Brian A. McCool, B.S., Ph.D. - US grants

A number of studies have implicated glutamate excitotoxcity in the neostriatal neuronal degeneration typical of Huntington's disease. It has also been noticed that both pre- and postsynaptic metabotropic glutamate receptors (mGluRs) may act to modify neuronal sensitivity to exitotoxic compounds in animal models of this disorder. Along these lines,, presynaptic Mglurs can be found on cortical projections into the stratum and are known to modulate glutamatergic transmission at these synapse, presumably via mechanisms which may involve calcium and/or potassium channels. Additionally, postsynaptic Mglurs are known to couple to phosphatydyl inositol metabolism and the mobilization of intracellular calcium in striatal neurons. Mglurs may therefore encourage either neuroprotective or neurodegenerative processes depending upon the signal transduction pathway to which they couple. To better understand the role of these receptors in the neostriatum and to assess their potential as targets for pharmacologic intervention in huntington's disease, the function of mGluR receptor subtypes will be assessed in both native and heterologous systems. This characterization may eventually allow for development of effective treatments for striatal degenerative disorders like Huntington's disease.

APPLICANT'S ABSTRACT: Ethanol's modulation of anxiety is a significant contributing factor to the abuse of this drug. For example, the punishment of withdrawal following chronic ethanol ingestion may help perpetuate abuse by the alcoholic individual. This intimate association between ethanol and anxiety is found in several species; and, the neural circuitry regulating fear and anxiety behaviors is also well conserved. Classic fear-conditional approaches have implicated the amygdala, a limbic forebrain area, as playing a pivotal role in the acquisition and expression of fear/anxiety behaviors. The amygdala is therefore a likely target for anxiety-related neuro-adaptive processes elicited by chronic ethanol abuse. Importantly, preliminary data suggests that chronic ethanol exposure causes facilitation of N-methyl-D-aspartate (NMDA) receptor function in dissociated amygdala neurons. Because amygdala NMDA receptors play an important role in fear-conditioned learning, we hypothesize that ethanol-induced adaptation in NMDA receptor function may result in an ethanol-dependent, 'chemical' conditioning of this brain region. This hypothesis will be tested by two specific aims. Specific Aim #1 will characterize the effects of chronic ethanol exposure on NMDA receptors in dissociated amygdala neurons using whole-cell patch clamp electrophysiology combined with single-cell reverse transcription/polymerase chain reaction. These studies will provide cellular and molecular insight into the mechanism of chronic ethanol-induced alterations in NMDA receptor physiology. Specific Aim #2 will determine the neurophysiologic consequences of increased NMDA-dependent synaptic plasticity within the amygdala to directly address chemical conditioning by chronic ethanol. This proposal provides a unique opportunity to examine the influences of chronic ethanol exposure on the molecular, cellular, and physiologic characteristics within the amygdala's fear/anxiety circuit. The proposed studies will also advance our knowledge of the fundamental neural mechanisms regulating ethanol abuse.

DESCRIPTION (provided by applicant): The anxiolytic properties of ethanol consumption and anxiogenic aspects of withdrawal from chronic exposure are well established in both humans as well as experimental animal models. Because these anxiety-related effects help potentiate alcohol abuse, it is important to gain a detailed understanding of the cellular and molecular mechanisms that underlie the ethanol-anxiety interaction. While numerous brain regions are known to interact during fear/anxiety, the amygdala, an "emotional" center in the limbic forebrain, is a central component of the neural anxiety circuitry. A delicate balance between excitatory and inhibitory neurotransmitter systems controls this system, with increases in amygdala excitation leading to enhanced fear/anxiety. Preliminary studies of using acutely isolated neurons from the lateral/basolateral amygdala indicate that chronic ethanol can alter NMDA-type ionotropic glutamate and inhibitory GABAA receptor function while sparing other neurotransmitter systems. Importantly, both receptor systems are sensitive to clinically relevant ethanol concentrations, indicating their functional adaptation during chronic exposure may be among the cellular substrates relating abuse with anxiety. Our central hypothesis, that chronic ethanol exposure and subsequent withdrawal differentially affect major amygdala neurotransmitter systems, will be tested by further examination of glutamate-gated ionotropic receptor and GABAA receptor expression and function within the rat lateral/basolateral amygdala. Specifically, we will utilize whole-cell patch clamp electrophysiology and single-cell reverse transcription/polymerase chain reaction (RT-PCR) with acutely isolated neurons. These studies will be done along with real-time RT-PCR to characterize cellular and molecular adaptations in the lateral/basolateral amygdala to chronic ethanol exposure and withdrawal. We will also assess the physiological ramifications of adaptations to chronic ethanol exposure by measuring post- and pre-synaptic function using whole-cell recordings within lateral/basolateral amygdala in in vitro slice preparations. Finally, we will test the association between receptor/synaptic function, anxiety, and withdrawal by integrating cellular, molecular, synaptic and behavioral measures during a withdrawal time course. Together, these studies will provide important insight into the mechanisms associated with chronic ethanol-induced adaptations related to fear/anxiety and eventually lead to a better understanding of the ethanol-anxiety interaction.

