Rueben A. Gonzales - US grants

Ethanol is thought to exert many of its behavioral and biochemical effects through a physical interaction with biomembranes. A primary function of plasma membranes in many cell types is the transduction of biochemical signals from outside the cell into intracellular messengers. This function underlies the phenomenon of synaptic transmission between neurons and hormonal activation of peripheral cells such as in the liver. The brain and liver are two organs which are clearly affected by the ingestion of ethanol acutely, and both organs are known to be damaged after chronic ethanol ingestion. Although membranes seem to be a primary locus for ethanol action, information is lacking about ethanol's effects on membrane signal transduction mechanisms. In particular, receptor-stimulated phosphoinositide (PI) hydrolysis has been implicated to be an important biochemical signal transduction system in both brain and liver. Neurotransmitters or hormones bind to specific cell surface receptors which results in the hydrolysis of phosphoinositides by a specific phospholipase C. Thus, inositol trisphospate and diacylglycerol are released and may act as second messengers inside the cell to release intracellular calcium and activate protein kinase C, respectively. This proposal will investigate the possibility that guanine nucleotide binding proteins are involved in the coupling of the receptor to the response in brain, and look at the proposed calcium releasing properties of inositol trisphosphate in brain membranes. Further experiments will then determine the effects of ethanol on the PI hydrolysis system in brain and liver. Preliminary experiments have established that ethanol in vitro and chronically in vivo can alter receptor-stimulated PI responses in brain and liver. Therefore, experiments are proposed to further characterize the mechanism of ethanol's effects on PI responses and test whether ethanol-induced alterations in receptor- stimulated PI responses lead to further changes in cell function (calcium mobilization in brain and liver, phosphorylase activation in liver). These experiments may lead to further knowledge of receptor functions in general, and may provide additional information concerning the pharmacology and/or toxicology of alcohol.

Ethanol is thought to exert many of its behavioral and biochemical effects through a physical interaction with biomembranes. A primary function of plasma membranes in many cell types is the transduction of biochemical signals from outside the cell into intracellular messengers. This function underlies the phenomenon of synaptic transmission between neurons and hormonal activation of peripheral cells such as in the liver. The brain and liver are two organs which are clearly affected by the ingestion of ethanol acutely, and both organs are known to be damaged after chronic ethanol ingestion. Although membranes seem to be a primary locus for ethanol action, information is lacking about ethanol's effects on membrane signal transduction mechanisms. In particular, receptor-stimulated phosphoinositide (PI) hydrolysis has been implicated to be an important biochemical signal transduction system in both brain and liver. Neurotransmitters or hormones bind to specific cell surface receptors which results in the hydrolysis of phosphoinositides by a specific phospholipase C. Thus, inositol trisphospate and diacylglycerol are released and may act as second messengers inside the cell to release intracellular calcium and activate protein kinase C, respectively. This proposal will investigate the possibility that guanine nucleotide binding proteins are involved in the coupling of the receptor to the response in brain, and look at the proposed calcium releasing properties of inositol trisphosphate in brain membranes. Further experiments will then determine the effects of ethanol on the PI hydrolysis system in brain and liver. Preliminary experiments have established that ethanol in vitro and chronically in vivo can alter receptor-stimulated PI responses in brain and liver. Therefore, experiments are proposed to further characterize the mechanism of ethanol's effects on PI responses and test whether ethanol-induced alterations in receptor- stimulated PI responses lead to further changes in cell function (calcium mobilization in brain and liver, phosphorylase activation in liver). These experiments may lead to further knowledge of receptor functions in general, and may provide additional information concerning the pharmacology and/or toxicology of alcohol.

