The lateral ventricles were identified bilaterally with a semi-automatic histogram segmentation operator, beginning 20 (.125 mm thick) slices rostral to the anterior commissure (AC) and extending 40 slices caudal to the AC. This segmented image was then registered to each of the remaining rats' baseline images using nonrigid coordinate transformations. Similarly, the baseline image from each animal was registered to its time2 images for parcellation of the ventricles. Comparison of the ratios of ventricular volume change (time2-time1/time1) revealed a profile of significant (p<.05, 2-tailed t-test) post-alcohol vapor exposure ventricular expansion in 9 contiguous slices, beginning .625 mm posterior to the AC (encompassing dorsolateral ventricles) and 7 contiguous slices, beginning 3.50 mm posterior to the AC (encompassing ventrolateral ventricles). This ventricular dilatation produced in wild-type rats with involuntary alcohol exposure successfully modeled ventricular expansion, a robust phenotype of human alcoholism. (Support: AA013521-INIA, AA005965, AA017168).
MAPPING OF ETHANOL DRINKING QUANTITATIVE TRAIT LOCI (QTL) THAT INDUCE AN OVER-DOMINANT EXCESSIVE DRINKING PHENOTYPE
T.J. Phillips; C. Reed; L.L. Brown; S. Burkhart-Kasch; C.H. Yu; M.C. Helms; B. Malmanger; R. Hitzemann; J.K. Belknap.
VA Medical Center, Portland Alcohol Research Center, Oregon Health & Science University, Portland, OR 97239
The goal of this project is to map genes that influence excessive alcohol drinking and produce an over-dominant drinking phenotype in the F1 cross of the C57BL/6J (B6) x FVB/J (FVB) progenitor strains (see Blednov et al., 2005, Alcohol Clin Exp Res 29:1949-58). Six hundred and nineteen C57BL/6J (B6) x FVB/J (FVB) F2 animals were phenotyped for 24-h, 2-bottle choice ethanol drinking (increasing concentrations from 3% to 30% ethanol; v/v), followed by limited access alcohol drinking in the dark (DID; 20% ethanol; v/v), using first a single bottle and then a 2-bottle choice DID procedure. Single nucleotide polymorphism (SNP) genotyping across the entire genome was performed for all phenotyped F2 mice. Data for the different ethanol drinking phenotypes were then analyzed for phenotypic correlations and quantitative trait locus (QTL) analysis was performed. The analysis concentrated on the mode of inheritance (additive, fully dominant, over-dominant, epistatic) for each QTL taken singly and also in pairwise combinations. Average ethanol consumption amounts for the F2 mice during the 24-h, 2-bottle choice procedure were 3.5 ± 0.1, 12.5 ± 0.3, 22.2 ± 0.5, 37.1 ± 0.7 and 27.4 ± 0.5 g/kg, for the 3%, 10%, 20%, 30% and the repeated evaluation of the 20% ethanol concentrations, respectively. For DID, mice consumed about 4.5 g/kg ethanol in 2 h when there was no water choice, and they consumed about 3.5 g/kg ethanol in 2 h when ethanol was offered vs. water. Blood ethanol concentrations obtained during the DID procedure were about 1.1 mg/ml for the no choice condition and 0.6 mg/ml for the choice procedure. There were strong phenotypic correlations for the two DID procedures, which were only weakly correlated with ethanol consumption during the 24-h choice procedure. QTL analyses identified three chromosomal regions where over-dominance was indicated for the 24-h choice, 2-bottle procedure and consumption of 30% ethanol, the trait for which over-dominance was most pronounced in the original F1 analysis. These QTL are on chromosomes 11 (78.86 Mb), 15 (71.6 Mb) and 16 (75.16 Mb). Expression analyses will be used to identify promising candidates in these regions for the excessive drinking phenotype. Support: Dept of Veterans Affairs and NIAAA (U01AA016655, U01AA13484; P60AA010760).
