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RSA 2003 Abstracts
University of Texas at Austin
A BIPHASIC RESPONSE TO ETHANOL OF DARPP-32 PHOSPHO-ISOFORMS IN THE NUCLEUS ACCUMBENS
SS Schreiber, RA Morrisett, RE MaldveCollege of Pharmacy, University of Texas-Austin, Austin TX, 78712
DARPP-32, a dopamine and cAMP-regulated phosphoprotein becomes a potent inhibitor of protein phosphatase-1 when phosphorylated at Thr34 by PKA. Additionally, DARPP-32 has three regulatory phosphorylation sites each activated by different protein kinases. Phosphorylation of Thr75-pDARPP-32 by Cdk5 inhibits PKA phosphorylation at Thr34 and conversely PKA activation inhibits phosphorylation at Thr75. In addition, phosphorylation of Ser137 by casein kinase 1 or Ser102 by casein kinase 2 enhances Thr34-pDARPP-32 by blocking its de-phosphorylation by PP-2B. We previously demonstrated DARPP-32 involvement in the regulation of the ethanol sensitivity of NMDA receptors (Maldve et al., 2002); however a comprehensive analysis of ethanol effects on specific DARPP-32 phospho-isoforms in the NAc has not been performed. Therefore, in this study, we examined the effects of ethanol on DARPP-32 phosphorylation and the possible mechanisms involved.Using immunoblot analysis, acute coronal slices were exposed to 25 mM ethanol for 0-20 min and evaluated for Thr34-, Thr75- and Ser137-pDARPP-32 expression. In response to ethanol, Thr34 and Thr75 were activated in a biphasic manner where a significant increase in Thr75 (~1.75-fold) was observed from 1-5 min, decreasing by ~40% at 10-20 min; whereas Thr34 expression was minimal at 1 min and increased 1.2-fold from 5-20 min. We did not detect any response to ethanol at Ser137 in the NAc; however in a companion presentation by Maldve et al. (RSA 2003 abstract), we demonstrate in the amygdala a response to ethanol for Thr75- and Ser137-pDARPP-32, but not for Thr34-pDARPP-32. These data suggest that isoforms of phospho-DARPP-32 are differentially regulated by ethanol in the NAc vs the amygdala.
Further experiments focused on mechanisms by which ethanol affects Thr34- and Thr75-pDARPP-32 in the NAc. A 20 min pretreatment with 1 mM H-89, a PKA antagonist, inhibited ethanol-induced Thr34-pDARPP-32 expression but tended to potentiate the Thr75-pDARPP-32 response. We therefore conclude that DARPP-32 phosphorylation in the NAc is differentially regulated by ethanol. Additionally, antagonism of PKA differentially modulates the response at the two phosphorylation sites suggesting alternative mechanisms for the ethanol effects on DARPP-32 activation. Supported by NIAAA grant AA13CR as part of the INIA to REM and UT Undergraduate Research Fellowship to SSS.
BLOCKADE OF THE LEPTIN-SENSITIVE PATHWAYS DECREASES ALCOHOL CONSUMPTION IN MICE
Y.A. Blednov; D. Walker; H. Alva; R.A. Harris
University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
The neuropeptide leptin links adipose stores with hypothalamic centers and serves as an endocrine signal involved in the regulation of appetite (Campfield et al., 1995) and possibly in the endorphinergic modulation of the drug reward system (Inui, 1999). Increased plasma leptin has been observed at the onset of alcohol withdrawal in humans and ethanol consumption after withdrawal was increased after injection of leptin in mice (Kiefer et al., 2001). We addressed the role of leptin in alcohol-related behaviors by studying ethanol consumption in two strains of spontaneously mutant mice which lack leptin (ob/ob) or the leptin receptor (db/db). Males and females of both mutant strains showed significantly lower preference for alcohol in two-bottle choice paradigm as compared with wild-type. Male ob/ob mice demonstrated slightly higher avoidance of bitter quinine solutions than wild type but mutant mice did not show altered preference for saccharin solutions in comparison with water. Injection of leptin (1 mg/kg i.p., daily for 8 days) failed to increase the preference for ethanol in ob/ob mice; i.e., we could not correct the effects of leptin deficiency by injection of leptin. These data show that blockade of the leptin pathway markedly decreases the preference for alcohol intake but this decrease may be the result of compensatory or developmental changes in other systems rather than a more direct effect of leptin on alcohol consumption. Supported by INAAA INIA Consortium and NIH grants AA06399 and AA13520.