Although alcohol is the most widely used and abused drug in our society today, the mechanisms which underlie its central pharmacological (intoxication, anesthesia) and toxicological (brain damage, memory deficits) effects are largely unknown. An area of emerging importance in understanding the neurochemical mechanisms which mediate or are consequent to ethanol use and abuse is that of excitatory amino acid (EAA) function. In particular, a subtype of EAA receptor, the N-methyl-D asparate (NMDA) receptor, is implicated in learning, memory, and neurodegenerative processes. Our preliminary data show that ethanol, at concentrations which are consistent with its pharmacological range achieved in vivo (10-60 mM), inhibits NMDA receptor function. The broad, long term objectives of this project are to investigate and gain further information about the interaction between EAA neurotransmission and ethanol in an in vivo system. The specific aims of this proposal are to determine (l) whether endogenous glycine agonists interact with the ability of acute ethanol to inhibit NMDA receptor function in vivo, (2) if acute ethanol administration alters extracellular excitatory amino acid concentrations in vivo, (3) if the mechanism of chronic ethanol-induced alteration of extracellular dopamine concentration in rat nucleus accumbens involves excitatory amino acid neurotransmission, and (4) whether ethanol interference with excitatory amino acid neurotransmission in the hippocampus contributes to the development of rapid tolerance to ethanol. The research design involves both in vivo and in vitro experiments. In vivo experiments will use intracerebral microdialysis to measure the effects of systemic ethanol on NMDA receptor function by monitoring the extracellular levels of dopamine and norepinephrine after perfusion of NMDA through the microdialysis probe. Both hippocampus and striatum will be investigated because in vitro data suggest that these areas are sensitive to ethanol. In addition, glycine agonist (glycine and D-serine) levels will be monitored in dialysates because these endogenous compounds may interfere with ethanol's effects on NMDA receptors. In vitro experiments will also investigate the regulation of release of these compounds by depolarization and ethanol. Further experiments will investigate the role that glutamate and aspartate may play in ethanol's mechanism of action by measuring extracellular levels of these BAA's after acute and chronic ethanol administration. Finally, behavioral measurements of rapid tolerance to ethanol's ataxic effects will be made while monitoring EAA levels in striatum and hippocampus during the behavioral test. The knowledge gained from these studies will contribute to our understanding of the role that BAA's play in the mechanisms of acute and chronic ethanol administration in vivo. This information may lead to new therapeutic approaches for the treatment of alcohol related problems.

The candidate for this RSDA application, Dr. Rueben Gonzales, obtained his Ph.D. in Pharmacology from the Univ. of Texas in 1983. After performing post-doctoral work for 3 years, Dr. Gonzales began to establish an independent research career as an academic scientist with a position at LSU Medical Center (2 yr) and currently is an Assistant Professor at the Univ. of Texas. The candidate has been productive in the alcohol field as evidenced by 36 publications and by obtaining 4 competitive extramural research grants. Dr. Gonzales' immediate goals are to firmly establish himself as an independent researcher in the alcohol field. Long term goals include continuing to contribute to knowledge of the basic biochemical mechanisms of ethanol's effects on brain function as an academic scientist, along with training pre- and post-doctoral students for careers in alcohol related research and helping with administrative duties of the University as a faculty member. The College of Pharmacy will support the research career development of the candidate by providing laboratory space and equipment and by relieving the candidate of a large teaching load during the period of the RSDA. The Dept. of Pharmacology is an excellent atmosphere for fostering the scientific growth and development of the candidate because of the possibility of collaboration with several notable senior faculty with strong research programs and with other neuroscientists through the Institute for Neuroscience. The RSDA will significantly enhance the candidate's research career development by allowing more time for research. The research project involves the investigation of the acute and chronic effects of ethanol on N-methyl-D-aspartate (NMDA) mediated responses. The NMDA receptor, a subtype of excitatory amino acid receptor, is implicated in learning, memory, and neurodegenerative processes. Preliminary data suggest that ethanol, at concentrations which are consistent with its pharmacological range achieved in vivo (10-60 mM), inhibits NMDA receptor function. The major aim of this proposal is to define the site and mechanism of ethanol's inhibitory effect on NMDA receptor function at the molecular level. Functional assays (NMDA-stimulated transmitter release and modulation of phosphoinositide hydrolysis in rat brain slices, and in vivo microdialysis), along with radioligand binding techniques, will establish whether ethanol is acting at one or more of the proposed sites which modulate NMDA receptor function (glycine, polyamine, zinc). The knowledge gained from these studies will contribute to our understanding of the molecular mechanisms of an important receptor system in the brain at a basic level. In addition, information will also be gained about the possible role of this system in ethanol-induced neuronal adaptation or damage and may lead to new therapeutic approaches for the treatment of alcohol related problems.