DESCRIPTION (provided by applicant): The anxiolytic properties of ethanol consumption and anxiogenic aspects of withdrawal from chronic exposure are well established in both humans as well as experimental animal models. Because these anxiety-related effects help potentiate alcohol abuse, it is important to gain a detailed understanding of the cellular and molecular mechanisms that underlie the ethanol-anxiety interaction. While numerous brain regions are known to interact during fear/anxiety, the amygdala, an "emotional" center in the limbic forebrain, is a central component of the neural anxiety circuitry. A delicate balance between excitatory and inhibitory neurotransmitter systems controls this system, with increases in amygdala excitation leading to enhanced fear/anxiety. Preliminary studies of using acutely isolated neurons from the lateral/basolateral amygdala indicate that chronic ethanol can alter NMDA-type ionotropic glutamate and inhibitory GABAA receptor function while sparing other neurotransmitter systems. Importantly, both receptor systems are sensitive to clinically relevant ethanol concentrations, indicating their functional adaptation during chronic exposure may be among the cellular substrates relating abuse with anxiety. Our central hypothesis, that chronic ethanol exposure and subsequent withdrawal differentially affect major amygdala neurotransmitter systems, will be tested by further examination of glutamate-gated ionotropic receptor and GABAA receptor expression and function within the rat lateral/basolateral amygdala. Specifically, we will utilize whole-cell patch clamp electrophysiology and single-cell reverse transcription/polymerase chain reaction (RT-PCR) with acutely isolated neurons. These studies will be done along with real-time RT-PCR to characterize cellular and molecular adaptations in the lateral/basolateral amygdala to chronic ethanol exposure and withdrawal. We will also assess the physiological ramifications of adaptations to chronic ethanol exposure by measuring post- and pre-synaptic function using whole-cell recordings within lateral/basolateral amygdala in in vitro slice preparations. Finally, we will test the association between receptor/synaptic function, anxiety, and withdrawal by integrating cellular, molecular, synaptic and behavioral measures during a withdrawal time course. Together, these studies will provide important insight into the mechanisms associated with chronic ethanol-induced adaptations related to fear/anxiety and eventually lead to a better understanding of the ethanol-anxiety interaction.

DESCRIPTION (provided by applicant): It is well established that acute ethanol consumption can decrease anxiety and that withdrawal from chronic ethanol exposure can increase anxiety. Furthermore, voluntary ethanol consumption can be regulated by exposure to anxiety/stress in both recovering alcoholics and experimental animal models. Rodent models have been especially important for understanding cellular and molecular adaptations to stress/anxiety and chronic alcohol ingestion. However, the extent to which findings in rodent models mimic those in human alcoholics is not well understood. Primate models of long-term ethanol self-administration may therefore provide valuable insight into the relationship between stress/anxiety and alcohol abuse that is potentially more relevant to the human condition. The regulation of anxiety and behavioral responses to stress involve complex interactions between numerous brain regions. The amygdala, a limbic forebrain area, is a pivotal brain region controlling such behaviors. Preliminary studies in our laboratory have utilized acutely isolated amygdala neurons from control Macaca fascicularis monkeys and from monkeys that have self-administered ethanol for 18 months. Results from these studies indicate that long-term ethanol self-administration causes alterations in amygdala GABA(A) receptor function and pharmacology that are distinct from those found in some rodent models. To better understand the interactions between anxiety/stress and chronic ethanol in primates, we will utilize whole-cell patch clamp electrophysiology in isolated amygdala neurons to characterize cellular adaptations of the GABA(A) receptor to peri-natal stress and long-term ethanol self administration in a related species, Macaca mulatta. These studies will be complemented by real-time RTPCR and western analysis of monkey amygdala tissue to identify molecular mechanisms underlying changes GABA(A) receptor function. Ultimately, our proposed studies will help lead to a better understanding of the ethanol-anxiety/stress interaction in primates.

