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RSA 2004 Abstracts

University of Texas at Austin

MICROARRAY ANALYSIS OF CHRONIC ALCOHOL RESPONSE IN BRAIN

A.E. Berman, S. E. Frost, J.A. Owen, R.D. Mayfield and S.E. Bergeson

Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology The University of Texas at Austin, Austin, TX 78712

Chronic alcohol intoxication and withdrawal affects gene expression in brain. To gain a better understanding of the molecular mechanisms of intoxication and withdrawal, a global microarray analysis was undertaken to identify significant changes in gene expression in response to chronic alcohol treatment. DBA/2J male mice were chronically exposed to alcohol inhalation or air alone for 72 hrs. Thermal control of the chamber was optimized to 29 oC to reduce alcohol-related hypothermia. A loading dose of 1.5 g/kg was administered i.p. The mice were then exposed to alcohol vapors using a controlled sigmoidal increase in chamber alcohol concentration monitored remotely by gas chromatography, resulting in an average retro orbital BEC of 1.5 mg/ml. Following optimization of thermal control and BEC, three consecutive chamber runs were completed. Mice exposed to alcohol vapor were sacrificed at 0, 6 and 24 hrs post alcohol ventilation, while control animals (72 hr in chamber with air only) were sacrificed at 0 hr (n=8/group). RNA was extracted from cerebellum, cortex, hippocampus, midbrain, olfactory bulb, and striatum and quantified by NanoDrop. Quality was verified by Agilent BioAnalysis. Custom printed cDNA microarrays containing 17,665 mouse brain gene targets were used. Experimental brain samples (n=5/group) were hybridized against a standard reference RNA utilizing Genisphere Array 350 Cyanine 3 and Cyanine 5 labeled oligodendrimers. Block-wise normalization was performed using software developed in our laboratory while cluster analysis was completed using the Longhorn Array Database. The statistical package SAM (Statistical Analysis for Microarrays) was utilized for initial analysis. False Discovery Rate, Bayesian and Partial Least Squares analyses were completed. Initial results for cerebellum and midbrain show striking changes in gene expression that significantly vary between control, intoxication, withdrawal and recovery states. Functional group analysis also shows that, as expected, multiple classes of genes are affected by chronic alcohol exposure. Supported by NIAAA grants T32AA07471, AA13182, AA13403, AA13475, and AA13518 (INIA Core, R. Harris).

NULL MUTANT MICE LACKING CHEMOKINES OR CHEMOKINE RECEPTORS SHOW DECREASED ETHANOL CONSUMPTION.

Y.A. Blednov; D. Walker; H. Alva; V. Ferreira, S. Bergeson, W. Kuziel; R.A. Harris.

University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712

Chemokines constitute a superfamily of small proteins (8-14 kDa) that induce chemotaxis and functional modulation of a wide variety of leucocytes during inflammation (Bajetto et al., 2002). Alcohol is a frequently abused drug that inhibits numerous immune functions of the host (Szabo, 1999). Gene micro-array studies in brains of human alcoholics (Lewohl et al., 2000; Mayfield et al., 2002) and cultured cells exposed to ethanol (Thibault et al., 2000) showed significant changes in expression of genes related to immune or inflammatory responses, including chemokines (MCP-1, MIP-1 alpha) and chemokine receptors (CCR5, CCR1 motif). To determine if chemokines may regulate ethanol consumption, we studied mutant mice with deletion of the CCR2, CCR5, MCP-1 or MIP-1 alpha genes. Deletion of CCR2, MCP-1 or MIP-1 alpha in mice resulted in lower preference for alcohol and consumption of lower amounts of alcohol in a two-bottle choice test (as compared with wild-type littermates). CCR5 (-/-) knockout mice showed slightly higher consumption of ethanol solutions, because they consumed more fluid. Ethanol treatment (2.5 g/kg, i.p.) induced stronger conditioned taste aversion in CCR2, MCP-1 or MIP-1 alpha null mutant mice than in controls. There were no differences in ethanol-induced loss of righting reflex (LORR) between null mutant (CCR2 and CCR5) and wild type mice. In contrast, mice lacking MCP-1 or MIP-1 alpha showed longer LORR than wild type mice. CCR2 and CCR5 null mutant mice were more sensitive to analgesic effect of morphine in hot-plate test. These data show that disruption of the chemokine network interferes with ethanol-induced behaviors, including reduction of ethanol consumption and an increase the aversive properties of alcohol. It is possible that some of these changes are due to changes in opioid systems. Supported by the NIAAA INIA Consortium program and NIH grants AA06399 and AA13520.