DESCRIPTION (provided by applicant): This is a proposal for the continuation of a training grant to support seven predoctoral and two postdoctoral fellows who will be broadly and intensively trained to conduct research on alcoholism. Training areas span the breadth of state-of-the-art approaches including molecular biology and genetics, electrophysiology, cellular imaging, neurochemistry, and behavior. Research models include both animal and human. The training program will promote and support collaborative research. Molecular biology training will encompass studies of the function of ligand-gated ion channels in cellular expression systems (Mihic, Harris), development of new transgenic animal models (Atkinson, Harris, Mihic), and identification of ethanol responsive genes (Iyer, Bergeson, Harris, Mihic, Atkinson). Genetics training will involve genotyping of human and animal subjects (Gonzales, Atkinson, Fromme, Harris, Bergeson, Iyer). Electrophysiological and microscopic imaging training will comprise experiments with whole cell and intracellular methods in single cells and in brain slices (Morikawa, Morrisett, Mihic, Harris). Training in neurochemistry will focus on intracellular and extracellular signaling mechanisms with an emphasis on phosphorylation (Morrisett), release and transport of glutamate, dopamine, and other neurotransmitters (Gonzales, Harris, Mayfield), and expression of synaptic proteins (Mayfield, Morikawa, Morrisett). Research training in the behavioral effects of ethanol (motor skills, reinforcement, anxiety, and withdrawal) will be done (Duvauchelle, Harris, Gonzales, Bergeson, Schallert). Training in psychosocial aspects of alcohol research (Fromme) and the interaction of genotype and alcohol drinking in humans (Fromme, Bergeson, Harris, Iyer) will also be done. Postdoctoral fellows will choose a project at the onset of their training. Predoctoral graduate training will rotate through the laboratories of selected faculty members prior to choosing an advisor for dissertation research. Predoctoral students will be required to complete a series of core course requirements covering ethanol's actions on the central nervous system, scientific ethics, experimental design, and statistical analysis. We will continue to focus on minority recruitment. The training program has an excellent record and will continue to focus on producing well qualified alcohol researchers.

The neurochemical mechanisms which underlie ethanol self-administration behavior are not well understood at present. Recent research has suggested that brain systems which mediate reinforcement of behavior are probably involved in the development and promotion of ethanol self- administration. One of the major pathways thought to be involved in reinforcement is the dopaminergic projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc). The goal of the present proposal is to test specific hypothesis about the involvement of dopamine in ethanol self-administration behavior in rats which have been trained to drink pharmacologically relevant amounts of ethanol. The major hypotheses to be tested are: (1) increased dopaminergic activity in the NAcc before or during ingestion underlies the control of ethanol and sucrose self-administration, (2) ethanol produces a different pattern of dopamine output in the NAcc compared with sucrose self- administration, (3) ethanol levels in brain produced by self- administration control, in part, dopamine levels in the NAcc along with the timing and pattern of subsequent ethanol drinking bouts, and (4) the mechanism for ethanol's effects on dopamine output is by increasing the release rather than inhibiting the uptake of dopamine. Five experiments will be carried out to determine (1) the relationship between extracellular dopamine in the NAcc during ethanol or sucrose self- administration in a limited access model, (2) the concentration-effect relationship between brain ethanol and extracellular dopamine during limited access self-administration, (3) if the change in dopamine during 6 hours of access to ethanol is the same as during 30 minute limited access to ethanol, (4) whether ethanol concentrations in brain are related to extracellular dopamine concentrations during 6 hour access to ethanol, and (5) if i.p. administered ethanol alters the in vivo recovery of dopamine or the true extracellular concentration using quantitative microdialysis methodology (point of no-net-flux). Together the results of these experiments will clarify the potential role of dopamine as a neurochemical mediator in the control of ethanol self- administration under conditions in which the ethanol is clearly reinforcing. In addition, the project will provide direct experimental support for the mechanism by which ethanol affects mesolimbic dopaminergic function in vivo. Increased understanding of the neurochemical mechanisms which underlie ethanol self-administration behavior may lead to new approaches for therapy of alcohol abuse and alcoholism.