INTEGRATION OF GENE EXPRESSION PROFILES ACROSS ANIMAL MODELS OF HIGH ALCOHOL DRINKING
I. Ponomarev.
Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712A large body of gene expression data has been and is being collected by INIA and non-INIA investigators using animal models of excessive alcohol consumption. Analysis across data sets for common patterns in gene expression provides more statistical power to detect small and reliable changes. The main goal of this project is to integrate brain gene expression data and to identify genes and biological pathways that show robust regulation across different INIA models of excessive alcohol consumption. This integration of data includes four steps. First, available and prospective mouse data sets are assigned into functional categories based on the nature of animal models used. Three domains have been created: “Genetics” includes data from alcohol-naïve selectively bred lines and inbred strains; “Binge” drinking includes data from mouse models based on Drinking In the Dark (DID) procedures and “Dependence” includes data from a variety of Withdrawal-Induced Drinking (WID) models. Next, data within each domain are analyzed using meta-analytic approaches to produce lists of genes consistently detected in each of the three categories. The second step identifies overlaps between any two or all three domains. The third step is proposed to further reduce candidate gene lists based on special interest “filters” that include data sets collected using alternative alcohol experimental procedures (other than DID or WID) and/or species (rat, human, fly). Finally, original and “filtered” gene lists are analyzed using bioinformatics tools. This integrative approach will identify biological entities (genes, pathways, etc.) likely to be functionally important for regulation of alcohol consumption. Top candidates will be posted at the INIA web site to solicit follow-up functional studies. Supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (UO1 AA013517; Pilot Project Component).
IN VIVO ETHANOL ADMINISTRATION INCREASES COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT (CART) IN THE MESOLIMBIC DOPAMINE SYSTEM OF C57BL6 MICE
A.G. Salinas and R.A. Morrisett
The University of Texas at Austin, College of Pharmacy, Austin, Texas 78712.CART, a putative neurotransmitter, was initially identified by PCR in rats treated with cocaine or amphetamine (Douglas et al, 1995). While several subsequent studies have characterized the role of CART in addiction, surprisingly few have examined the role of this neuropeptide in alcoholism. The current study was undertaken to corroborate and to expand upon our prior findings in male Sprague Dawley rats which were the first to demonstrate that ethanol administration can indeed elicit CART transcription in a dose-dependent manner (Salinas et al, 2006).
Male C57BL6 mice were acclimated to a reversed light schedule for two to three weeks and weighed every 2-3 days. For the each of four days before the experimental day, the mice were sham injected with PBS and on the fifth day the mice were administered saline or 1, 2 or 3 g/kg ethanol ip. For measures of CART transcription via PCR analysis, the mice were sacrificed and the nucleus accumbens (NAc) was microdissected. Total RNA was isolated, reverse transcribed and the resultant cDNA was then analyzed using a multiplex PCR reaction with primers for CART and 18S rRNA as a control. For immunohistochemical analysis of CART peptide itself, 1 hr following saline or ethanol administration, the mice were transcardially perfused with 4% paraformaldehyde. The fixed brains were subsequently sectioned and processed for CART immunoreactivity imaging with a DAB chromagen and light microscopy. Trunk bloods were collected from the PCR study animals to verify the blood alcohol content (BAC) for each animal and for use as a covariate. To account for the diurnal rhythm of CART all of the experimental procedures were carried out during a two hour window.
We observed an increase in CART mRNA in the NAc in mice treated with 2 or 3 g/kg ethanol (323±109% and 281±70% of control, respectively) but not in the 1 g/kg ethanol-treated mice. We also observed similar ethanol-induced increases in CART immunoreactivity in the NAc core and shell with a 2 g/kg dose (164±27% and 204±32%, respectively). We observed a significant correlation between ethanol treatment dose and BAC. These data indicate that ethanol modulates CART expression in the NAc across rodent species and therefore support the concept that CART is responsive not only to psychomotor activating agents but ethanol as well. This work was supported by NIH grants R01AA15167 and U01AA16651 to RAM.
FOS B IMMUNOREACTIVITY IN SELECT BRAIN REGIONS OF ALCOHOL-PREFERRING (P) RATS OPERANTLY RESPONDING FOR ETHANOL, SACCHARIN OR WATER.
W.N. Strother; Z.A. Rodd, Z. Gu, V. McQueen, W.J. McBride.
Institute of Psychiatric Research, Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, 46202.
The objective of the present study was to determine the effects of chronic ethanol (EtOH), saccharin (SACC) and water operant self-administration on FosB immunoreactivity (ir) in select brain regions of adult P rats. P rats were randomly assigned to 1 of 3 groups; 15% EtOH (n=11), 0.0125% SACC (n=11) or water (n=8). Rats were self-trained in daily 1-hr operant sessions in standard 2-lever chambers for 6 wks to respond concurrently for EtOH (FR5) vs water (FR1), SACC (FR5) vs water (FR1), or water (FR5) vs water (FR1). During the last 2 wks prior to perfusing the rats, responses/session averaged ~300 + 10 for the EtOH group, ~500 + 20 for the SACC group, and ~20 + 2 for the water group. EtOH intakes averaged ~1.5 g/kg/session during these 2 wks. Rats were sacrificed 1 hr after completing the operant session. Rats were transcardially perfused with 4% paraformaldehyde and post-fixed over night. Coronal 40 µm sections were collected in phosphate buffer using a freezing microtome. FosB-ir (Santa Cruz @ 1:1k) was detected using a modified avidin-biotin-immunoperoxidase protocol with diaminobenzidine as the chromogen. Mean cell counts were calculated as FosB-ir cells/mm2. There were no significant differences in FosB-ir between any of the groups (mean # Fos B cells/mm2 for water control group given in parentheses) in the nucleus accumbens core (~1,700) or shell (~1,100), lateral septum (~400), ventral tegmental area (~6), globus pallidus (~50), or the caudate putamen (~1,200). It is possible that the lack of an effect by EtOH is a result of the limited-access operant paradigm not producing sufficient blood ethanol concentrations to meet the thresholds necessary for FosB accumulation. In addition, the results suggest that activity and learning associated with the operant task do not alter FosB expression in certain CNS regions. (AA07611, AA10721, AA16652 [INIA]).