STRUCTURE AND TEMPORAL PATTERN OF VOLUNTARY ETHANOL INTAKE IN C57Bl/6 MICE.

M. Brown; Y.A. Blednov; R.A. Harris.

University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712

Despite the wide use of daily intake as a measure of ethanol self-administration by laboratory animals, it is clear that an understanding of the mechanisms for regulation of ethanol intake require a more detailed examination of patterns of ethanol consumption. Recently, a volumetric drinking monitor (Columbus Instruments) was introduced and we used it to monitor water and ethanol intake in C57Bl/6 female mice given access to water and a sequence of increasing concentrations (5%, 10% and 15%) of ethanol. Consistent with earlier results from rats using operant techniques (Samson et al., 1991; Boyle et al., 1997), mice drink ethanol and water in discrete bouts. There were quantitative, but not qualitative, differences between day and night ethanol consumption. Mice consumed significantly higher amount of ethanol and water in higher number of bouts during the dark phase. However, the size of bouts and preference for ethanol were similar for light and dark phases. One week after presentation of the highest concentration of ethanol (15%), mice were given two repeated 4 days accesses to the 10% of ethanol divided by a one week period of deprivation. This repeated presentation of 10% ethanol revealed highly variable responses. There was a strong correlation between preference for alcohol during reinstatement and preferences for alcohol (5%, 10% or 15%) as well as number of ethanol bouts (frequency of drinking) during initial periods of consumption. Thus, several parameters of initial intake of 15% ethanol (amount of ethanol ? g/kg; size of ethanol bout ? g/kg/bout; preference and number of ethanol bouts) may serve as predictors for amount of ethanol (g/kg) consumed during reinstatement. These results suggest that initial patterns of alcohol consumption may predict later development of alcohol consumption and aversion. Supported by the NIAAA INIA Consortium program and NIH grants AA06399 and AA13520.

CHRONIC ETHANOL CONSUMPTION INCREASES ACTIVATION OF CHOLINERGIC NEURONS IN THE SHELL NUCLEUS ACCUMBENS IN ETHANOL-PREFERRING (P) RATS

M.C. Camp, R.D. Mayfield, A.A. Alcantara

The University of Texas at Austin, Austin, Texas 78712

In a previous study we investigated the acute effects of ethanol on cellular activation, as measured by Fos-immunoreactivity, in brain areas that have been implicated in the reinforcing and anxiolytic effects of substance abuse and dependence. Specifically, we reported an increase in neuronal activation of cholinergic cells in the shell nucleus accumbens. Building on the identification of cellular substrates involved in initial ethanol exposure the present study focused on cellular activation following chronic self-administration. We examined the effects of ethanol self-administration on the activation of cholinergic interneurons of the nucleus accumbens in ethanol-preferring (P) rats. Neuronal activation was measured by Cdk5 labeling in cholinergic cells using dual-labeling immunocytochemical procedures. Ethanol was administered using a two-bottle choice paradigm in which experimental animals (n=10) were offered 10% (v/v) ethanol and water with unlimited access for 23 h/day for 28 days. On average 6 g/kg of ethanol per day was consumed. Control animals (n=12) received the same experimental manipulations except that both bottles contained water. The number of Cdk5-immunoreactive cholinergic neurons in the shell ?limbic? compartment of the nucleus accumbens was significantly increased in ethanol treated animals compared to controls (~51%). Chronic ethanol consumption did not produce a significant difference in cholinergic cells of the core ?motor? region of the nucleus accumbens. The present study indicates that cholinergic cells of the shell nucleus accumbens, but not of the core, are activated following chronic ethanol consumption. By identifying brain regions and cellular microcircuits involved in chronic ethanol consumption, we can begin to better understand the underlying substrates that mediate substance abuse, and therefore the potential circuits that should be targeted by improved pharmaceutical and behavioral programs designed to treat alcohol abuse and alcoholism. Supported by the National Institute on Alcohol Abuse and Alcoholism and the Integrative Neuroscience Initiative on Alcoholism (NIAAA/INIA) (AA13497-02), the Waggoner Center for Alcohol and Addiction Research, and The Morton Meyerson Family Foundation.

