Friedbert Weiss, Ph.D. - US grants

The present proposal is designed to extend previous work in this laboratory aimed at identifying neuronal circuitries and neurochemical substrates involved in ethanol reward and dependence. It is the purpose of this project to use the intracranial microdialysis technique to monitor the synaptic concentration of several forebrain transmitters previously implicated in ethanol seeking behavior and reward, and to establish a "profile" of basal and ethanol-induced neurotransmitter activity for both unselected Wistar and alcohol-preferring (P-1) rats. Transmitter release will be monitored before and during oral ethanol self-administration in a two-lever free-choice (water/ethanol 10%w/v) condition, using dependent rats undergoing ethanol withdrawal. Additionally, high and low responders for ethanol intake. Dialysate will be collected from the nucleus accumbens, caudate nucleus and ventral pallidum, and will be assayed for dopamine (DA), 5-hydroxytryptamine (5- HT) or gamma-aminobutyric acid (GABA). These data will serve to identify differences in basal DA, 5-HT and GABA levels between two strains of rats differing in their preference for ethanol, establish the neurochemical effects of self-administered ethanol on the release of these transmitters, and will help determine their role in ethanol reward and dependence. Finally, the effects of ethanol on neurotransmitter activity will be compared to those of other drugs of abuse including cocaine and heroin, These studies have important implications with regard to the understanding of ethanol actions on central neurotransmitter systems and the identification of neural substrates for ethanol reward. Such issues are of critical importance for the development of effective strategies for the prevention and treatment of ethanol abuse and dependence.

This proposal is designed to establish a reliable animal model of cocaine dependence for the evaluation of the therapeutic efficacy of potential treatment drugs. The animal model will permit assessment of treatment drug effects on separate aspects of cocaine dependence, including the rewarding value of cocaine, craving, and propensity to relapse. Initially, behavioral parameters of responding for cocaine under different reinforcement contingencies will be established using progressive ratio, concurrent, and multiple schedules . Repeated extinction and spontaneous recovery tests in the presence or absence of environmental cues associated with cocaine availability will provide information about the persistence of cocaine-seeking behavior. The characteristic patterns of responding observed in these behavioral tests will serve as measures of (1) reward magnitude, operationally defined as the "breaking point" for responding for cocaine on a progressive ratio schedule, (2) degree of preference for cocaine in a drugs vs. food choice situation, (3) craving, operationally defined as the magnitude and duration of extinction responding on a non-reinforced multiple schedule component, and (4) propensity to relapse, operationally defined as the facilitation of spontaneous recovery after introduction of a conditioned stimulus. The behavioral parameters serve as reference points against which the effects of potential treatment drugs will be evaluated. Initial compounds to be tested include agents that act on monoamine and opioid neurotransmission. As other agents become available, subsequent compounds will include neuropeptide analogues and drugs that act on non-monoaminergic systems. These studies have important clinical implications with regard to new drugs that may be effective in treating or preventing cocaine abuse and dependence, and may aid in determining which of the various neuropharmacological actions of cocaine are involved in different components of cocaine dependence.

This proposal requests funds to extend preliminary work on the development of new reliable microdialysis and analytical methods for the recovery and detection of neuropeptides in freely-moving rats during cocaine withdrawal. The work in this proposal will focus on two neuropeptides of high-molecular weight: corticotropin-releasing factor (CRF) and neuropeptide Y (NPY). These neuroactive peptides are involved in physiological, emotional, and behavioral responses to stress and are present in high concentrations in limbic brain regions that are also densely innervated by ascending dopaminergic neurons, and that have been previously implicated in psychostimulant reinforcement, dependence, and withdrawal. Thus, their functional role as well as their localization suggests that these neuropeptides may independently, or by interaction with dopaminergic neurotransmission, contribute to the cocaine withdrawal syndrome. To test this hypothesis, CRF and NPY levels will be monitored in the centrolmedial amygdala, bed nucleus of the stria terminalis, and shell of the nucleus accumbens (i.e., the "extended amygdala") of freely moving rats during withdrawal from cocaine after unlimited-access intravenous cocaine self-administration. To accomplish these goals, we will initially refine recently developed original microdialysis and radioimmunochemical methods with which detectable basal and stimulated release of CRF was obtained in the mediobasal hypothalamus of awake rats. These procedures will then be extended to NPY with careful and extensive in vitro and in vivo validation. These experiments will provide important insights about the role of stress-related limbic neuropeptides in the behavioral and neurochemical effects of cocaine withdrawal.