DESCRIPTION (provided by applicant): It is well established that acute ethanol consumption can decrease anxiety and that withdrawal from chronic ethanol exposure can increase anxiety. Furthermore, voluntary ethanol consumption can be regulated by exposure to anxiety/stress in both recovering alcoholics and experimental animal models. Rodent models have been especially important for understanding cellular and molecular adaptations to stress/anxiety and chronic alcohol ingestion. However, the extent to which findings in rodent models mimic those in human alcoholics is not well understood. Primate models of long-term ethanol self-administration may therefore provide valuable insight into the relationship between stress/anxiety and alcohol abuse that is potentially more relevant to the human condition. The regulation of anxiety and behavioral responses to stress involve complex interactions between numerous brain regions. The amygdala, a limbic forebrain area, is a pivotal brain region controlling such behaviors. Preliminary studies in our laboratory have utilized acutely isolated amygdala neurons from control Macaca fascicularis monkeys and from monkeys that have self-administered ethanol for 18 months. Results from these studies indicate that long-term ethanol self-administration causes alterations in amygdala GABA(A) receptor function and pharmacology that are distinct from those found in some rodent models. To better understand the interactions between anxiety/stress and chronic ethanol in primates, we will utilize whole-cell patch clamp electrophysiology in isolated amygdala neurons to characterize cellular adaptations of the GABA(A) receptor to peri-natal stress and long-term ethanol self administration in a related species, Macaca mulatta. These studies will be complemented by real-time RTPCR and western analysis of monkey amygdala tissue to identify molecular mechanisms underlying changes GABA(A) receptor function. Ultimately, our proposed studies will help lead to a better understanding of the ethanol-anxiety/stress interaction in primates.

DESCRIPTION (provided by applicant): Stress-alcohol interactions clearly contribute to consequences of alcohol abuse, including increased anxiety during withdrawal from chronic exposure. However, little is known about the biological basis for these complex relationships. Animal models are likely to make significant contributions to our understanding of these issues. For example, comparisons between genetically-defined inbred mouse strains would provide a unique opportunity to understand the relevant genetic mechanisms regulating adaptations to chronic ethanol and withdrawal. Likewise, subjecting these strains to defined environmental stressors would yield important insight into the 'experience-dependent'mechanisms that influence the behavioral consequences (e.g. anxiety) related to this alcohol exposure. Although numerous brain regions are known to govern fear/anxiety behaviors, the lateral/basolateral amygdala is a central component of 'anxiety'circuitry. Because amygdale GABAA receptors regulate anxiety behavior, ethanol- and stress-dependent adaptations in this system may be important for the behavioral consequences related to their interactions. Our preliminary results suggest that the C57BL/6 (B6) and DBA/2J (D2) inbred mouse lines differ markedly in their basal anxiety as well as the functional and molecular expression of GABAA receptors expressed in lateral/basolateral amygdale (BLA). Preliminary findings also indicate that chronic ethanol exposure differentially alters the function of BLA GABAA receptors expressed by B6 and D2 neurons. Therefore, this application will specifically test the central hypothesis that lateral/basolateral amygdala GABAA receptor expression/function is a phenotypic marker for BLA GABAA the relative anxiety-related liability of stress-alcohol interactions. We will test our central hypothesis using housing manipulations and chronic ethanol inhalation to investigate the interactions between environmental stressors, amygdala GABAergic expression/function, and the neurobiological consequences of withdrawal. We will specifically integrate behavioral, molecular biological, and electrophysiological experimental approaches to better understand the stress-ethanol interactions related to anxiety-like behaviors. Ultimately, we hope our approach will allow us to eventually characterize the specific genetic mechanisms regulating the molecular and neurophysiological adaptations associated with these relationships.