REAL-TIME TWO-PHOTON IMAGING OF ETHANOL FACILITATION OF AGONIST-INDUCED CLUSTERING OF DOPAMINE D1 RECEPTORS IN VIABLE MEDIUM SPINY ACCUMBAL NEURONS.

LM Diaz, Y Han, RA Morrisett.

The College of Pharmacy and The Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas 78712

The mesolimbic pathway is an important structure which mediates drug reinforcement, and an important component of this pathway is the D1 receptor (D1DR). D1DRs desensitize by several mechanisms including a process involving clustering and internalization. This synaptic alteration, by removing post-synaptic D1DRs, may constitute a molecular mechanism underlying ethanol neuroadaptation and/or acute tolerance. In order to measure D1DRs in real-time in viable NAc neurons, we constructed a mammalian viral expression system. This construct utilized a sindbis RNA virus expressing the D1DR containing an eGFP tag. This sindbis virus expressed D1DRs in several medium spiny neuronal preparations including ventral striatal primary cultures, organotypic cultures that included the NAc and VTA and in the NAc in vivo. Functionality was verified by measuring cAMP production which was dose dependently increased in response to DA and was inhibited by the D1DR antagonist SCH23390. We used a custom built two-photon microscope (Olympus Fluoview BX50WI) to repeatedly image the localization of eGFP-D1DRs in cultured medium spiny neurons. Prolonged z-series projection constructs in control neurons demonstrated stable and homogenous fluorescence throughout the neuronal soma and processes. Prolonged treatment of neurons with DA (100 _M) induced receptor clustering as seen by coalescence of fluorescence puncta defined by an increase in maximum intensity of 20% or more for a region of interest. Agonist induced clustering appeared to be significantly facilitated with ethanol pretreatment. Neurons treated with ethanol (100 mM) for 30 minutes prior to application of DA (50 _M) for an additional 30 minutes displayed clustering of receptors while cells not pretreated with ethanol with the same exposure to DA (50 _M) showed little to no clustering. The findings indicate that DA induces alterations in D1DR localization which occur rapidly and are potentiated by ethanol. Therefore, this synaptic restructuring of D1DR receptors may contribute to ethanol neuroadaptation.

(Supported by F31AA14307 to LMD and U01AA13497, TCADA and the Waggoner Center to RAM).

DIFFERENCE IN ETHANOL PREFERENCE, BUT NOT ETHANOL-STIMULATED DOPAMINE RELEASE, IN TWO SUBSTRAINS OF MALE C57BL/6 MICE.

M.O. Job; V.S. Ramachandra; R.A. Gonzales

The University of Texas at Austin, Division of Pharmacology, College of Pharmacy, Austin, TX 78712.

C57BL/6 mice are a commonly used model for ethanol drinking studies because this strain is well known to prefer ethanol in a two bottle choice paradigm. However, several substrains are now available, and previous indications in the literature suggest that ethanol preference may vary between specific substrains. Accumbal dopamine release has been suggested to play a role in ethanol reinforcement, but this response has not been examined in specific C57BL/6 substrains. The purpose of this study was to directly compare two substrains (C57BL/6J and C57BL/6N) with respect to ethanol preference and ethanol-stimulated dopamine release from the ventral striatum. C57BL/6J mice were originally obtained from Jackson Laboratories and a colony was maintained at the University of Texas. C57BL/6N mice were obtained from Charles River. Ethanol preference was assessed using a two bottle choice procedure in which singly housed male mice had access to either water or ethanol solutions (5% or 10%, v/v). The volume of liquid consumed was measured every 2-3 days. The C57BL/6J mice had significantly higher ethanol preference (n=4) for both 5% and 10% ethanol compared with the C57BL/6N mice (n=6). A separate group of mice were prepared for microdialysis from the ventral striatum, and the dopamine response to 2.0 g/kg ethanol (i.p.) was determined. Ethanol concentrations in the dialysates were also measured following the ethanol injection. The ethanol injection significantly increased dialysate dopamine concentrations in both substrains (n=4-5), but there was no difference between the two substrains in the magnitude or time course of the dopamine response. The C57BL/6N mice had significantly higher ethanol concentrations following the injection compared with the C57BL/6J mice. Overall the results suggest that the genetic drift between the two substrains has differentially affected the ethanol preference phenotype rather than a neurochemical phenotype that is thought to be associated with ethanol reinforcement. The difference in ethanol appearance in the accumbal dialysates suggests that ethanol absorption and distribution may be affected by the genetic drift between substrains (AA13486, AA11852).