The purpose of the proposed project is to continue to identify and establish the pharmacological profile of agents with potential protective effects against multiple triggers for relapse and selectivity for cocaine-seeking behavior without producing general suppressant effects on motivated behavior. Data generated during the previous funding period have identified the mGlu^ agonist LY379268, the mGlu5 antagonist MTEP, the orexin/hypocretin (Orx/Hcrt) Hcrt-1 antagonist SB334867, and the 01 receptor antagonist BD1047 as candidates meeting one or more of these criteria. The present extension of this project represents an essential next step for defining the potential of these receptors as treatment targets, and to begin to provide understanding of the neurobiological basis for the therapeutic effects of agents acting at these receptors. The research plan is to first establish the profile of action for LY379268, MTEP, SB334867, and BD1047 on cocaine-seeking in animal models of cue- and stress-induced relapse under clinically relevant conditions, including (a) examination of the efficacy of these agents for reversingcue- and stress-induced reinstatement with repeated treatment and (b) examination of the effects and shifts in the dose-response profiles of these agents for reversing cue-and stress-induced reinstatement in rats with a history of escalated cocaine self-administration. These studies will be followed by examination of the potential of these agents to reverse negative affect associated with cocaine withdrawal, a condition that next to cue-induced craving and stress has been implicated as a critical risk factor for relapse. These studies will be conducted in rats with a history of escalated cocaine self-administration, using the defensive burying animal model of anxiety. A final objective is to establish the neurocircuitry basis for the behavioral effects of these agents using inducible transcription factor (i.e., Fos, Zif-268, Arc)neural mapping. It is expected that these studies will advance understanding of novel, potentially highly promising treatment targets for relapse prevention;and of the neuroscience of cocaine-seeking behavior and addiction, in general.

The present proposal is designed to extend previous work in this laboratory aimed at identifying neuronal circuitries and neurochemical substrates involved in ethanol reward and dependence. It is the purpose of this project to use the intracranial microdialysis technique to monitor the synaptic concentration of several forebrain transmitters previously implicated in ethanol seeking behavior and reward, and to establish a "profile" of basal and ethanol-induced neurotransmitter activity for both unselected Wistar and alcohol-preferring (P-1) rats. Transmitter release will be monitored before and during oral ethanol self-administration in a two-lever free-choice (water/ethanol 10%w/v) condition, using dependent rats undergoing ethanol withdrawal. Additionally, high and low responders for ethanol intake. Dialysate will be collected from the nucleus accumbens, caudate nucleus and ventral pallidum, and will be assayed for dopamine (DA), 5-hydroxytryptamine (5- HT) or gamma-aminobutyric acid (GABA). These data will serve to identify differences in basal DA, 5-HT and GABA levels between two strains of rats differing in their preference for ethanol, establish the neurochemical effects of self-administered ethanol on the release of these transmitters, and will help determine their role in ethanol reward and dependence. Finally, the effects of ethanol on neurotransmitter activity will be compared to those of other drugs of abuse including cocaine and heroin, These studies have important implications with regard to the understanding of ethanol actions on central neurotransmitter systems and the identification of neural substrates for ethanol reward. Such issues are of critical importance for the development of effective strategies for the prevention and treatment of ethanol abuse and dependence.

