INIA - RSA Abstracts 2004

RSA 2004 Abstracts

Oregon Health & Science University

CONSECUTIVE DAYS OF PASSIVE ETHANOL INFUSION RESULT IN HIGHER LEVELS OF ETHANOL SELF-INFUSION THAN DO ALTERNATE DAYS OF PASSIVE ETHANOL INFUSIONS IN RATS

T. L. Fidler, B. G. Oberlin, S. S. Watson & C. L. Cunningham

Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, 97239.

Using a model of ethanol self-infusion via intragastric (IG) catheter we have previously shown that rats that received 3-4 passive infusions of ethanol every day (Massed) via IG catheter subsequently self-infused more ethanol than rats with no prior ethanol experience. The present study examined the effect of inserting a 24 hour recovery period between each day of passive infusion (Spaced). We hypothesized that intensity of withdrawal might be enhanced by the Spaced infusion procedure, resulting in greater ethanol self-infusion. Sprague Dawley rats were implanted with IG catheters and allowed to recover. Following habituation to the apparatus, rats received passive infusions of 10% ethanol. Groups received the same number of passive infusions with the dose/infusion individually determined based on daily intoxication ratings. During the first 2 days of self-infusion, rats had access to a single flavor of Kool-Aid and licks were paired with ethanol infusions (No-Choice days). On remaining days, the rats also had access to a second Kool-Aid flavor (Choice days). Licks on the second flavor were paired with water infusions. Rats in the Massed Group self-infused a significantly higher ethanol dose than did rats in the Spaced Group on Choice days. Groups did not differ during No-Choice days. Contrary to our hypothesis, rats that had been exposed to the massed procedure during the passive infusion phase self-administered higher doses of ethanol than those rats that had experienced the Spaced Procedure. This finding suggests that self-administration may have been enhanced because the massed procedure was more effective for inducing tolerance to aversive ethanol effects that normally limit self-infusion. [Supported by INIA Consortium Grant AA13479]

GLUTAMATE WITHIN THE NUCLEUS ACCUMBENS IS DIFFERENTIALLY AFFECTED FOLLOWING ETHANOL ADMINISTRATION IN C57BL/6J VS DBA/2J MICE

C.K. Meshul; H.K. Homer; N. Yoneyama; A. Wetzel; C. Moore; D.A. Finn.

VA Medical Center and the Dept. of Behavioral Neuroscience, OHSU, Portland, OR 97239

The neurotransmitter glutamate may play a role in the rewarding properties of ethanol (ETOH), however, reports on changes in glutamate levels within the nucleus accumbens (NAC) following systemic administration of ETOH have been inconsistent. Since C57BL/6J (B6) and DBA/2J (D2) mice differentially sensitize to the activating effects of ETOH, the goal of this study was to determine the effects of 10 days of ETOH administration on glutamate levels in these two strains of mice. Alterations in glutamate within the NAC were quantified using both in vivo microdialysis and the density of nerve terminal glutamate immunolabeling using immuno-gold electron microscopy. We find that following 10 days of ETOH administration (2 g/kg/d, i.p.), there was a significant increase in the basal extracellular levels of glutamate within the NAC in the B6 mice but a decrease in D2 mice when microdialysis was conducted 1 day after the termination of ETOH injections compared to the control group. In D2 mice, 10 days of withdrawal from ETOH resulted in a continued decrease in the extracellular level of NAC glutamate. There was a trend towards an increase in the density of nerve terminal glutamate immunolabeling within the shell of the NAC in the B6 group administered ETOH for 10 days when compared to the control group. In D2 mice administered ETOH for 10 days, there was a trend toward a decrease in the density of glutamate immunolabeling. Following 2-bottle choice, 2 hour limited access to ETOH (10%) for 10 days or 10d on/10d off, we find that B6 mice will drink over 2 g/kg and that the extracellular level of glutamate within the NAC is increased in both ETOH drinking groups (higher in the 10d on/10d off vs. 10d on group) compared to the control group. Overall, these data suggest that B6 and D2 mice are differentially sensitive to the effects of ETOH on glutamate levels in the NAC. Supported by NIAAA INIA Consortium Grant AA13478, NIAAA grant AA10760 and the Dept. of Veterans Affairs.

MODIFIED FLUID RESTRICTION PARADIGM DETECTS GENETIC DIFFERENCES IN HIGH ETHANOL INTAKE

D.A. Finn; N. Yoneyama; A.R. Wetzel; J.C. Crabbe

VAMC Research, Portland Alcohol Research Center & Dept. of Behavioral Neuroscience, OHSU, Portland, OR 97239 USA.