CHOLINERGIC INTERNEURONS IN THE SHELL COMPARTMENT OF THE NUCLEUS ACCUMBENS ARE ACTIVATED BY ETHANOL
B.E. Herring, R.D. Mayfield, M.L. Berlanga, M.C. Camp, R.A. Morrisett, A.A. AlcantaraThe University of Texas at Austin, Austin, Texas 78712
The present study focused on the effects of ethanol on cholinergic interneurons of the shell compartment of the nucleus accumbens in Sprague-Dawley rats. The nucleus accumbens is the primary target of the mesolimbic dopamine pathway, which plays a critical role in reward and reinforcement. The shell compartment of the nucleus accumbens in particular receives extensive afferent projections from the ventral tegmental area and is involved primarily in the rewarding and hedonic effects of alcohol and other drugs of abuse. This study sought to determine the effects of varying doses of an acute, intraperitoneal injection of ethanol (0.5, 1, and 2 g/kg) on the activation of cholinergic interneurons located in the shell region of the nucleus accumbens. Neuronal activation was measured by Fos labeling in cholinergic cells using dual labeling immunocytochemical procedures. The administration of 1 g/kg (but not 0.5 or 2 g/kg) produced an increase in the percent of Fos immunoreactive cholinergic neurons (p <0.05, n=29) 2 hours following injections. Preliminary findings in a separate study conducted on ethanol-preferring (P) and -nonpreferring (NP) rats (n=8) yielded similar findings. In both P and NP rats the administration of 1 g/kg of ethanol, produced increases in the number of Fos labeled cholinergic interneurons in the shell compartment of the nucleus accumbens. Taken together, these findings demonstrate that accumbal cholinergic interneurons are involved in the initial stage of ethanol exposure. These findings establish the groundwork for future studies that will investigate the specific role that cholinergic interneurons play in the establishment and maintenance of alcohol abuse and alcoholism. This work was supported by NIAAA grant AA13497-02 and the Waggoner Center for Alcohol and Addiction Research.
DELETION OF METABOTROPIC GLUTAMATE RECEPTORS (mGluR4 and mGluR5) DID NOT CHANGE ETHANOL CONSUMPTION BUT ABOLISHED THE MOTOR STIMULATION BY ETHANOL
K. Creech; D. Walker; H. Alva; R.A. Harris; Y.A. Blednov
University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712
Metabotropic glutamate receptors play an important role in the generation of locomotion as well as in the behavioral effects of psychostimulants (Vezina, Kim, 1999; Swanson, Kalivas, 2000). Mice lacking mGluR5 gene show the absence of the reinforcing and motor stimulant effects of cocaine (Chiamulera et al., 2001). The activation of mGluR4 receptor modulates the strength of primary sensory transduction through the umami or other taste pathways (Dingledine, Conn, 2000). We addressed the role of two metabotropic glutamate receptors in alcohol related behavior by studying the ethanol consumption and ethanol-induced locomotion in mutant mice with deletion of mGluR5 and mGluR4 genes. There were no significant differences between wild type and knockout strains in ethanol consumption or preference in two-bottle and four-bottle choice experimental paradigms. Total fluid consumption was significantly higher in mGluR5 as well as in mGluR4 null mutant mice than in wild type mice in both tests. Low doses of ethanol (1.5 g/kg for mGluR4 and 2.0 and 2.5 g/kg for mGluR5) stimulated motor activity of wild type mice in both colonies. There was no effect of ethanol in mGluR4 knockout mice (1.0 – 2.5 g/kg). In contrast, mGluR5 null mutant mice were more sensitive to sedative effects of ethanol. These data show that mGluR5 and mGluR4 are important for mediation of motor stimulant but not motivational effects of ethanol. Supported by the NIAAA INIA Consortium program and NIH grants AA06399 and AA13520.
DELETION OF α-SYNUCLEIN DECREASES ETHANOL CONSUMPTION IN MICE
C. Miranda; 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 -synuclein is involved in a range of neurodegenerative diseases including Parkinson disease and serves as a regulator of DA synthesis. We found that -synuclein gene expression is increased in the frontal cortex of human alcoholics. Gene expression is also increased in alcohol preferring iP rats (Liang et al., this meeting). To determine if -synuclein regulates ethanol consumption, we studied mutant mice with deletion of the -synuclein gene. Null mutant females showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. Total fluid intake was similar in mutant and wild type mice. There was no significant difference in consumption of sweet or bitter solutions. In a four-bottle choice paradigm with simultaneous presentation of water and three different concentrations of alcohol (5%, 10% and 15%), null mutant females showed higher avoidance of 10% and 15% solutions of alcohol. Total amount of consumed alcohol and total preference for alcohol solutions were also lower than in wild type control mice. These data show that -synuclein promotes ethanol consumption in mice and is remarkably consistent with data from rats and humans suggesting a role for -synuclein in excessive alcohol consumption. Supported by the NIAAA INIA Consortium program and NIH grants AA06399 and AA13520.