The neurochemical mechanisms which underlie ethanol self-administration behavior are not well understood at present. Recent research has suggested that brain systems which mediate reinforcement of behavior are probably involved in the development and promotion of ethanol self- administration. One of the major pathways thought to be involved in reinforcement is the dopaminergic projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc). The goal of the present proposal is to test specific hypothesis about the involvement of dopamine in ethanol self-administration behavior in rats which have been trained to drink pharmacologically relevant amounts of ethanol. The major hypotheses to be tested are: (1) increased dopaminergic activity in the NAcc before or during ingestion underlies the control of ethanol and sucrose self-administration, (2) ethanol produces a different pattern of dopamine output in the NAcc compared with sucrose self- administration, (3) ethanol levels in brain produced by self- administration control, in part, dopamine levels in the NAcc along with the timing and pattern of subsequent ethanol drinking bouts, and (4) the mechanism for ethanol's effects on dopamine output is by increasing the release rather than inhibiting the uptake of dopamine. Five experiments will be carried out to determine (1) the relationship between extracellular dopamine in the NAcc during ethanol or sucrose self- administration in a limited access model, (2) the concentration-effect relationship between brain ethanol and extracellular dopamine during limited access self-administration, (3) if the change in dopamine during 6 hours of access to ethanol is the same as during 30 minute limited access to ethanol, (4) whether ethanol concentrations in brain are related to extracellular dopamine concentrations during 6 hour access to ethanol, and (5) if i.p. administered ethanol alters the in vivo recovery of dopamine or the true extracellular concentration using quantitative microdialysis methodology (point of no-net-flux). Together the results of these experiments will clarify the potential role of dopamine as a neurochemical mediator in the control of ethanol self- administration under conditions in which the ethanol is clearly reinforcing. In addition, the project will provide direct experimental support for the mechanism by which ethanol affects mesolimbic dopaminergic function in vivo. Increased understanding of the neurochemical mechanisms which underlie ethanol self-administration behavior may lead to new approaches for therapy of alcohol abuse and alcoholism.

DESCRIPTION (provided by applicant): Our goals are to identify alterations in neurochemistry that occur in the extended amygdala following chronic alcohol administration and examine the hypothesis that endogenous K-opioid receptor (KOR) systems attenuate the neurochemical and behavioral effects of alcohol. We postulate that activation of KOR systems by alcohol is a key homeostatic mechanism that opposes the development of alcohol dependence, and that dysregulation of KOR systems results in altered vulnerability to the reinforcing effects of alcohol and to alcohol addiction. We will test these postulates pharmacologically in wild-type mice, and by using mice with constitutive deletion of the gene encoding the KOR-1 receptor or dynorphin, the endogenous KOR ligand. Specific Aim 1 will characterize alterations in dopamine, GABA and glutamate neurotransmission that occur in the nucleus accumbens (Acb) and central nucleus of the amygdala during abstinence from chronic alcohol administration and determine whether hypofunction of KOR systems exacerbates these effects. The no net flux method of quantitative microdialysis will be used to monitor extracellular dopamine concentrations and changes in dopamine uptake and release. Conventional dialysis will be used to measure basal and stimulus (alcohol, KCI)- evoked dopamine, GABA and glutamate efflux. Specific Aim 2 will determine whether hypofunction of KOR systems alters adaptations in behavior that occur following chronic alcohol administration. We will measure somatic signs of alcohol withdrawal and sensitization of the withdrawal response that occurs after repeated bouts of alcohol intoxication and withdrawal. Specific Aim 3 will determine whether KOR system hypofunction results in increased sensitivity to the rewarding effects of ethanol and whether dopamine dynamics are altered in the Acb and amygdala of mice self-administering alcohol. Operant oral alcohol self- administration will be used to characterize the rewarding effects of alcohol and microdialysis will be used to monitor neurotransmitter dynamics in self-administering mice. The data derived from these studies will delineate neuroadaptations that occur in the extended amygdala following both context-dependent and independent alcohol administration and the role of endogenous opioid systems in modulating the neurochemical effects of alcohol, alcohol dependence, and reward.