The goal of the current application is to understand the relationship between alcohol exposure, withdrawal from chronic alcohol exposure, and the neurobiological mechanisms regulating anxiety-like behavior. The proposed experiments will utilize a rat model of chronic ethanol exposure and will integrate electrophysiological, cellular and molecular biological, and behavioral experimental approaches to examine the role of a specific brain region, the lateral/basolateral amygdala (BLA), in these processes. This brain area has been extensively implicated as an important regulatory component of the neural circuitry controlling anxiety-like behavior. Furthermore, findings from the previous funding period have shown extensive neurophysiological adaptations in both the glutamatergic and GABAergic neurotransmitter systems in the BLA. The objectives of the current proposal are therefore to 1) understand the neurobiological and cellular mechanisms governing these alterations and 2) define the role of BLA neurotransmitter systems in regulating anxiety-like behavior associated with chronic ethanol exposure and subsequent withdrawal. Our proposed experiments will specifically test the central hypothesis that anxiety during withdrawal from chronic ethanol exposure is caused by functional alterations within the major amygdala neurotransmitter systems. Specific Aim 1 will test this hypothesis by characterizing neurophysiological and molecular alterations in glutamate neurotransmission following a chronic intermittent ethanol inhalation exposure in rats. Experimental approaches include in vitro slice patch-clamp electrophysiology to examine the function of BLA glutamatergic afferents and biochemical analysis of glutamate receptor expression and localization. Specific Aim 2 will examine chronic ethanol- and withdrawal-related alterations in rat BLA GABAergic neurotransmission and receptor expression. In vitro slice whole-cell electrophysiology will be used in this aim as well. In addition, real-time RT-PCR will be employed to analyze molecular adaptations in the BLA GABAergic system. Specific Aim 3 will address our central hypothesis by directly examining BLA glutamatergic and GABAergic alterations in the context of enhanced expression of anxiety-like behavior following chronic ethanol exposure. The proposed experiments will specifically examine the evolving relationship between exposure and withdrawal time course and neurophysiological function. Additionally, traditional behavioral pharmacological approaches will be used to better understand how changes in this neurobiological function contribute to the expression of withdrawalrelated anxiety. Ultimately, the proposed experiments will better define the specific neurobiological contributions of the amygdala to anxiety-like behavior expressed following chronic alcohol exposure and withdrawal. These studies could therefore provide insight into the cellular mechanisms governing anxietyassociated relapse in human alcoholics.

DESCRIPTION (provided by applicant): The goal of the current application is to understand the relationship between chronic alcohol exposure, withdrawal from this exposure, and the neurobiological mechanisms regulating anxiety-like behavior. The proposed experiments will utilize a rat model of chronic ethanol exposure and will integrate electrophysiological, cellular and molecular biological, and behavioral experimental approaches to examine the role of a specific brain region, the lateral/basolateral amygdala (BLA), in these processes. This brain area has been extensively implicated as an important regulatory component of the neural circuitry controlling anxiety-like behavior in rodents. Furthermore, findings from the previous funding period have shown extensive neurophysiological adaptations in both the glutamatergic and GABAergic neurotransmitter systems in the BLA. The objectives of the current proposal are therefore to 1) understand the neurobiological and cellular mechanisms governing these alterations and 2) define the role of BLA neurotransmitter systems in regulating anxiety-like behavior associated with chronic ethanol exposure and subsequent withdrawal. Our proposed experiments will specifically test the central hypothesis that anxiety during withdrawal from chronic ethanol exposure is caused by functional alterations within the major amygdala neurotransmitter systems. Specific Aim 1 will test this hypothesis by characterizing neurophysiological and molecular alterations in glutamate neurotransmission following a chronic intermittent ethanol inhalation exposure in rats. Experimental approaches include in vitro slice patch-clamp electrophysiology to examine the function of BLA glutamatergic afferents and biochemical analysis of glutamate receptor expression and localization. Specific Aim 2 will examine chronic ethanol- and withdrawal-related alterations in rat BLA GABAergic neurotransmission and receptor expression. In vitro slice whole-cell electrophysiology will be used in this aim as well. In addition, cell and molecular biological approaches will be employed to analyze molecular adaptations in the BLA GABAergic system. Specific Aim 3 will address our central hypothesis by directly examining the BLA glutamatergic and GABAergic systems in the context of enhanced expression of anxiety-like behavior following chronic ethanol exposure. The proposed experiments will specifically examine the evolving relationship between exposure and withdrawal time course and neurophysiological function. Additionally, traditional behavioral pharmacological approaches will be used to better understand how changes in this neurobiological function contribute to the expression of withdrawal-related anxiety. Ultimately, the proposed experiments will better define the specific neurobiological contributions of the amygdala to anxiety-like behavior expressed following chronic alcohol exposure and withdrawal. These studies could therefore provide insight into the cellular mechanisms governing anxiety-associated relapse in human alcoholics. PUBLIC HEALTH RELEVANCE The focus of this research project is to understand the impact of chronic ethanol exposure and withdrawal on neurophysiological processes that regulate emotions like anxiety. Once the project is completed, we will better understand how chronic ethanol exposure and withdrawal alter the brain.