This application was prepared in response to RFA AA-94-06 and is designed to address the need for "more basic research using animal models on the cellular and molecular mechanisms by which alcohol acts". The principal objective will be to identify neurochemical substrates of alcohol-seeking behavior as determined by genetic and environmental variables, and thereby, to establish a "rational basis" for subsequent treatment drug selection and testing. Intracranial microdialysis combined with operant behavioral tests will be employed to study the significance of normal or abnormal function of limbic dopamine, serotonin and opioid peptide release in ethanol preference, reinforcement, and ethanol-seeking behaviors. Specific Aim 1 seeks to elucidate the neurochemical basis of genetically- determined initial ethanol preference by correlating voluntary intake with parameters of basal function and the ethanol-stimulated response profile of forebrain dopamine, serotonin, and opioid peptide release, and by contrasting results in two alcohol preferring lines of rats to that of genetically heterogeneous Wistar rats. These studies will be complemented by examination of changes in neurotransmitter function linked to preference that are induced by daily ethanol administration, as well as functional adaptations that contribute to the "switch" from the non- dependent to the dependent state. Specific Aim 2 will identify neurochemical bases of ethanol-maintained reinforcement by establishing the extent to which the neurochemical effects of ethanol in the context of voluntary self-administration differ from its direct pharmacological actions, whether a common neurochemical basis exists for the reinforcing effects of ethanol across lines of alcohol preferring rats, and whether differences in neurotransmitter responsivity to self-administered alcohol can account for differences in voluntary ethanol intake between alcohol preferring and heterogeneous Wistar rats. Specific Aim 3 will identify neurotransmitter substrates of conditioned motivational processes presumed to be involved in ethanol-seeking behavior of nondependent and postdependent animals, and to determine the neurochemical basis of differences in ethanol-seeking behavior between alcohol preferring and Wistar rats. The goal here is to identify the behavioral and neurochemical significance of environmental stimuli predictive of alcohol reinforcement and to determine whether effects of stimuli associated with the positive reinforcing actions of ethanol differ from those paired with ethanol availability during withdrawal. The neurochemical information provided by these studies can then be employed for the selection as well as the behavioral and neurochemical evaluation of promising treatment drugs. The results of this work are expected to advance the understanding of the neurochemical basis of ethanol preference as well as motivational and reinforcement processes that play important roles in sustained ethanol abuse. More importantly, this information will have direct implications for the development of effective pharmacotherapies for the treatment and prevention of ethanol abuse.

DESCRIPTION: This proposal request funds to develop an ultrasensitive mass spectrometric procedure for the analysis of neurosteroids in tissue samples and microdialysates of select brain areas of freelymoving rats after acute and chronic administration of ethanol, and during alcohol withdrawal. The work in this proposal will first focus on extending the gas chromatographic-mass spectrometric mode of negative chemical ionization of polyfluorinated derivatives to the analysis of the neurosteroids allopregnenolone, pregnenolone and dehydroepiandrosterone in small milligram quantities of tissue from select areas of the brains of control and alcohol treated rats. These neurosteroids have been demonstrated to be synthesized de novo in the rat brain from cholesterol, such that their overall levels in brain are different from, and can be independent of circulating levels of these steroids. Subsequently, these neurosteroids have been implicated in the physiological, emotional, and behavioral responses to stress. Moreover, the anxiolytic and anticonvulsant effects of both ethanol and these neurosteroids have been hypothesized to be mediated in part by elective interactions within the complex family of gamma-aminobutyric acid GABA receptors in the brain. These receptors control the flux of chloride ions in neural membranes, where GABA acts as the primary inhibitory neurotransmitter in the mammalian brain. Neurosteroids and ethanol both augment GABAergic neurotransmission, and because of their common neuropharmacologic actions, it is postulated that neurosteroids influence the development of alcohol abuse and alter the effects of alcohol withdrawal. The results of this research will provide important insights about the role of neurosteroids in alcohol abuse and in the behavioral and neurochemical effects of alcohol withdrawal.