DESCRIPTION (provided by applicant): Our goals are to identify alterations in neurochemistry that occur in the extended amygdala following chronic alcohol administration and examine the hypothesis that endogenous K-opioid receptor (KOR) systems attenuate the neurochemical and behavioral effects of alcohol. We postulate that activation of KOR systems by alcohol is a key homeostatic mechanism that opposes the development of alcohol dependence, and that dysregulation of KOR systems results in altered vulnerability to the reinforcing effects of alcohol and to alcohol addiction. We will test these postulates pharmacologically in wild-type mice, and by using mice with constitutive deletion of the gene encoding the KOR-1 receptor or dynorphin, the endogenous KOR ligand. Specific Aim 1 will characterize alterations in dopamine, GABA and glutamate neurotransmission that occur in the nucleus accumbens (Acb) and central nucleus of the amygdala during abstinence from chronic alcohol administration and determine whether hypofunction of KOR systems exacerbates these effects. The no net flux method of quantitative microdialysis will be used to monitor extracellular dopamine concentrations and changes in dopamine uptake and release. Conventional dialysis will be used to measure basal and stimulus (alcohol, KCI)- evoked dopamine, GABA and glutamate efflux. Specific Aim 2 will determine whether hypofunction of KOR systems alters adaptations in behavior that occur following chronic alcohol administration. We will measure somatic signs of alcohol withdrawal and sensitization of the withdrawal response that occurs after repeated bouts of alcohol intoxication and withdrawal. Specific Aim 3 will determine whether KOR system hypofunction results in increased sensitivity to the rewarding effects of ethanol and whether dopamine dynamics are altered in the Acb and amygdala of mice self-administering alcohol. Operant oral alcohol self- administration will be used to characterize the rewarding effects of alcohol and microdialysis will be used to monitor neurotransmitter dynamics in self-administering mice. The data derived from these studies will delineate neuroadaptations that occur in the extended amygdala following both context-dependent and independent alcohol administration and the role of endogenous opioid systems in modulating the neurochemical effects of alcohol, alcohol dependence, and reward.

DESCRIPTION (provided by applicant): The neurochemical and cellular mechanisms that contribute to the control of ethanol drinking behavior are not fully understood, but the involvement of dopamine in the mesolimbic system has been hypothesized for some time. Many neurochemical systems are known to regulate the activity of the mesolimbic dopamine system, e.g., the opiate peptides and their receptors. Naltrexone, a clinically effective drug used to reduce relapse in alcoholism, is a broad spectrum opiate receptor antagonist, but its mechanism is not known in detail. Mu opiate receptors are anatomically located in the ventral tegmental area (VTA) and are known to control VTA dopamine neuron activity through the inhibition of GABA interneurons. However, the role of this mechanism in ethanol's regulation of dopamine release, and the role of particular opiate receptor subtypes has not been firmly established. Our preliminary studies show that mu opiate receptors are involved in the mechanism by which ethanol stimulates dopamine release in the ventral striatum, one of the terminal areas of the mesolimbic system. We propose to determine whether genetic deletion or irreversible blockade of the mu opiate receptor alters the dose-response curve for ethanol-stimulated dopamine release in the ventral striatum (aim 1). The genetic model we propose to use will be congenic mu receptor knockouts and wild-type controls that have been established on a C57BL/6 background. These studies will be complemented by investigation of effects of irreversible blockade of mu receptors with the use of naloxonazine. We also will determine whether the deletion of the mu receptor or irreversible blockade alters ethanol consumption using a two bottle choice procedure (aim 2). Moreover, we propose to determine whether mu receptors in the ventral tegmental area or the ventral striatum are involved in the inhibitory effects of loss of mu receptor function on ethanol stimulated dopamine release (aim 3). This will be done by microinjection of naloxonazine into either area and measuring the ethanol response. The role of mu receptors in the control of VTA-dopamine neurons will be examined using electrophysiological measures of cellular and synaptic activity in GABAergic interneurons (aim 4). These studies will utilize both the genetic and pharmacological approaches as described above. Together, the proposed experiments will elucidate the role of mu opiate receptors in the modulation of mesolimbic dopamine activity by ethanol.