Homer2: An active regulator of vulnerability to high alcohol intake
Karen K. Szumlinski
Dept Psychology and the Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA, 93110The Homer family of post-synaptic scaffolding proteins has emerged as a critical regulator of neuroplasticity, including that produced by various drugs of abuse. Repeated alcohol exposure, via intraperitoneal injection, free-access consumption or intermittent, binge-like consumption up-regulates the protein expression of the Homer2 isoform within the nucleus accumbens - a brain region highly implicated in goal-directed behavior and motivation for drug. Converging evidence from behavioral and neurochemical genetic studies involving inbred and selectively bred rodent lines, Homer2 knock-out and related transgenic mice, as well as investigations into the effects of virus-mediated Homer2 over- and under-expression provide compelling evidence that idiopathic or alcohol-induced increases in accumbens Homer2 expression facilitate alcohol-induced neuroplasticity within the accumbens to promote an alcoholism-like behavioral phenotype. This work was supported by NIAAA grants AA015351 and AA016650 (INIA-West), as well as a Santa Barbara Cottage Hospital research award.
CAN TOLERANCE CONTRIBUTE TO PREDISPOSITON TO ALCOHOLISM?
B. Tabakoff, W. Hu, L. Saba, S. Bhave, P. Hoffman
University of Colorado Denver School of Medicine, Aurora, CO 80045Tolerance to ethanol’s actions can be evidenced within a single drinking session and such “acute tolerance” is, many times, operationally incorporated in measures of ‘sensitivity” to ethanol’s actions. Schuckit has well demonstrated the predictive value of low “sensitivity” to alcohol’s actions in unraveling the contribution of family history of alcoholism to increased propensity for the development of alcohol dependence in family history positive (FHP) males. Newman performed a meta-analysis of various studies on alcohol sensitivity of FHP subjects. The meta-analysis indicated that the low sensitivity to ethanol’s actions may be due primarily to rapid development of acute tolerance in FHP individuals. We performed a series of genetic/genomic studies in mice to identify genes whose levels of expression may be associated with propensity for development of acute tolerance. Thirty-two strains of recombinant inbred (RI) mice, twenty stains of inbred mice and two replicate lines of selectively bred mice (HAFT1, HAFT2, LAFT2 and LAFT2) were used in our work. Meta-analysis of correlations between gene-expression levels and development of acute tolerance across strains and lines of mice was used to generate results. Our approach for narrowing gene lists to the most appropriate candidates involves the premise that differentially expressed genes which correlate with a complex trait should be regulated in cis or trans manner from a phenotypic (p) QTL mapped for the complex trait of interest. QTL mapping for acute tolerance produced QTL’s on mouse chromosomes 1, 2, 4, 5 and 6. The expression (e) QTL’s for 50 genes from the list of correlated genes fell within the acute tolerance p QTL’s. Further filtering was accomplished by measures of heritability and careful probe sequence alignment. Eight candidate genes remained after all filters were applied. These eight candidate genes were Epb4.1, Kcnb1, Wnk1, Hlf, Cute1, Glccl1 an Mtch2. The candidate genes can be grouped in pathways involving glutamatergic, dopamine and adenosine neurotransmission and membrane polarization. Brain locations which have high levels of expression of these genes are the cerebral cortex and areas of the hippocampus (e.g. dentate gyrus) inferring parallels between acute tolerance and LTP. Interestingly, the p-QTL for acute tolerance in mice are syntenic for certain QTL’s for alcohol dependence mapped by the COGA research group. Supported by NIAAA and Banbury Fund.