DISTINCT PATTERNS OF GENE EXPRESSION IN HUMAN FRONTAL CORTEX DISCRIMINATE ALCOHOLIC FROM NONALCOHOLIC INDIVIDUALS.

Jianwen Liua , Joanne M. Lewohlc, R. Adron Harrisa , Peter R. Doddc, Patrick K. Randallb, Vishwanath Iyer, and R. Dayne Mayfielda

aWaggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, TX 78712; bDepartment of Psychology, University of Texas at Austin, Austin, TX 78712; cDepartment of Biochemistry, University of Queensland, St Lucia 4072, Australia.

Neuroadaptations that underlie the effects of chronic alcohol abuse are likely due to altered gene expression. Previous gene expression studies using human post-mortem brain demonstrated that genes belonging to functionally-related families of genes were altered by alcohol abuse. However, these changes in gene expression were small and it was not clear if gene expression profiles had sufficient power to discriminate control from alcoholic individuals. Gene expression from the superior frontal cortex of individual control and alcoholic cases was examined using cDNA microarrays consisting of ~47,000 elements. Partial least squares (PLS) statistical procedures as well as automated biological theme-based databases were used in the gene classification process. Following PLS analysis, significantly changed genes were identified which accurately distinguished between control and alcoholic samples. In addition, approximately 160 genes were identified which were present in controls but absent in alcoholics suggesting that genes are more likely to be "turned off" than "turned on" in response to long-term alcohol abuse. The genes identified in this study belong to diverse biological themes based on molecular and biological processes such as protein kinase activity, regulation of transcription, and growth regulation. These results demonstrate that combining high-density cDNA arrays with a PLS statistical analysis allows use of small and diverse changes in gene expression to distinguish accurately alcoholic from non-alcoholic individuals. Supported by NIH AA12404 and AA13518.

MICROARRAY ANALYSIS OF ACUTE ALCOHOL RESPONSE

K.H. Lodowski, A.E. Berman, J.A. Owen, S.E. Frost, R.D. Mayfield and S.E. Bergeson

Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology, The University of Texas at Austin, Austin, TX 78712

In order to study genetically determined and sex specific alcohol-induced gene expression in brain, the well characterized inversely responsive C57BL/6J and DBA/2J inbred strains of mice were treated with 4 g/kg i.p. ethanol. Following cervical dislocation at 6 hours post injection, brains were quickly removed and immediately frozen in liquid nitrogen. RNA was extracted from whole brain and quality was verified by the Agilent BioAnalyzer and quantified by Nanodrop. Custom printed cDNA microarrays containing 17,665 mouse brain gene targets were used. Direct comparisons were completed in a 2x2 design for strain and treatment for each sex. Comparisons for sex differences were completed across arrays. A total of thirty hybridizations (n=5/group) utilizing Genisphere Array 350 Cyanine 3 and Cyanine 5 labeled oligodendrimers were performed. Block-wise normalization was performed using software developed in our laboratory while cluster analysis was completed using LAD (Longhorn Array Database). The statistical package SAM (Statistical Analysis for Microarrays) was utilized for initial analysis. False Discovery Rate, Bayesian and Partial Least Squares analyses were completed. Our results indicate that approximately 1000 genetic differences in gene expression occur between the two strains, which validate differences previously found in other studies. In addition, important alcohol-specific and alcohol-related sex differences were identified. A better understanding of gene expression related to the genetic components of alcoholism as well as alcohol-induced and sexually dimorphic neuroadaptation should increase the likelihood of developing better treatments for this disease of considerable importance. Supported by NIAAA grants T32AA07471, AA13182, AA13403, AA13475, and AA13518 (INIA Core, R. Harris).