DESCRIPTION (provided by applicant): This proposal for a T32 training grant is to provide support for pre and postdoctoral trainees in the field of alcohol research at Wake Forest University School of Medicine. We are a group of outstanding, well-funded alcohol investigators with a solid funding base in basic, clinical, human populations research that has been successful for fourteen years in training young scientists to become independent investigators. Our research is carried out in a highly collaborative, multidisciplinary manner so that trainees not only receive the training necessary to become independent investigators, but also as members of interdisciplinary teams of the kind that will increasingly characterize their future research careers. The research training faculty do work that is highly translational. First, virtually all of our studies employ alcohol self-administration. It also spans the scale from mice and rats, through monkeys and individual humans, to human populations. Many of our studies examine the same end points and employ the same behavioral models in multiple species. Training consists of rigorous didactic work along with intensive laboratory-based research. It is augmented by a robust curriculum in career development, including teaching and writing courses, and ethics. There are multiple opportunities for students to hone their presentation skills, including journal clubs and required research seminar presentations at least twice or more each year, depending on which graduate program they are in. We require our students to apply for individual NRSA support both to free up slots on the training grant and to help them hone their skills in grant writing. Training also benefits from distinct school resources that include a Translational Science Institute that offers courses in translational Research, a primate center with several populations of monkeys including a fully pedigreed and genotyped vervet colony, and state of the art imaging. We have also formed a partnership with a local treatment center that will give our students clinical exposure to enhance their understanding of the disease of alcoholism and ground their research in he real world.

DESCRIPTION (provided by applicant): Withdrawal stress following chronic ethanol exposure results in heightened anxiety-like behaviors that contribute to relapse in abstinent alcoholics. However, very little is known about the neurobiological mechanisms controlling these outcomes. Animal models can make significant contributions towards understanding these issues. For example, we have shown that the rat glutamatergic and GABAergic synaptic function in the lateral/basolateral amygdala (BLA) are dramatically regulated by chronic ethanol exposure and withdrawal. Similarly, the rat BLA CRF/urocortin system appears to enhance excitatory BLA responses. Chronic ethanol-related alterations in glutamate, GABA, and CRF/urocortin all potentially contribute to withdrawal-anxiety. But, the precise contributions of any individual alteration are confounded by their fundamental contributions in ethanol-naive animals. The C57BL/6J (B6) and DBA/2J (D2) mouse offer an alternative approach. These inbred lines differ dramatically with respect to a number of different ethanol related behaviors, including their behavioral sensitivity to chronic ethanol exposure and withdrawal. This is extended to withdrawal-anxiety by preliminary evidence provided in the current application that shows greater D2 sensitivity. Furthermore, our published and preliminary findings suggest that BLA GABAergic and potentially glutamatergic synaptic function in these two mouse lines are markedly distinct. This strongly suggests that chronic ethanol exposures producing substantial withdrawal-anxiety in one strain but not the other can be used to highlight individual neurophysiological changes with the greatest behavioral impact. The proposed experiments will therefore test the central hypothesis that the greater withdrawal-related increases in anxiety in D2 mice will be reflected by more significant increases in BLA excitatory neurotransmitter systems, like glutamate and CRF/urocortin. Our primarily electrophysiological analysis of these systems as well as GABAergic function will be integrated with both behavioral measures of anxiety during withdrawal-stress in B6, D2, and genetically modified mice. The proposed experiments are significant because they offer a unique opportunity to define specific BLA neurobiological targets for future therapies.