DESCRIPTION: (Adapted from the Investigator's Abstract) This proposal seeks to continue to investigate the neurochemical basis of ethanol-seeking behavior, with a shift in emphasis toward the investigation of the neurobiological basis of relapse. Alcoholism is a chronic relapsing disorder and vulnerability to relapse following withdrawal presents a great challenge for the treatment of alcohol addiction. The clinical literature suggests that one of the primary factors underlying high rates of relapse in detoxified alcoholics are conditioned responses elicited by ethanol-related environmental stimuli that lead to craving or the compulsion to consume ethanol. Yet, experimental studies of cue-induced alcohol-seeking behavior and its neurobiological basis are rare. Moreover, pertinent information is restricted to nondependent rats and, thus, provides only a limited heuristic basis for the understanding of the stimulus control of ethanol-seeking behavior and its neurobiological substrates in human alcoholics. The objective of this proposal is to investigate experimentally the role of ethanol-associated environmental stimuli in the initiation of ethanol-seeking behavior after abstinence, and to identify neuroanatomical, neurochemical, and neuropharmacological substrates for these conditioned effects of ethanol cues. Specific Aim 1 will utilize an operant response-reinstatement model of relapse developed during the previous funding period to establish the time course and resistance to extinction to the behavioral effects of ethanol-associated environmental stimuli, and to compare their effects in nondependent rats to those in rats with a history of dependence. Specific Aim 2 will focus on the identification of neurobiological substrates of cue-induced reinstatement of ethanol-seeking behavior by examining the effects of ethanol cues on neural activation within brain reward regions using Fos immunohistochemistry and by characterizing the effects of ethanol cues on extracellular dopamine (DA) levels within brain reward regions as well as on the release of the stress-regulatory neuropeptide corticotrophin-releasing factor (CRF) with the central nucleus of the amygdala. Experiments in Specific Aim 3 will examine whether the response-reinstating actions of ethanol cues are sensitive to pharmacological manipulation, and to further isolate critical brain regions by combining tests of the effects of ligands for opioid, dopamine, and serotonin receptors on ethanol-seeking behavior with measurements of alterations in cue-induced Fos expression by these agents. By increasing understanding of the neurobiological basis of relapse, these studies are expected to have direct implications for the development of pharmacotherapeutic treatments for the treatment and prevention of compulsive ethanol-seeking behavior and relapse.

This proposal seeks to extend the focus of studies conducted during the previous funding period which were concerned with neural mechanisms of cocaine reinforcement and acute cocaine withdrawal, to an investigation of the neurobiological basis of protracted cocaine withdrawal and relapse. The proposed studies will employ a multidisciplinary research strategy to identify enduring post-cocaine changes at the neurochemical, neuroendocrine, and molecular level and to relate these perturbations to changes in the vulnerability to relapse as measured by the reinstatement of extinguished cocaine-seeking behavior elicited by cocaine-associated stimuli, and footshock stress. The overall hyposthesis is that a predictive relationship exists between the severity of neurobiologic changes and the susceptibility to relapse in one or both of these behavioral models. The proposed studies will focus on forebrain dopamine and serotonin transmission, stress systems including extrahypothalamic corticotropin-releasing factor (CRF) function and pituitary-adrenocortical hormones, as well as on intracellular signal transduction systems including the MAP- kinase pathway and other signal transduction intermediates. The overall experimental plan is to first identify abnormalities in the targeted neurobiological systems throughout a 4 month protracted withdrawal phase in rats with a history of cocaine self-administration that mimics human cocaine binge abuse (Specific Aim 1). The behavioral significance of these disturbances will then be established in Specific Aim 2 by examining whether these changes, or their remission over the course of protracted abstinence, are paralleled by changes in susceptibility to the response-reinstating actions of cocaine cues and stess. Specific Aim 2 will also seek to identify specific neurobiological systems that mediate the effects of cocaine cues and stress, and whether functional abnormalities in these systems observed in Specific Aim 1 alter their response cocaine cues and stress. The role in relapse of neurobiological systems identified in Specific Aims 1 and 2 will then be verified in Specific Aim 3 by testing whether appropriate pharmacological manipulations can inhibit cocaine-seeking behavior induced by cocaine cues and stress. By increasing understanding of the neurobiological basis of protracted abstinence and relapse, these studies will have direct implications for the development of pharmacotherapeutic strategies for treatment of cocaine dependence and prevention of relapse.