MU OPIATE RECEPTOR MODULATION OF ETHANOL-STIMULATED DOPAMINE RELEASE IN THE VENTRAL STRIATUM: GENDER AND STRAIN EFFECTS IN MICE

V.S. Ramachandra, M.Job, F.S. Hall, G.R. Uhl, R.A. Gonzales and A. Tang

The University of Texas at Austin, Division of Pharmacology, College of Pharmacy, Austin, TX 78712

Ethanol stimulates dopamine activity in the ventral tegmental area ? nucleus accumbens pathway, and this is thought to play a role in ethanol reinforcement. Previously we showed that genetic deletion of the mu opiate receptor (MOR) in a mixed background mouse strain (C57BL/6 x 129SvEv) abolished ethanol-stimulated accumbal dopamine release in females, but not males. This study investigated the effect of pharmacological antagonism of MORs with a mu1 selective antagonist, naloxonazine, to determine the potential involvement of developmental compensation in the previous work. We also investigated the ethanol response in males and females of the congenic strain of MOR knockout mice (C57BL/6). Microdialysis was used to determine the dopamine response to 2.0 g/kg ethanol (i.p.) in the ventral striatum. Naloxonazine (15 mg/kg, i.p., 20 hr prior to the dialysis experiment) pretreatment decreased the ethanol-stimulated accumbal dopamine response compared with their respective controls, although females showed a larger effect in the mixed background mice. This result confirms that the previous sex difference in ethanol response we observed in the MOR knockouts on the mixed background was not due to developmental compensation. Ethanol-induced accumbal dopamine responsiveness was also reduced in the congenic MOR knockout colony (C57BL/6), but this reduction was similar in both males and females. The lack of sex difference in the congenic MOR knockout model (C57BL/6) suggests that the parental strain, 129SvEv, is the major contributor to the sex difference observed in the naloxonazine experiment in the mixed background mice (C57BL/6 x 129SvEv). Additionally, no differences in the time course of dialysate ethanol concentrations were found in any of the experiments. We conclude that the MOR is a critical factor in the mechanism by which ethanol stimulates accumbal dopamine activity, and this may contribute to the reinforcing effects of ethanol. (AA13486, AA11852)

VISUALIZING ALTERATIONS IN DARPP-32 PHOSPHORYLATION USING IMMUNOBLOT ANALYSIS AND CONFOCAL MICROSCOPY

SS Schreiber, G Reyna, RA Morrisett, RE Maldve

The College of Pharmacy and the Waggoner Center for Alcohol and Addiction Research, University of Texas-Austin, Austin TX, 78712