DESCRIPTION (provided by applicant): The overall goal of the current application is to understand the neurobiological mechanisms that help confer pathological behaviors like enhanced negative affect following ethanol physical dependence. We will accomplish this goal by utilizing a rat model of chronic ethanol exposure and by integrating optogenetic, synaptic neurophysiology, and behavioral experimental approaches to examine adaptations glutamatergic and GABAergic neurotransmission in a specific brain region, the lateral/basolateral amygdala (BLA). This brain area has been extensively implicated as an important regulatory component of the neural circuitry controlling both anxiety-like behavior during withdrawal from chronic ethanol exposure as well as reward-seeking in drug- naive and -exposed animals. Findings from the previous funding period have demonstrated that the extensive glutamatergic and GABAergic synaptic adaptations occur within specific pre- and postsynaptic compartments and potentially within specific afferent systems. The objectives of the current proposal are therefore to understand the neurobiological and cellular mechanisms governing the specificity of these alterations. Our proposed experiments will test the central hypothesis that specific alterations in synaptic function at distinct BLA afferents following chronc ethanol lead to the development and expression of withdrawal-related anxiety. Specific Aim 1 will test this hypothesis by defining the regional origin for pre- and post-synaptic alterations expressed following chronic ethanol exposure and withdrawal. We will utilize optogenetic approaches to control synaptic transmission arising from specific afferents along with in vitro slice patch-clamp electrophysiology. These studies are significant because they will first implicate specific brain regions involved in BLA alterations during ethanol physical dependence. Second, these afferents carry unique forms of information; so any region-specific involvement will identify for the first time how information processing may be disrupted by chronic ethanol exposure. Specific Aim 2 will examine the functional and behavioral relationships between BLA neurophysiology and the plasticity-like state resulting from chronic intermittent ethanol/withdrawal. In this case, we will address our central hypothesis by directly examining BLA glutamatergic and GABAergic synaptic alterations using in vitro slice recordings interpreted in the context of enhanced expression of anxiety-like behavior following chronic ethanol exposure. The proposed experiments will specifically examine the evolving relationship using exposure and withdrawal time courses. These studies are significant because they will identify the precise cellular and synaptic mechanisms leading to ethanol conditioning in the BLA. Ultimately, the application will better define specific neurobiological contributions by the amygdala to enhanced anxiety-like behavior following chronic alcohol exposure and withdrawal. These studies will provide insight into potential cellular mechanisms governing abuse and relapse in human alcoholics.