ETHANOL INDUCES REGIONAL AND ISOFORM SPECIFIC PHOSPHORYLATION OF DARPP-32 IN THE AMYGDALA
RE Maldve, PA Guerrero and RA Morrisett
College of Pharmacy, University of Texas, Austin, TX 78712
Alterations in the mesolimbic dopaminergic neurons arising from the VTA may trigger amygdala processing of anxiety and angst associated with ethanol abstinence. Many of the actions of dopamine are mediated by D-1 receptors and activation of an intracellular mediator, DARPP-32. This dopamine and cAMP-regulated phosphoprotein (32 kD) is phosphorylated by PKA at Thr34 and functions to inhibit activation of protein phosphatase-1. In addition to Thr34, DARPP-32 is phosphorylated at three alternate sites by different protein kinases. DARPP-32 has been well characterized in the neostriatum and NAc, and we previously described its role in the ethanol sensitivity of the NMDA receptor in the NAc (Maldve et al, 2002). However, there is no information on DARPP-32, its phospho-isoforms or direct ethanol effects in the amygdala. Therefore, in this study, we identify and characterize DARPP-32 phosphorylation and acute ethanol effects in the amygdala.
Using confocal microscopy, we observed an increase in the relative fluorescence of Thr34-pDARPP-32 in neurons of the central and basolateral amygdala from acute coronal slices treated with 1 mM forskolin for 1 and 10 min. We also detected Thr75-pDARPP-32 fluorescence using the same treatment paradigm. Additional experiments used immunoblot analysis to measure changes in specific DARPP-32 phospho-isoforms in acute coronal slices treated with the D1 receptor agonist SKF 38393. In slices exposed to 1 mM SKF 38393 for 0 - 20 min, Thr34-pDARPP-32 increased by ~ 3-fold at 10 min and 20 min. Thr75-pDARPP-32 increased similarly following SKF treatment; however D1 receptor activation did not affect Ser137-pDARPP-32 expression. We have evidence that suggests that ethanol effects on DARPP-32 phospho-isoforms are brain region specific. In a companion presentation by Schreiber et al. (RSA 2003 abstract), we demonstrate in the NAc that ethanol increases Thr34- and Thr75-pDARPP-32 in a biphasic manner but has no effect on Ser137-pDARPP-32. However, in the amygdala there was no definitive ethanol effect on Thr34, yet Thr75-pDARPP-32 expression increased 3- to 4-fold within 1-20 min and Ser137-pDARPP-32 increased 1.5 and 2-fold by 10 and 20 min, respectively. There was no change in total DARPP-32 expression in all experiments performed. We surmise that distinct phospho-isoforms of DARPP-32 are expressed in the amygdala and that these phosphorylation sites may be differentially regulated by ethanol. Supported by UO1-AA13CR and TCADA to REM.
GENETIC AND “ALCOHOL DEPRIVATION-SPECIFIC” REGIONAL TRANSCRIPTOME ANALYSIS IN INBRED ALCOHOL-PREFERRING RATS
M.K. Mulligan1, J.A. Owen1, M.B. Warner1, R.L. Bell2, W.N. Strother2, W.J. McBride2 and S.E. Bergeson1
1Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology, University of Texas, Austin, TX 78712
2Depts. of Psych. and Med. Indiana Univ. School of Medicine, Indianapolis, IN 46202
Relapse is a prevalent problem in human alcoholics. Using a rat model of alcohol relapse, an mRNA differential display analysis has been initiated to test the “Two-Hit” hypothesis that both genetic and environmental factors contribute to excess alcohol intake. After chronic free-choice drinking followed by a prolonged period of alcohol deprivation, iP rats temporarily consume a greater amount of alcohol when they are next given access. This increase in ethanol intake is called the alcohol deprivation effect (ADE). Regional brain expression differences in alcohol naïve iP and inbred alcohol-non-preferring (iNP), and in iP rats that were on continuous alcohol access or were repeatedly deprived, were screened. The transcriptome from specific brain regions from these four groups was isolated and analyzed by mRNA Differential Display PCR. Total RNA was extracted from 8 brain regions including the amygdala, caudate putamen, hippocampus, hypothalamus, anterior and posterior cortex, septum and nucleus accumbens. iP/iNP genetic expression differences, iP long-term continuous alcohol drinking changes, and iP alcohol deprivation-specific expression were compared across all 8 brain regions. Preliminary results indicate that brain gene expression differences exist between iP and iNP animals, and between naive, continuous alcohol exposed, or repeated alcohol-deprived iP rats. A better understanding of the brain regions involved and the genes that contribute to either the genetic propensity toward, and/or underlying neuroadaptation should increase the likelihood of developing successful treatments for alcoholism. Supported by NIAAA INIA program grants AA13475, AA13521, AA13522 and NIAAA training grant AA07471-13.
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