DESCRIPTION (provided by applicant): Neuroadaptive changes induced by chronic ethanol have been implicated in symptoms of protracted withdrawal, and relapse risk during abstinence. Studies during the previous funding by this lab and other groups at the TSRI Alcohol Research Center suggest that dysregulation of extrahypothalamic corticotropin-releasing factor (CRF) systems that mediate behavioral and emotional responses to stress may be a neuroadaptive consequence of chronic ethanol use that contributes importantly to affective changes and ethanol-seeking behavior during acute and protracted withdrawal. The present proposal is designed to extend these studies to a systematic multidisciplinary investigation of functional abnormalities in extra hypothalamic CRF systems at different stages of ethanol withdrawal, and to establish the behavioral significance of these changes. The experimental plan is to first characterize the nature and time course of ethanol-related behaviors and their modification by stress over a 12-week withdrawal period. Subsequent studies will determine whether behavioral changes identified in these experiments coincide with specific abnormalities in pre or postsynaptic CRF function in the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST). Finally, the relevance of functional abnormalities in these CRF systems for behavioral changes that accompany protracted ethanol withdrawal will be confirmed using site-specific administration of CRF-R1 and CRF-R2 receptor antagonists and manipulation of CRF receptor expression by targeted viral vector approaches. The experiments will be guided by the hypothesis that ethanol self-administration, ethanol-seeking behavior, anxiety-like behavior, as well as the augmentation of these behaviors by stress will be greater in rats with a history of dependence than in nondependent rats. Moreover, it is expected (a) that behavioral differences between non-dependent and post-dependent rats will be related to differences in CRF function within the CeA and/or BNST, and (b) that some predictive relationship exists between time-dependent changes in ethanol-related behaviors over course of protracted withdrawal and specific alterations in CRF function in the CeA and/or BNST. A final prediction is that the exacerbation of anxiety, ethanol-seeking and intake in postdependent rats is sensitive to modification by manipulation of CRF neurotransmission in the CeA or BNST. By increasing understanding of the neurobiological basis of acute and protracted withdrawal, these studies are expected to have direct implications for the development of pharmacotherapeutic treatment strategies for the prevention of ethanol-seeking behavior and relapse at different stages of withdrawal and abstinence.

DESCRIPTION (provided by applicant): Previous data generated by the applicants suggest that chronic exposure to drugs of abuse leads to long-lasting perurbations in the functioning of peptidergic stress-regulatory systems in the brain. Evidence in this regard is strongest in the case of extrahypothalamic corticotrophin-releasing factor (CRF) systems. More recently, a second neuropeptide system implicated in stress-regulatory functions, the nociceptin/orphanin FQ (N/OFQ) system, was shown to be stronlgy dysregulated following chronic exposure to drugs of abuse. The pathological status of these peptide systems following chronic drug use may play a critical role in abnormal responsiveness to stress and long-lasting vulnerability to relapse associated with exposure to stressful events. The proposed studies will, therefore, test the hypothesis that chronic, high-dose cocaine exposure leads to abnormally enhanced sensitivity to stress, that this state persists over prolonged periods of abstinence and represents a major factor for vulnerability to relapse. This hypothesis is directly linked to the expectation that these behavioral changes are the result of disruptions produced by chronic cocaine in the functioning of extrahypothalamic CRF and brain N/OFQ peptide systems that can be traced to the presynaptic, postsynaptic, or gene expression level within specific brain sites. To test these hypotheses, alterations in stress sensitivity will be measured at three post-cocaine intervals over a 12-week period at the behavioral and neurobiological level. SPECIFIC AIM 1 will examine whether rats with a history of escalated cocaine self-administration show enhanced sensitivity to acute stress compared to rats with only limited-access self-administration experience or cocaine-naive rats using several tests of altered stress-sensitivity. These include direct measures of susceptibility to relapse induced by acute stress and the exacerbation by stress of reinstatement produced by cocaine-related environmental stimuli, as well as measures of "allostatic load" reflected by stress-induced exacerbation of anxiety-like behavior in two behavioral models of anxiety, the elevated plus maze and defensive burying test. SPECIFIC AIMS 2 and 4 (focusing respectively on extrahypothalamic CRF and the N/OFQ system) will then determine whether relationships exist between behavioral hypersensitivity to stress challenges and specific abnormalities in the functioning of the CRF and N/OFQ stress-regulatory systems. Experiments in SPECIFIC AIMS 3 and 5 will employ site-specific microinjection of pharmacological agents to confirm that specific abnormalities in CRF and N/OFQ functioning are responsible for exacerbated stress responses and vulnerability to relapse identified in Specific Aim 1. In addition to providing essential neurobiological information on the relationship between drug-induced alterations of stress-regulatory systems and their relevance for future addictive behavior, these studies are expected to have important implications for treatment drug development aimed at ameliorating chronic cocaine-induced dysregulation of brain stress systems and reducing relapse risk associated with exposure to stressful events.