DESCRIPTION (provided by applicant): This renewal application is for a T32 training grant which provides support for five predoctoral and three postdoctoral trainees in basic alcohol research at Wake Forest School of Medicine. The objectives of the training program are to 1) provide multi-disciplinary training in the neurobiology of alcohol abuse and addiction and 2) produce independent, productive scientists for the alcohol research community. Our rationale for the proposed training arises from our diverse training faculty - a group of well-funded alcohol investigators with solid expertise in basic, clinical, and human-population research - who have been successful for almost twenty years in training young scientists. Our environment is highly collaborative and multidisciplinary such that our trainees gather the necessary skills to become independent investigators and to operate within interdisciplinary teams that will increasingly characterize the future of biomedical research. The alcohol research that characterizes our training environment is also highly translational. First, virtually all of our training labs employand publish in alcohol self-administration; and this has characterized the training program from its inception. The training program also spans experimental model systems from mice and rats, through monkeys and individual humans, to human populations. And many of our training faculty employ overlapping experimental approaches, examine some of the same end points, and/or employ the some of the same behavioral models across multiple species. Our training design therefore consists of both rigorous didactic instruction as well as intensive laboratory-based research. It is augmented by a robust curriculum in career development, including teaching and grant- and manuscript-writing courses, and ethics. There are multiple opportunities for students and postdoctoral fellows to hone presentation skills including journal clubs, data clubs, and required research seminar presentations at least twice or more each year. As a condition for reappointment to the training program, we require our all trainees apply for individual NRSAs (or independent private support) during their training and to publish in peer-reviewed journals. This requirement helps sharpen grant writing skills that are critical in today's research environment. We also attempt to limit predoctoral trainees to less than three years of support on the training grant. This time-limit ensures that the training grant provides support to the best, brightest, and most productive students and fellows at Wake Forest School of Medicine. Our program also benefits from distinct school resources that include a Translational Science Institute which offers courses in translational research, a nationally-recognized primate center with several different populations of monkeys including a fully pedigreed and genotyped vervet colony, and state-of-the-art imaging facilities that can accommodate humans, non-human primates, and rodents. Finally, we partner with resident psychiatrists and psychologists to give our students clinical exposure to current and long-abstinent patients. This enhances their understanding of alcohol abuse disorders and grounds their research in the real world.

? DESCRIPTION (provided by applicant): The overall goal of the current application is to understand the neurobiological mechanisms that help confer pathological behaviors like enhanced negative affect following ethanol physical dependence. Recent studies suggest that intrinsic lateral/basolateral amygdala (BLA) GABAergic neurons tightly control the expression of negative emotions including those expressed during withdrawal following chronic ethanol exposure. Dopaminergic inputs from the ventral tegmentum/substantia nigra pars compacta have been shown to regulate this GABAergic system and disinhibit BLA principal neurons which drive the expression of anxiety-like behaviors. Based on strong preliminary evidence, our proposed experiments will test the central hypothesis that ethanol dependence dis-inhibits BLA output by dysregulating dopaminergic modulation of these GABAergic neurons. We will test our central hypothesis and accomplish our overall goal by utilizing a well- established rat model of chronic ethanol exposure and by integrating optogenetic, pre- and post-synaptic dopamine neurophysiology, and behavioral experimental approaches. The BLA has been extensively implicated as an important regulatory component of the neural circuitry controlling both anxiety-like behavior during withdrawal from chronic ethanol exposure as well as reward-seeking in drug-naïve and -exposed animals. Specific Aim 1 will test our central hypothesis hypothesis by examining presynaptic dopamine function during withdrawal from ethanol dependence. We will directly measure DA release and reuptake in vitro by integrating in vitro fast-scan cyclic voltammetry with optogenetic control of DA release and chronic ethanol exposure. We hypothesize that, based on our previous publications, chronic ethanol exposure will differentially modulate basal or `tonic' DA levels and phasic DA release to ultimately enhance DA signaling. Specific Aim 2 will examine how postsynaptic DA receptor function is altered in the BLA using in vitro whole cell patch clamp electrophysiology with innovative optogenetic approaches to measure postsynaptic DA receptor signaling. Our working hypothesis is that ethanol physical dependence will increase postsynaptic D1- and D2-like DA receptor signaling such that DA-mediated inhibition of GABAergic function is up-regulated. Specific Aim 3 will place the detailed cellular effects of dependence on DA signaling within a whole-animal context by integrating optogenetic control of DA release with in vivo measures of BLA-dependent behaviors. Our working hypothesis is that dependence-related changes in dopamine neurotransmission and signaling ultimately control withdrawal-dependent anxiety-like behavior. The proposed work employs a unique and highly integrated experimental approach to provide unparalleled insight into the neurobiological mechanisms governing the negative reinforcing effects of chronic ethanol exposure. Ultimately, these studies will provide insight into potential cellular mechanisms governing abuse and relapse in human alcoholics.

