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RSA 2006 Abstracts
University of Texas at Austin
REDUCED ETHANOL CONSUMPTION AND PALATABILITY IN MICE LACKING PREPRODYNORPHIN: D. Walker; Y. A. Blednov; R. A. Harris. University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712, USA.Many studies suggest a role for endogenous opioid peptides and their receptors in regulating ethanol intake. Thus, µ- and κ-opioid receptor knockout mice do not self-administer alcohol (Roberts at al., 2000; Kovacs et al., 2005). Lack of preproenkephalin also leads to reduction of ethanol consumption (Blednov et al., 2004), whereas ethanol oral self-administration is increased in mutant mice with decreased beta-endorphin expression (Grisel et al., 1999). Our goal was to study ethanol-related behaviors in mutant mice lacking preprodynorphin (Sharifi et al., 2001). Null mutant female mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of short 4-hours access (light phase of the light/dark cycle) null mutant mice did not show any differences in response to saccharin but they showed significantly reduced response to sucrose. To determine the possible cause for this reduction of ethanol intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild type mice in ethanol-induced (3.4 g/kg and 3.8 g/kg) loss of righting reflex, acute ethanol (4 g/kg) withdrawal, ethanol-induced (2 g/kg) conditioned place preference or conditioned taste aversion to ethanol (2.5 g/kg). These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake probably caused by decreased alcohol palatability. Supported by the NIAAA INIA Consortium program and NIH grants AA13520 and AA06399.
INCREASED ETHANOL CONSUMPTION AND PALATABILITY IN MICE LACKING NEURONAL NITRIC OXIDE SYNTHASE (nNOS): M. Martinez; Y. A. Blednov; R. A. Harris. University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712, USA.
Evidence from previous studies has implicated the neuronal nitric oxide synthase (nNOS)-NO pathway CNS-mediated drug effects. Thus, inhibition of NOS has been shown to influence the development of tolerance (Khanna et al., 1993, 1995; Kolesnikov et al., 1993, 1997) and sensitization (Itzhak et al., 1998; Itzhak and Martin, 2000) to several drugs of abuse, including alcohol. Our goal was to study ethanol-related behavior in mutant mice lacking nNOS (Huang et al., 1993). Null mutant mice of both sexes showed significantly higher preference for alcohol and consumed larger amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. There were no differences between null mutant and wild type mice in preference for saccharin or quinine solutions. However, knockout mice consumed larger amounts of saccharin and water than wild type mice. In contrast, under conditions of short 4-hours access (light phase of the light/dark cycle) null mutant mice showed a stronger response to saccharin as well as for sucrose compared with wild type mice of both sexes. There were no differences between null mutant and wild type mice in ethanol-induced (3.4 g/kg and 3.8 g/kg) loss of righting reflex, acute ethanol (4 g/kg) withdrawal or conditioned taste aversion to ethanol (2.5 g/kg). These results indicate that deletion of nNOS substantially increases alcohol intake, probably by increasing alcohol palatability. Sup
INCREASED ETHANOL CONSUMPTION AND PALATABILITY IN MICE LACKING GLUTAMIC ACID DECARBOXYLASE (GAD65): Y. A. Blednov; D. Walker, M. Ramirez, R. A. Harris. University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712, USA.
γ-Aminobutyric acid (GABA) is synthesized by two isoforms of glutamic acid decarboxylase, GAD65 and GAD67(Erlander et al., 1991). Morphological (Martin et al., 1991) and genetic (Asada et al., 1996;Kash et al., 1999) studies suggest that the two isoforms have distinct roles. GAD67 is thought to be responsible for the maintenance of basal GABA levels (Erlander et al., 1991). On the other hand, GAD65 seems to be rapidly activated in times of high GABA demand (Erlander et al., 1991). Our goal was to study ethanol-related behavior in mutant mice lacking GAD65 (Asada et al., 1996). Three different colonies of mutant mice maintained on C57Bl/6, 129N1 (1 generation of backcross on 129/SvJ) and 129N2 (2 generations of backcross on 129/SvJ) have been tested. Only null mutant male mice maintained on 129N1 and 129N2 backgrounds showed significantly higher preference for alcohol and consumed larger amounts of alcohol in a two-bottle choice test as compared with correspondent wild-type littermates. There were no differences between mutant and wild type mice of all 3 different genetic backgrounds in preference for sweet and bitter compounds. To determine the possible cause for this increase of ethanol intake, we studied other ethanol-related behaviors in mice lacking GAD65 gene maintained only on 129N2 genetic background which showed the strongest ethanol drinking phenotype. Under conditions of short 4-hours access (light phase of the light/dark cycle), null mutant mice did not show any differences in response to saccharin but they showed significantly increased response to sucrose. There were no differences between null mutant and wild type mice in ethanol-induced (3.4 g/kg and 3.8 g/kg) loss of righting reflex, or conditioned taste aversion to ethanol (2.5 g/kg). However, null mutant male mice showed significantly reduced severity of ethanol-induced (4 g/kg) acute withdrawal. These results indicate that deletion of GAD65 leads to substantial increase of alcohol intake probably caused by increase of alcohol palatability as well as reduction of severity of ethanol withdrawal. Supported by the NIAAA INIA Consortium program and NIH grants AA13520 and AA06399.