DESCRIPTION (provided by applicant): The Research Society on Alcoholism (RSA) Annual Meeting is the premier scientific meeting involving all areas of alcohol research. In addition to biomedical aspects, it also covers medical/clinical, and psychosocial research. The RSA meeting is held annually, and previous meetings (31) have been held mostly in the continental US, although some meetings have also been held in Hawaii and Canada (Vancouver, Montreal). For the 32nd meeting in 2009, the meeting will be held in San Diego, California (June 19-24, 2008). The 33rd meeting in 2010 will be in San Antonio, TX. The location of these meetings will provide an attractive opportunity for scientists from around the U.S.A. along with others around the world to present and discuss their research. The meetings will have a large impact on alcohol research in the U.S., and the outcome of the research presentations and discussion will advance the field. The program for each meeting will feature topics that are highly relevant to the alcohol research community and provide opportunities for discussion of existing and new collaborative research. The research presentations will be in the form of symposia, roundtables, and workshops that will be proposed by the membership of RSA. The RSA Program Committee will select the proposals with the highest scientific merit and innovation. In addition, numerous poster sessions will be held. The RSA has made an effort to recruit and retain new investigators in the field by highlighting their work as well as providing professional development activities, and this will continue for the 2009 and 2010 meetings. This application requests funds to offset expenses for young scientists (predoctoral and new investigators within 4 years of their terminal degree) to attend these important meetings. Funds are requested for travel and registration fees. An effort will be made to specifically target underrepresented groups. Some administrative costs are also requested.

? DESCRIPTION (provided by applicant): The Research Society on Alcoholism (RSA) Annual Meeting is the premier annual scientific meeting involving all areas of alcohol research. In addition to biological/preclinical aspects, it also covers medical/clinical, and psychosocial research. The RSA meeting is held annually, and previous meetings (37) have been held mostly in the continental US, although some meetings have also been held in Hawaii and Canada (Vancouver, Montreal). For the 38th meeting in 2015, the meeting will be held in San Antonio, TX (June 20-24, 2015). The 39th meeting in 2016 will be in New Orleans, LA. The location of these meetings will provide an attractive opportunity for scientists from around the U.S.A. along with others around the world to present and discuss their research. The meetings will have a large impact on alcohol research in the U.S., and the outcome of the research presentations and discussion will advance the field. The program for each meeting will feature topics that are highly relevant to the alcohol research community and provide opportunities for discussion of existing and new collaborative research. The research presentations will be in the form of symposia, roundtables, and workshops that will be proposed by the membership of RSA. The RSA Program Committee will select the proposals with the highest scientific merit and innovation. In addition, numerous poster sessions will be held. The RSA has made an effort to recruit and retain new investigators in the field by highlighting their work as well as providing professional development activities, and this will continue for the 2015 and 2016-2020 meetings. Since RSA is a stable, established organization with annual meetings, this application is for 5 years of funding (support for the 2016-2020 meetings). Planning for the next three meetings is well underway, but we expect similar planning for years 4-5 of this application to begin soon. This application requests funds to offset expenses for young scientists (predoctoral and postdoctoral trainees within 4 years of their terminal degree) to attend these important meetings. Funds are requested for travel and registration fees. An effort will be made to specifically target underrepresented groups. Some administrative costs are also requested. The specific aim of this grant is to provide travel and registration assistance for students and young investigators to attend the annual RSA meeting. The overall goal is to provide information to those individuals who are interested in learning more about current research in the alcohol field with the possibility of pursuing alcohol research as a career.