The acute sensitivity and long-term effects of ethanol on a wide variety of postsynaptic receptors has been well documented. And, presynaptic facilitation of GABAergic synapses by acute ethanol has been described in many brain regions. But, acute ethanol does not appear alter presynaptic glutamate release. Several labs, including our own, have shown that chronic ethanol exposure robustly up-regulates presynaptic glutamate release at some synapses. But, the presynaptic mechanism responsible for this chronic up-regulation remains undefined. We recently showed that acute ethanol can robustly modulate presynaptic function at glutamate synapses in the lateral/basolateral amygdala. Like many studies, we did not observe any direct effect of ethanol on glutamate release at these synapses. However, using high frequency stimulation to deplete readily- releasable vesicle pool, we found that acute ethanol robustly inhibited synaptic responses represented by the recycling pool of synaptic vesicles. This data thus potentially defines an entirely novel presynaptic effect ethanol: inhibition of synaptic vesicle recycling. Our data also show that ethanol modulation of vesicle recycling is frequency-dependent. This suggests unique contributions by distinct presynaptic recycling pathways. Finally, we present preliminary data showing that the presynaptic Munc proteins, which regulate both vesicle priming and recycling, modulate ethanol inhibition of these recycling pathways. We thus propose the central hypothesis that Munc13-1 and Munc13-2 mediate ethanol inhibition of distinct vesicle recycling pathways. We will directly test this hypothesis by accomplishing the two specific aims. The first specific aim will test the working hypothesis that ethanol inhibits two independent vesicle recycling pathways, clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Published evidence shows differential, strain-specific ethanol modulation of recycling at both moderate stimulation frequencies, dominated by clathrin-dependent endocytosis, and during high-frequency stimulation, when activity- dependent bulk endocytosis controls recycling. To test this working hypothesis, we will integrate shRNA- mediated knockdown of essential presynaptic proteins within each pathway, selective pharmacological modulation, and whole-cell patch clamp electrophysiology in vitro. The second specific aim will test the working hypothesis that Munc13-1 and Munc13-2 differentially regulate ethanol inhibition of distinct vesicle recycling pathways. Our rationale here is that genetic variations in Munc13-1 and Munc13-2 are associated with the differential, mouse strain-specific ethanol sensitivity of low- and high-frequency responses. We will test our working hypothesis for this aim by again integrating shRNA-mediated knockdown of Munc13-1 or Munc13-2 with in vitro electrophysiology. The proposed work is significant because it will characterize both a novel presynaptic effect of acute ethanol and contributions by unique presynaptic proteins.

SUMMARY The neurobiological mechanisms controlling the transition from alcohol use to abuse are poorly understood. Our overall approach is to examine vulnerable populations to highlight specific cellular/molecular pathways involved in this transition. For example, human adolescents exposed to heavy alcohol use are at much greater risk for the development of alcoholism as adults. Recent studies suggest similar liabilities for adolescent animals including rodents. Our published work indicates adolescent chronic ethanol exposure differentially modulates both glutamatergic and GABAergic neurotransmission in the lateral/basolateral amygdala (BLA), a `node' within circuits critical for the integration cognitive and sensory information during emotional responses, in an input-specific fashion. Our data also suggest a critical role for mammalian target of rapamycin (mTOR)- dependent signaling cascades in synaptic strengthening. Further, we provide preliminary data suggesting that many of these synaptic effects are absent or greatly diminished in adult animals. This suggests that mTOR- dependent signaling directly regulates synaptic modulation during adolescent ethanol exposure. The overall goal of the current project is to therefore use a well-established ethanol vapor exposure in adolescent rats to understand the long-term impact of this exposure in adult animals by integrating cellular, molecular, and behavioral methodologies. The proposed work includes three specific aims: Aim 1 will characterize the effects of adolescent ethanol exposure on adult BLA glutamatergic and GABAergic neurotransmission using whole- cell patch clamp electrophysiology; Aim 2 will describe the effect of adolescent ethanol exposure on mTOR signaling in both postsynaptic and presynaptic compartments in the BLA; and, Aim 3 will examine pharmacological intervention along the mTOR-signaling pathway and its impact on the long-term behavioral consequences of adolescent ethanol exposure. Together these aims are significant because they leverage a vulnerable population (adolescents), innovative technical and conceptual approaches, and the substantial expertise of our research team to help identify specific cellular signaling processes governing the impact of ethanol exposure across multiple levels of analysis. We will directly test if these signaling processes represent potential therapeutic targets for treatments designed to interrupt the transition from ethanol use to abuse.