WHY DO MICE DRINK OR NOT DRINK ALCOHOL? LESSONS FROM NULL MUTANTS: Y. A. Blednov; R. A. Harris. University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712.
Screening of ethanol-behavioral phenotypes in mutant mice was carried out in 36 different mouse mutants using 6 behavioral tests: voluntary ethanol consumption in 2-bottle choice paradigm; motor response in novel situation; ethanol-induced loss of righting reflex; ethanol-induced acute withdrawal; limited access to alcohol and conditioned taste aversion (Blednov et al., 2001 a,b; 2002; 2003 a,b,c; 2004 a,b; 2005; Boehm et al., 2003, 2004 a,b,c; Findlay et al., 2002; Hill et al., 2003). From 36 mutants significant changes in alcohol drinking phenotypes were shown for males of 20 lines and for females from 19 mutant lines; among male mice, 14/20 colonies showed a reduction of alcohol consumption and 6/20 showed increased alcohol drinking; among female mice, 17/19 colonies showed reduction of alcohol consumption and only 2/19 showed increased alcohol drinking. Thus, most mutations decrease alcohol intake and the role of withdrawal severity and stress responses in these changes in drinking will be presented. Using this database we carried out correlational analysis among different behavioral phenotypes in wild type and mutant mice. Preliminary results showed that preference for alcohol in the voluntary drinking paradigm in 36 different colonies of wild type mice (mostly heterogeneous mixed genetic backgrounds) is correlated with two general mechanisms – positive (preference for sweet taste and stress response) and negative (severity of acute ethanol withdrawal and conditioned taste aversion). The correlations were dependent on the concentrations of ethanol test in the two bottle test. Thus, for modest concentrations of ethanol (6% and 9%) preference for alcohol was negatively correlated with withdrawal severity and with conditioned taste aversion. On the contrary, there was a significant positive correlation between preference for alcohol and motor response to novelty (stress response) and preference for sweet solution of saccharin. In addition to the negative mechanisms described above, preference for a high concentration of ethanol (12%) was negatively correlated with duration of ethanol-induced loss of righting reflex and preference for bitter solution of quinine (only for male mice). Stress response was also positively correlated with preference for sweet taste creating a tightly linked cycle of 3 positively correlated behaviors. In turn, positive correlation between conditioned taste aversion and ethanol withdrawal suggests that sensitivity to negative hedonic states associated with ethanol lead to low drinking. Similar correlation analysis has been carried out in 36 colonies of mutant mice. Positive control of preference for sweet taste still exists in mutant mice but this correlation is substantially reduced compare with wild type mice. However, no other correlations were found in mutant mice. We hypothesize that mutations reduce or eliminate the fine regulation of alcohol consumption observed in normal mice. In wild type mice, the regulation of voluntary ethanol intake by multiple positive and negative pathways serves to prevent excessive drinking. Mutations destroy these fine interconnections providing at least a theoretical possibility of mutant mice with excessive drinking. Supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (AA U01-13520 - INIA Project; AA06399).ONE RECEPTOR – DIFFERENT OUTCOMES: COMPARING GENE EXPRESSION PROFILES OF GABAA RECEPTOR α1 AND β2 SUBUNIT MUTANTS:
I. Ponomarev; S. Park; Y. A. Blednov; R. A. Harris; R. D. Mayfield. Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712.GABAA receptors play an important role in ethanol actions. The α1, β2 and γ2 subunits constitute the major type of these receptors in the brain. Despite losing about 50% of GABAA receptors, both α1 and β2 subunit null mutants are viable and fertile but demonstrate distinct physiological and behavioral alterations, indicating a unique role of each subunit in GABAA receptor function. In particular, α1 mutants have slightly reduced baseline activity and a marked ethanol-induced locomotor activation, while β2-deficient mice show an increased baseline locomotion and no stimulatory effects of ethanol. To investigate molecular mechanisms underlying these differential responses we used cDNA microarrays to compare transcriptional changes in the cerebellum of α1 and β2 null mutants. The deletion of either α1 or β2 receptor subunits resulted in the expression of different pools of genes, suggesting different mechanisms of cellular adaptation to altered inhibition in each mutant. Genes involved in GABA and glutamate transmission, such as the GABA transporter (GAT1), GABA vesicular transporter (VGAT) and glutamate receptors (Gria3, Grm7, Grm8) were among differentially regulated transcripts. Our results indicate that the deletion of different subunits of the same receptor activated different transcriptional mechanisms that may underlie different ethanol-related phenotypes of α1 and β2 subunit-deficient mice. Supported by grants from National Institute of Alcohol Abuse and Alcoholism, NIH (AA UO1 13520, AA UO1 13518; INIA Projects).