DARPP-32 becomes an inhibitor of PP1 when phosphorylated at Thr34 by PKA, and D1 receptor activation is a primary pathway of PKA activation. Phosphorylation of Thr75 by Cdk5 inhibits PKA, and conversely PKA activation inhibits Thr75 phosphorylation. We have previously demonstrated that DARPP-32 is important in modulating the ethanol sensitivity of the NMDA receptor. However the direct effects of ethanol on DARPP-32 have not been demonstrated. We initiated our studies by evaluating the effects of several doses of ethanol on Thr34 and Thr75 DARPP-32 phosphorylation in slices of the NAc by western blot analysis. Thr34 DARPP-32 expression was increased significantly at 1 min (23%?6% ,p>0.05, n=3-4) following 25 mM EtOH exposure. However, by 5 and 10 min, the response had returned to baseline levels. Conversely, Thr75 DARPP-32 expression was reduced at 1 min, but increased steadily over 5 and 10 min and was elevated as compared to Thr34 expression. At 50 and 100 mM doses of ethanol, phosphorylation of Thr34 was reduced as compared to thr75 at all time points examined. We conclude that elevated ethanol concentrations may alter dopamine/PKA/DARPP-32 signaling in the NAc. Furthermore, we have developed a method for use of phospho-DARPP-32 antibodies with confocal microscopy and used this method to analyze changes induced by D1 receptor activation as well as ethanol treatment. Using confocal microscopy, coronal slices of the NAc were prepared from 25-30 day old Sprague Dawley rats and treated with 10?M SKF38393 for 0-5 min (alone or after a 20 min pretreatment with 10?M SCH23390) or with 25mM EtOH for 0-20 min. Slices were fixed for immunofluorescence and incubated with pThr34-DARPP-32 or pThr75-DARPP-32 antibody. Treatment with SKF38393 increased pThr34 fluorescence by 32% within 5 min. This increase was inhibited 24% by pretreatment with SCH23390. Treatment with SKF38393 inhibited pThr75 fluorescence by 25% within 1 min, and treatment with SCH23390 released this inhibition such that fluorescence levels reached 24% over control. Treatment with 25mM EtOH increased pThr34 fluorescence by 45% by 5 min, with no observable changes in pThr75 fluorescence. These complementary methods demonstrate that EtOH induces complex changes in the phosphorylation of DARPP-32. Supported by NIAAA grant AA13CR to REM, U01AA13497 to RAM, and UT Undergraduate Fellowship to SSS.

MORE SEVERE ETHANOL WITHDRAWAL AND REDUCED ETHANOL CONSUMPTION IN MICE LACKING a-SYNUCLEIN.

D. Walker; H. Alva; Y.A. Blednov; R.A. Harris.

University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712

The presynaptic protein a-synuclein is involved in a range of neurodegenerative diseases including Parkinson disease and serves as a regulator of DA synthesis. Earlier in our lab we found that mutant mice with deletion of the a-synuclein gene showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates (Miranda et al., 2003). These data are consistent with data from rats and humans suggesting a role for a-synuclein in excessive alcohol consumption (Liang et al., 2003; Mayfield et al. 2002).To determine the possible cause for reduction of ethanol intake we studied some other types of ethanol-related behavior in mutant mice lacking a-synuclein gene. There were no differences between null mutant and wild type mice in ethanol-induced (4.0 g/kg) loss of righting reflex, motor stimulation by ethanol (1.0, 1.5, 2.0 and 2.5 g/kg) and conditioned taste aversion to alcohol (1.5 and 2.5 g/kg). However, null mutant mice showed significantly more severe acute ethanol withdrawal (4.0 g/kg) than wild type mice. Importantly, that difference between knockout and wild type mice in severity of acute ethanol withdrawal as well as in voluntarily ethanol intake have been found only in females but not in males. These data show that a-synuclein regulates ethanol consumption in mice via modification of severity of alcohol withdrawal consistent with other genetic data linking alcohol consumption and withdrawal severity (Metten et al., 1999). Supported by the NIAAA INIA Consortium program and NIH grants AA06399 and AA13520

RT-PCR ANALYSIS OF COCAINE AND AMPHETAMINE-RELATED TRANSCRIPT (CART) FOLLOWING IN VIVO ETHANOL ADMINISTRATION

JD Wilde, Y Han, RA Morrisett, RE Maldve, The College of Pharmacy and the Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX, 78735.