Beyond the GPCR’s: Effector Characteristics and Ethanol’s Action
Boris Tabakoff, Sergey Pronko, Maria Couppis, Elisabetta Maragnoli, and Paula Hoffman
Although pharmacology has given elevated status to knowledge regarding receptors for neurotransmitters and hormones, receptors (and particularly GPCR’s) are only one of a multitude of components necessary to transduce a signal at a chemical recognition site to a signal recognizable to a cells interior. CRF signals originate through the interaction of CRF with the CRF Receptors (CRFR’s) 1 and 2. The CRFR 1’s are coupled through the Gs heteromeric proteins to adenylyl cyclase (AC). The identity of AC (10 isoforms) to which the CRFR 1’s are coupled makes a profound difference in the integration of intracellular responses to CRF and the biologic manifestations of fear and anxiety. We previously showed that the type 7 isoform of AC was the most sensitive to ethanol. We have modified the expression of the type 7 AC (AC7) in brains of mice by transgenic and homologous recombination (knockout ) technology and studied the behavioral/hormonal and electrophysiologic characteristics of these animals. Studies of behavioral manifestations of the genetic modulation of AC7 levels in brain showed lower levels of “anxiety” (plus-maze) in the transgenic AC7 male mice but this response was not seen in females. Conditioned fear responses also distinguished the AC7 transgenic mice from their WT littermates. The AC7 transgenic male mice showed an exaggerated in-vivo response to ethanol in terms of ACTH release into the circulation and the subsequent corticosterone response. Anterior pituitary slices from theAC7 transgenic mice showed a greater ACTH release in the presence of CRF and ethanol and the knock-down mice showed a lower response than the WT animals. Ethanol accentuated the cyclic AMP response to CRF in pituitary slices of the AC7 transgenic mice. Similarly, ethanol accentuated the cyclic AMP response in slices of amygdala taken from brains of AC7 transgenic mice. Electrophysiologic examination of GABA mediated MEPP’s in slices of central amygdala indicated that change in the levels of AC7 in brain altered the effect of ethanol on GABA release in this preparation. Measure of CRFR1 receptors indicated no significant differences between wild-type and transgenic and knockdown mice. Our data indicate that changes in effector levels/function can alter cellular, organ and behavioral responses to ethanol without significant changes in GPCR’s but through changes in coupling of the receptor to the rate limiting effector. Supported by NIAAA and Banbury Fund.
HISTORY OF ALCOHOL DEPENDENCE SHOWS LONG-LASTING EFFECTS ON CIRCADIAN BODY TEMPERATURE RHYTHMS IN MICEJ. L. Trujillo, A. J. Roberts, M. R. Gorman.
The Scripps Research Institute, Molecular & Integrative Neurosciences Department, La Jolla, CA 92037, USAThe purpose of this study was to examine the influence of ethanol dependence and withdrawal on circadian body temperature rhythms in mice. Relapse in human alcoholics is correlated with withdrawal-induced sleep dysfunction, which is one indicator of circadian function. This suggests that circadian dysfunction is related to alcohol dependence and that disruptions in circadian rhythmicity may contribute to relapse susceptibility. As body temperature is regulated by the circadian pacemaker and may be monitored continuously and non-invasively, it represents an attractive index of changes in circadian rhythmicity. It is well known that body temperature is affected by both ethanol withdrawal and intake, but there is little information about how alcohol induces enduring changes in body temperature, a practical question relating to humans. To address this issue, we monitored circadian body temperature rhythms in mice prior to and following induced alcohol dependence and withdrawal. Adult male C57BL/6J mice were implanted with telemeters and allowed recovery time before baseline rhythms were obtained. Following this, mice experienced a standard protocol for induced alcohol dependence including 3 consecutive days with 16 hours of vaporized alcohol per day. Blood samples were taken following each vapor session to measure blood alcohol levels. Immediately following the third (last) day of vapor, mice were returned to cages where rhythms were monitored for two weeks, within which time rhythms returned to a pre-dependence state. The vapor exposure procedure was repeated and again rhythms were monitored. Under these conditions, we found lasting alterations in body temperature rhythms. Interestingly, two bouts of vapor have been shown to produce robust and lasting increases in ethanol drinking behavior in the same strain of mice. Following multiple bouts of alcohol dependence, mice showed increased variability in their circadian body temperatures. Preliminary analyses show that mice exhibit lowered temperature values during the rhythm’s trough (or lower lows) compared to control mice exposed to similar experimental procedures. These results indicate that experimentally induced alcohol dependence can lead to lasting changes in circadian function. Supported by INIA-West (AA013523).