Recent work in our laboratory found that scheduling periods of fluid availability produced high, stable ethanol (EtOH) intake (? 2 g/kg in 30 min) and ataxia in an inbred strain that exhibits preference (C57BL/6; B6) and in genetically heterogeneous mice. Thus, the purpose of the present experiments was to test genotypes, which differ in voluntary EtOH intake, on our modified fluid restriction paradigm. B6, DBA/2 (D2), B6D2F2, and mice selectively bred for high (STDRHI) and low (STDRLO) voluntary EtOH intake received a total of 7 EtOH sessions (5% solution, 30 min) every 3rd day. Mice initially had 4 hrs of fluid/day for the 1st 3 EtOH sessions, which increased by 2 hrs with each subsequent EtOH session until the amount of fluid/day was 10 hrs (last 2 EtOH sessions). EtOH intake in B6 mice was significantly greater than in D2 mice, with EtOH intake intermediate in the F2 cross of the 2 progenitor strains. Notably, EtOH intake in the D2 mice significantly decreased once total fluid/day was increased to 8-10 hrs. The short-term selected lines differed in EtOH intake during the 1st 2 EtOH sessions (STDRHI > STDRLO), but this initial difference disappeared with subsequent EtOH sessions. Blood EtOH concentration following the final EtOH session was significantly positively correlated with the EtOH dose consumed (r = 0.75, n = 36, p < 0.0001). Currently, we are using this model to examine EtOH intake in the genetically heterogeneous mice that will be the founding population for a selective breeding project (HS/Npt) to produce an animal model of high EtOH intake. The pattern of distribution of EtOH intake in genetically heterogeneous and B6D2F2 mice suggest that we will have no difficulty picking breeders for high EtOH intake. Supported by NIAAA INIA Consortium Grants AA13478 and AA13519 and the Dept. of Veterans Affairs.

NEUROSTEROID SPECIFICITY IN THE MODULATION OF ETHANOL INTAKE PATTERNS IN MALE C57BL/6J MICE

M.M. Ford; N. Yoneyama; T.J. Phillips; D.A. Finn

Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR 97239.

Previous work demonstrated that the neurosteroids allopregnanolone (ALLOP) and epipregnanolone (EPIPREG) transiently increased and persistently decreased limited-access ethanol intake, respectively, in C57BL/6J (B6) mice. ALLOP is a positive allosteric modulator at GABA-A receptors whereas EPIPREG is a partial agonist/antagonist, interactions that likely underlie the ability of these neurosteroids to alter ethanol-related behaviors. The purpose of this study was to assess the impact of additional GABAergic neurosteroids on consumption patterns in order to reach a better understanding of GABA-A receptor involvement in the regulation of ethanol intake. Systemic administration of the agonist pregnanolone (PREG) and the antagonist pregnenolone sulfate (PS) were evaluated. Male B6 mice (n=24) were acclimated to a reverse light/dark schedule and permitted 2-hr access to lickometer chambers containing one 10% ethanol (10E) bottle and one water bottle at the beginning of the dark phase. Drinking patterns were monitored with lickometer circuits attached to each fluid sipper. A saccharin fading procedure was used to establish stable baseline 10E intake. All mice were habituated to vehicle injections (20% w/v b-cyclodextrin) for 4-5 days, and then received 3-day injection regimens of either vehicle or neurosteroid (10 mg/kg PREG or 50 mg/kg PS) immediately prior to the drinking session. Total session intakes (g/kg/2hrs) were slightly reduced by PS (-16%), but were unaltered by PREG versus respective vehicle controls. However, PREG elicited a robust increase (+109%) in 10E lick responses during the initial 20 min of access whereas PS elicited only a moderate elevation in this measure (+33%) this measure when compared to vehicle controls. These initial lick responses were associated with 44% and 13% reductions in the latency to first 10E bout in PREG- and PS-treated mice, respectively. Consistent with earlier findings, neurosteroids with various GABA-A receptor activity profiles differentially modulate ethanol intake patterns. Supported by: The Department of Veterans Affairs and grants AA13478, AA12439, AA10760, and DA07262.