A putative peptide neurotransmitter, cocaine and amphetamine regulated transcript (CART), has been implicated in drug reward and reinforcement mechanisms. Several studies have demonstrated CART mRNA expression in the NAc increases significantly following in vivo administration of cocaine or amphetamine. CART mRNA and peptide forms are present in the shell and core of the NAc and colocalized with GABA in a specific subset of medium spiny neurons. Bi-directional interactions between CART and DA have also been demonstrated as icv injections of a CART peptide increased the DA metabolite, DOPAC, within the NAc. We asked whether CART expression may be responsive to ethanol administration in vivo. We examined the effects of in vivo ethanol injections (ip) on CART mRNA expression in the NAc. Habituated rats received ip injections of either saline or ethanol and were sacrificed 1 hr later. Total mRNA was isolated from the ventral striatum tissue and quantified by RT-PCR using CART specific primers. Compared to control habituated rats, ethanol treatment (1.0 g/kg or 3.5 g/kg) dose-dependently increased CART mRNA expression in the NAc (by 13"12%, n=7 and 40"16%, n=6 (p? 0.05), respectively). While a relationship between CART and DA has been established, the mechanisms underlying their interaction have yet to be defined. CART expression may be negatively regulated by D3 receptors; indeed, D3 receptors appear to contribute to ethanol reward. Accordingly, we explored the relationship between D3 activity and CART expression in the NAc in the presence of ethanol to determine whether the ethanol-induced enhancement of CART expression might be additive with or occluded by D3 receptor blockade. Habituated rats received ip injections of saline, ethanol (3.5 g/kg), or ethanol in the presence of raclopride, a D2/D3 antagonist. Interestingly, the co-administration of ethanol and raclopride produced a 69"9% (n=4, (p? 0.005) decrease in CART expression as compared to CART induced by ethanol treatment. These data indicate that an intoxicating dose of ethanol significantly increases CART expression which may be due to a novel dependence on D3 receptor activation. These findings are the first demonstration of CART expression enhanced by ethanol and suggest that this peptide might be generally responsive to a variety of reinforcing drugs. (Supported by NIAAA grant AA13CR to REM and U01AA13497 to RAM and the Waggoner Center for Addiction Research)

UP-REGULATION OF DOPAMINE D1 RECEPTORS IN THE CENTRAL NUCLEUS OF THE AMYGDALA (CEA) REDUCES ALCOHOL CONSUMPTION IN C57BL/6J (B6) 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, 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.

MULTIPLE SITES OF ETHANOL MODULATION OF NMDA RECEPTOR-MEDIATED SYNAPTIC TRANSMISSION IN THE NUCLEUS ACCUMBENS

TA Zhang, AW Hendricson, and RA Morrisett

College of Pharmacy and Waggoner Center for Alcohol & Addiction Research; The University of Texas at Austin, Austin, TX, 78712

The nucleus accumbens (NAc) integrates excitatory glutamatergic inputs from prefrontal cortex and dopaminergic inputs from the ventral tegmental area and plays an important role in drug reinforcement. Previously we have shown that dopamine receptor-1 (D1) activation strongly decreases ethanol inhibition of NMDA receptor function in the NAc through the D1/cAMP/PKA/DARPP-32/PP-1 signaling pathway (Maldve et al., 2002). To address such interactions in greater detail, we describe here analysis of electrically?evoked NMDA mEPSCs in medium spiny neurons from acute NAc slices from 12-20 day rats. We used Sr++ (4 mM) to supplant Ca++ and support densely populated asynchronous release in this preparation in order to maximize event yield for frequency/amplitude analysis. NMDA mEPSCs were inhibited by (DL)-APV and typically ranged from 5 to 40 pA (mean amplitude: 10?0.1 pA; mean inter-event interval: 16?0.2 msec; >7000 events/12 neurons). Ethanol (25-75 mM) exhibited dose-dependent inhibitory effects on amplitude, frequency and decay kinetics of asynchronous NMDA mEPSCs. Ethanol significantly suppressed NMDA mEPSC amplitude at 50 and 75 mM (p<0.05, n=5; p<0.01, n=12; Student?s t test). Significant inhibition of frequency of the NMDA mEPSCs was observed at 75 mM ethanol (p<0.01, Student?s t test, n=12). We also measured paired-pulse ratio of facilitation which was increased from 1.5?0.1 to 1.9?0.1 by 75 mM ethanol (p<0.05, n=5). Finally, the decay time constant of NMDA mEPSCs was shortened about 35% by 75 mM ethanol (p<0.05, n=7). We conclude that ethanol exerts multiple inhibitory effects at excitatory synapses on medium spiny neurons of the NAc. NMDA mEPSC amplitude displayed the greatest sensitivity to ethanol whereby the release process and termination of the synaptic event were inhibited at greater ethanol concentrations. Taken together, these data indicate that ethanol modulates transmission at the synapse via at least three distinct mechanisms. (Supported by R01AA09230 to RAM, a Jones Fellowship to TAZ and F32 AA14068 to AWH).

 

 

 

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