NEUROIMAGING OF ANIMAL MODELS OF ALCOHOLISM: SECOND REPORT FROM THE INTEGRATIVE NEUROSCIENCE INITIATIVE ON ALCOHOLISM
INTRODUCTION: E. V. Sullivan
PRESENTATION 1 - MRI ANALYSIS OF NEUROANATOMICAL CHANGES INDUCED BY LONG-TERM ETHANOL SELF-ADMINISTRATION IN MONKEYS.
K. A. Grant:
We have undertaken a longitudinal series of experiments to quantify neuroanatomical changes that accompany ethanol self-administration and withdrawal within separate groups of male and female cynomolgus monkeys. Following one year of ethanol self-administration, T1-weighted images were acquired (TR=2.5s, TE=4.4ms, flip angle=12°, 0.5mm-sided isotropic voxel resolution, 6 averages). Preliminary analyses indicate this strategy will enable direct comparisons between ethanol exposure and brain structural changes. Increased lateral ventricle anterior horn volume, and/or decreased hippocampal volume is observed following high levels of daily ethanol consumption (5.17g/kg average daily intake) relative to moderate levels of daily consumption (2.15g/kg average daily intake). Future efforts will be directed at quantifying volumetric differences as a function of sex (male v female), species (rhesus v cynomolgus macaques) and response to an HPA challenge (high v low ACTH to dexamethasone).PRESENTATION 2 -PROTON MAGNETIC RESONANCE SPECTROSCOPY OF INTRAVENOUSLY-ADMINISTERD ETHANOL IN THE CYNOMOLGUS MACAQUE BRAIN.
Christopher Kroenke:
Magnetic resonance (MR) spectroscopy measurements can be used to quantify brain ethanol levels in vivo. Using a chemical shift imaging technique under conditions of substantial T2-weighting (TE=150ms), we observe consistent regional patterns in the ethanol 1H methyl resonance intensity in non-human primate subjects following intravenous infusions. Signals in voxels within 1 cm of the midline are approximately 40% larger than voxels in immediately more lateral locations. This experimental strategy enables the systematic study of regional variability of the ethanol 1H methyl T2 value in primate brain, and will provide a means for investigating the relationship between the molecular environment of ethanol and its mechanism of pharmacological action.PRESENTATION 3 - VENTRICULAR EXPANSION AFTER ALCOHOL EXPOSURE BY VAPOR CHAMBER IN WISTAR RATS.
Adolf Pfefferbaum:
For an animal model to parallel human alcoholism, excessive alcohol exposure should produce widespread brain damage, manifest as tissue shrinkage and complementary ventriculomegaly. Blood alcohol levels (BAL) of ~125 mg/dl with voluntary consumption in P-rats produced only modest brain dysmorphology on structural MRI. To enhance BALs, MRI was conducted before and after 16 weeks of escalating vaporized alcohol exposure (average BALs=292mg/dl). MRI, quantified with atlas-based parcellation, revealed a profile of significant post-alcohol vapor exposure ventricular expansion in 9 contiguous slices, beginning .625 mm posterior to the AC (encompassing dorsolateral ventricles) and 7 contiguous slices, beginning 3.50 mm posterior to the AC (encompassing ventrolateral ventricles). This ventricular dilatation produced in wild-type rats with involuntary alcohol exposure successfully modeled ventricular expansion, a robust phenotype of human alcoholism. (Support: AA013521-INIA, AA005965, AA017168).PRESENTATION 4 - LONG-TERM ALCOHOL EXPOSURE BY VAPOR CHAMBER ALTERS MRS DETECTABLE METABOLITES IN RAT STRIATUM.
Natalie M. Zahr:
In vivo MRS can be used in longitudinal studies to track alterations in brain metabolites during the development of alcohol dependence in animal models. We exposed wild-type Wistar rats to vaporized alcohol using a rodent inhalation system. After 16 weeks of escalating EtOH exposure, average blood alcohol levels (BALs) were 292mg/dl (range: 124-475mg/dl). Four of 8 EtOH rats showed neurological signs. Eight more weeks of EtOH exposure resulted in average BALs of 445mg/dl; all 8 EtOH rats exhibited neurological signs. Major proton metabolite signals were quantified and referenced to water. The groups did not differ at baseline, but after 16 weeks of EtOH exposure, the EtOH group had higher total choline (Cho) levels (p=.0004) than Ctrls. After 24 weeks of greater EtOH exposure, the EtOH group had higher glutamate (Glu; p=.026), Glu+glutamine (Glx; p=.007) and Cho (p=.002) than the Ctrl group. This pattern of metabolic changes suggests onset of neurotoxicity in response to EtOH and comports with findings in actively drinking alcoholic humans where Cho levels are above normal in parietal gray matter. (Support: AA013521-INIA, AA005965, AA017168).DISCUSSANT: Antonio Noronha