DESCRIPTION (provided by applicant): This is a proposal for the renewal of a training grant, the primary purpose of which is to provide stipends for seven predoctoral and two postdoctoral fellows who will be broadly and intensively trained to conduct research on alcoholism. This program has a long history of training predoctoral and postdoctoral students in alcoholism research to help the national effort of producing the next generation of independent scientists with a focus on alcohol research. Training opportunities span the breadth of state-of-the-art approaches including molecular biology and genetics, electrophysiology, cellular imaging, neurochemistry, and behavior. Research models include both animal and human. The training program will promote and support collaborative research across multiple departmental units and Ph.D. programs including Clinical Psychology, Behavioral Neuroscience, Pharmacology, Neuroscience, and Cellular and Molecular Biology. Molecular biology training will encompass 1) studies of the function of excitatory and inhibitory ion channels in cellular expression systems (Mihic, Harris), and 2) development of new transgenic animal models (Harris, Mihic, Pierce-Shimomura, Atkinson), and 3) identification of ethanol responsive genes (Atkinson, Harris, Mihic, Pierce-Shimomura). Genetics training will involve genotyping of human and animal subjects (Fromme, Harris, Mayfield). Electrophysiological and microscopic imaging training will comprise experiments with whole cell and intracellular methods in single cells and in brain slices (Morrisett, Mihic, Harris, Atkinson, Pierce-Shimomura). Training in neurochemistry will focus on 1) intracellular and extracellular signaling mechanisms with an emphasis on phosphorylation (Morrisett, Mayfield), 2) release and transport of glutamate, dopamine, and other neurotransmitters (Gonzales, Harris), and 3) expression of proteins (Harris, Mayfield, Morrisett). Research on the behavioral effects of ethanol will include its influence on motor skills, reinforcement, anxiety, and withdrawal (Harris, Gonzales). Training in psychosocial aspects of alcohol research (Fromme) and the interaction of genotype and effects of alcohol drinking in humans (Fromme, Harris) will also be done. The training program will continue to emphasize professional development including improvement of oral and written communication skills. Predoctoral students will be required to complete a series of core course requirements, scientific ethics, experimental design, and statistical analysis, and grant writing, and will end up with a Ph.D. degree. Postdoctoral training will be for three years and consist of focused alcohol research guided by a faculty mentor. We will continue to focus on minority recruitment. The training faculty has an excellent history of collaboration with a primary focus on alcohol research. The research laboratories are well equipped with the latest instrumentation for neurochemical and behavioral testing.

PROJECT SUMMARY/ABSTRACT This is a proposal for the renewal of a training grant, the primary purpose of which is to provide stipends for five predoctoral and three postdoctoral fellows who will be broadly and intensively trained to conduct research on the neurobiological and behavioral effects of alcohol. This program has a long history of training predoctoral and postdoctoral students in alcoholism research to help the national effort of producing the next generation of independent scientists with a focus on alcohol research. Training opportunities span the breadth of state-of- the-art approaches including molecular biology and genetics, electrophysiology, cellular imaging, neurochemistry, and behavior. Research models include both animal and human. The training program will promote and support collaborative research across multiple departmental units and Ph.D. programs including Clinical Psychology, Behavioral Neuroscience, Pharmacology, Neuroscience, and Cellular and Molecular Biology. Molecular biology training will encompass 1) studies of the function of excitatory and inhibitory ion channels in cellular expression systems (Mihic, Harris), 2) development of new transgenic animal models (Messing, Harris, Mihic, Pierce-Shimomura, Atkinson), and 3) identification of ethanol responsive genes (Atkinson, Harris, Mihic, Pierce-Shimomura). Genetics training will involve genotyping of human and animal subjects (Fromme, Harris, Mayfield, Ponomarev). Electrophysiological and microscopic imaging training will comprise experiments with whole cell and intracellular methods in single cells and in brain slices (Morrisett, Mihic, Harris, Atkinson, Pierce-Shimomura, Messing, Marinelli). Training in neurochemistry will focus on 1) intracellular and extracellular signaling mechanisms with an emphasis on phosphorylation (Morrisett, Mayfield), 2) release and transport of glutamate, dopamine, and other neurotransmitters (Gonzales, Harris, Dominguez), and 3) expression of proteins (Harris, Mayfield, Morrisett, Messing). Research on the behavioral effects of ethanol will include its influence on motor skills, reinforcement, anxiety, and withdrawal (Harris, Gonzales, Dominguez, Morrisett). Training in psychosocial aspects of alcohol research (Fromme) and the interaction of genotype and effects of alcohol drinking in humans (Fromme, Harris) will also be done. The training program will continue to emphasize professional development including improvement of oral and written communication skills and grant writing. Predoctoral students will be required to complete a series of core course requirements, scientific ethics, experimental design, and statistical analysis, and will end up with a Ph.D. degree. Postdoctoral training will be for three years and consist of focused alcohol research guided by a faculty mentor. We will continue to focus on minority recruitment. The training faculty has an excellent history of collaboration with a primary focus on alcohol research. The research laboratories are well equipped with the latest instrumentation for neurochemical and behavioral testing.