META-ANALYSIS OF BRAIN: I. Ponomarev.
Selectively bred lines and inbred strains are two principal genetic approaches that have been used to study mechanisms of excessive alcohol consumption in mice. In attempt to identify a subset of genes that consistently differentiate high-drinking from low-drinking animals we analyzed brain transcription profiles from four genetic models: three pairs of selected lines and a panel of 6 isogenic strains known to differ markedly in voluntary alcohol consumption. For each transcript, a magnitude of the difference between high-drinking and low-drinking genotypes was estimated by calculating a measure of effect size (Cohen’s d value). The effect size values generated for the four genetic models of alcohol preference were than averaged and a z-test was used to test significance of deviation of the mean effect size from zero. This meta-analysis identified 3,800 unique genes significantly and consistently changed between all high and low alcohol consuming models.
ONE RECEPTOR – DIFFERENT OUTCOMES: COMPARING GENE EXPRESSION PROFILES OF GABAA RECEPTOR α1 AND β2 SUBUNIT MUTANTS:
I. Ponomarev; S. Park; Y. A. Blednov; R. A. Harris; R. D. Mayfield. Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712.GABAA receptors play an important role in ethanol actions. The α1, β2 and γ2 subunits constitute the major type of these receptors in the brain. Despite losing about 50% of GABAA receptors, both α1 and β2 subunit null mutants are viable and fertile but demonstrate distinct physiological and behavioral alterations, indicating a unique role of each subunit in GABAA receptor function. In particular, α1 mutants have slightly reduced baseline activity and a marked ethanol-induced locomotor activation, while β2-deficient mice show an increased baseline locomotion and no stimulatory effects of ethanol. To investigate molecular mechanisms underlying these differential responses we used cDNA microarrays to compare transcriptional changes in the cerebellum of α1 and β2 null mutants. The deletion of either α1 or β2 receptor subunits resulted in the expression of different pools of genes, suggesting different mechanisms of cellular adaptation to altered inhibition in each mutant. Genes involved in GABA and glutamate transmission, such as the GABA transporter (GAT1), GABA vesicular transporter (VGAT) and glutamate receptors (Gria3, Grm7, Grm8) were among differentially regulated transcripts. Our results indicate that the deletion of different subunits of the same receptor activated different transcriptional mechanisms that may underlie different ethanol-related phenotypes of α1 and β2 subunit-deficient mice. Supported by grants from National Institute of Alcohol Abuse and Alcoholism, NIH (AA UO1 13520, AA UO1 13518; INIA Projects).
A META-ANALYTIC APPROACH TO STUDY TRANSCRIPTIONAL REGULATION OF ALCOHOL DRINKING: I. Ponomarev; M. K. Mulligan. Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, TX 78712.
Selectively bred lines and inbred strains are two principal genetic approaches that have been used to study mechanisms of excessive alcohol consumption in mice. In attempt to identify a subset of genes that consistently differentiate high-drinking from low-drinking animals we analyzed brain transcription profiles from four genetic models: three pairs of selected lines and a panel of 6 isogenic strains known to differ markedly in voluntary alcohol consumption. Initial comparison of the four models revealed striking similarity in the direction of transcriptional differences between alcohol preferring and non-preferring mice. For each transcript, a magnitude of the difference between high-drinking and low-drinking genotypes was estimated by calculating a measure of effect size (Cohen’s d value). The effect size values generated for the four genetic models of alcohol preference were than averaged and a z-test was used to test significance of deviation of the mean effect size from zero. This meta-analysis identified 3,800 unique genes significantly and consistently changed between all high and low alcohol consuming models. Importantly, this approach allowed identification of genes with small and moderate effect that have previously been undetected by single studies. Functional group and transcription factor analyses highlighted numerous cellular pathways thus illustrating the complexity of mechanisms involved in regulation of voluntary alcohol consumption. Many of the genes and functional groups revealed by the current study were not anticipated by previous work, indicating the opportunity to obtain new knowledge through large scale genomic and meta-analytic approaches. Supported by NIAAA/INIA.
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