VOLUNTARY ETHANOL INTAKE IN 22 INBRED MOUSE STRAINS

N. Yoneyama, J.K. Belknap, J.C. Crabbe, A. Wetzel, D.A. Finn

VAMC Research & Dept. of Behavioral Neuroscience, OHSU, Portland, OR 97239

Several inbred strains are known to drink an ethanol (EtOH) solution while others do not. There are several advantages to using inbred strains. An inbred strain is genetically stable through time and laboratories, which allows scientists to create a record of phenotypic behaviors. Genetic correlations of ethanol consumption can also be estimated by comparing multiple inbred strains. Therefore, the purpose of this experiment was to examine genetic differences in voluntary consumption of solution containing EtOH, EtOH plus 0.2% saccharin or 0.2% saccharin in a total of 22 inbred strains of mice. Male and female mice were individually housed and had unlimited access in a two-bottle preference choice paradigm to one bottle containing tap water or one containing increasing concentrations of EtOH (3%, 6%, 10%), 0.2% saccharin, and then increasing concentrations of EtOH (3%, 6%, 10%) plus 0.2% saccharin. Mice were given access to each different solution for a total of 4 days, with a side change every other day. Consistent with previous studies, C57BL/6J(B6) mice consumed an EtOH dose of > 10g/kg/day while DBA/2J (D2) mice consumed <2g/kg/day. Among the other strains tested, no strain voluntarily consumed greater doses of EtOH than the B6 mice. However, strains C58/J and C57BLKS/J showed levels of EtOH consumption close to that of the B6 mice. Strains that consumed relatively low levels of EtOH similar to D2 mice were the BUB/BnJ and BTBR+Tff/tf mice. Interestingly, the addition of 0.2% saccharin to the varying EtOH solutions increased EtOH intake for all strains and altered the strain distribution pattern. A strong positive correlation (r ? 0.83) was shown between the EtOH concentrations, but the correlation with saccharin consumption was considerably lower (rs= 0.35-0.42). In the future we will be able to conduct genetic correlations to explore the genetic relationships between voluntary EtOH consumption and other EtOH phenotypes. Supported by NIAAA INIA Consortium Grants AA13478 and AA13519 and the Dept. of Veterans Affairs.

UP-REGULATION OF DOPAMINE D1 RECEPTORS IN THE CENTRAL NUCLEUS OF THE AMYGDALA (CEA) REDUCES ALCOHOL CONSUMPTION IN C57BL/6J MICE

Weiran Wu, Laurea Diaz, Yunging Han, Richard Morrisett and Robert J. Hitzemann

Department of Behavioral Neuroscience, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201 and Institute for Neuroscience and Waggoner Center, University of Texas, Austin, TX 78712-0125.

Previously we have demonstrated that bilateral 6-OHDA lesions of the CeA in B6 mice had no effect on alcohol consumption and preference. In order to further investigate the role of CeA dopaminergic systems in regulating alcohol consumption, we have utilized a Sindbis virus vector containing both the dopamine D1 receptor (Drd1) and the green fluorescent protein (GFP) genes. B6 mice were allowed to establish a stable drinking pattern (6-8 days) using a 3 bottle choice paradigm (water, 10% ethanol, 20% ethanol). The mice were then bilaterally injected with the Drd1 vector into the CeA, focusing the injection to the lateral division (CeL). Control animals were injected with vector that did not contain the Drd1 gene. Ethanol preference and consumption was then measured for an additional 6-8 days. The administration of the vector containing the Drd1 gene, decreased ethanol consumption and preference 60 to 70%; the sham vector had no effect on these parameters. Injection sites were verified by the presence of GFP. Microscopic examination revealed no necrosis at the sites of injection. Overall, these data illustrate for the first time that Drd1 over-expression can produce significant reductions in both ethanol consumption and preference. Supported in part by AA 13484 (RH), AA 13497 (RM), AA 14307 (LD) and the Waggoner Center.

ANALYSIS OF GENE EXPRESSION DATA: RELEVANCE OF THE METHOD USED TO THE DETECTION OF ETHANOL-RELATED QUANTITATIVE TRAIT GENES (QTGS)

R. Hitzemann, S. McWeeney, B. Malmanger, M. Lawler, C. Reed and J.Belknap.

Departments of Behavioral Neuroscience and Public Health and Preventive Medicine, Oregon Health & Science University, Portland, OR 97239-3098 and Research Service, Veterans Affairs Medical Center, Portland, OR 97201.

There are now at least 15 quantitative trait loci (QTLs) repeatedly detected for ethanol related phenotypes at LOD scores of > 3 and several with LOD scores of > 10. We have argued that the one approach to the conversion of these QTLs to QTGs will involve the integration of functional genomics (gene array expression analysis) into the research strategy (Belknap et al. 2001; Hitzemann et al. 2002,2003). This integration is highly dependent on the sensitivity of the Alow level A analysis to detect differences in brain gene expression among the inbred strains used to form the intercrosses for the detection of the QTLs. The question addressed in this study is how (when using Affymetrix U74Av2 and MOE 430 arrays) the analysis strategy used affects the detection of differential gene expression within well defined QTL intervals. We compared 5 analysis methods (RMA, GC-RMA, MBEI, MAS5, PDNN) to detect differences for the U74 array and the first four methods to detect differences for the 430A array. For the U74 array, the order of sensitivity is PDNN>RMA>MAS5=MBEI=GC-RMA. For some QTL intervals, PDNN (Position Dependent Nearest Neighbor [Zhang et al. 2003]) detected up to 500% more differentially expressed genes when compared to the default analysis, MAS5. Regardless of the method used, the 430A array detected ~ 100% more differentially expressed genes (on a percentage basis) than the U74 array. The most sensitive method was RMA (Robust Microarray Average [Irizarry et al. 2003]) although MAS5 would be an adequate substitute. A strategy will be outlined that uses the data from all methods to form the domain of candidate QTGs and will include a strategy to interrogate this domain to find the best candidates for further analysis. Supported in part by AA 11034, 13484, AA 06243 and grants from the Department of Veterans Affairs.

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