DESCRIPTION (provided by applicant): This application seeks to continue an established research program concerned with understanding the compulsive nature of ethanol-seeking behavior at the neuropharmacological and neurochemical level. Efforts during the previous funding period were dedicated to developing animal models of relapse and implementation of these models for investigations of the neuropharmacological and neurochemical mechanisms responsible for the resumption of ethanol seeking following abstinence. In this work, several promising leads emerged on novel neural systems that participate in the regulation of ethanol-seeking and relapse. Among these is modulation of glutamatergic neurotransmission by the Group II family of metabotropic glutamate receptors (mGluRs). These findings, along with an emerging literature implicating Group II mGluRs in several neurobehavioral effects of ethanol, strongly suggest that mGluR-mediated neural events participate in the control of ethanol-seeking elicited by exposure to drug-related environmental stimuli and stress. The purpose of this proposal is to test this hypothesis at the behavioral, neurocircuitry, and neurochemical level. The research plan is to first establish, via pharmacological manipulations, the significance of Group II mGluRs in ethanol-seeking associated with alcohol cue exposure and stress using operant response-reinstatement models of relapse (SPECIFIC AIM 1). These studies will be followed by investigation of the role of Group II mGluRs in susceptibility to relapse in ethanol-dependent rats. This question will be approached from two perspectives. The first will focus on the consequences of a history of repeated ethanol withdrawal on Group II mGluR receptor function and the regulation of ethanol-seeking by these receptors (SPECIFIC AIM 2). The second approach will be to examine whether Group II mGluRs play a specific role in enhanced susceptibility relapse that develops as a result of experience with ethanol during withdrawal - an event that introduces learning about a previously latent incentive dimension of ethanol's rewarding potential, namely its capacity to alleviate physically or emotionally aversive states (SPECIFIC AIM 3). These studies of the behavioral pharmacology of Group II mGluRs as it relates to ethanol-seeking and relapse will be complemented by experiments designed to elucidate the neurocircuitry details and neurochemical mechanisms by which Group II mGluR-regulated glutamate signaling attenuates cue and stress-induced reinstatement of ethanol-seeking behavior (SPECIFIC AIM 4). The proposed research will generate original information on a novel neural mechanism relevant for understanding the neurobiological basis of long-lasting vulnerability to relapse in alcoholics. As well, the results will provide information directly relevant for evaluating the potential of Group II mGluRs to serve as treatment drug targets for relapse prevention.

[unreadable] DESCRIPTION (provided by applicant): Alcohol addiction is a chronically relapsing disorder characterized by compulsive drug-seeking and use. A major factor contributing to this profile of behavior is the process of associative learning whereby environmental stimuli paired with alcohol consumption acquire incentive-motivational value. Little is known about the processes leading to the development of the compulsive-like nature of ethanol-seeking. The objective of this proposal is to elucidate these processes with emphasis on identifying neural substrates responsible for the distinctly compulsive nature of alcohol-seeking as opposed to those mediating behavior motivated by natural reward essential for survival, well being and "healthy" hedonic pursuits. To accomplish this, a novel behavioral model will be employed based on the observation that alcohol-seeking elicited by ethanol (EtOH) -associated contextual cues (conditioned reinstatement) shows remarkable persistence whereas the motivating effects of stimuli conditioned to highly potent natural reward (PNR) decay rapidly. This differential persistence of conditioned reinstatement will serve as the central model to dissociate "compulsive- like" from "normal" seeking behavior. Based on preliminary data, these studies will target several primary neural systems: The hypothalamic orexin/hypocretin (Orx/Hcrt) and CART (cocaine and amphetamine-related transcript) systems, as well as Group II metabotropic glutamate receptors. A second objective will be to identify novel neural systems that regulate compulsive ethanol-seeking. The research plan employs three converging approaches for detecting and verifying a role of specific brain sites and neural systems in the differential persistence of reward-seeking that characterizes reinstatement induced by EtOH-associated contextual cues vs. stimuli conditioned to PNR: Initially, (in SPECIFIC AIM I), a systematic neural mapping approach will be employed to differentiate brain sites activated by an EtOH cue from those responsive to a PNR cue, using inducible transcription factors (ITF) as markers. This strategy will be complemented in SPECIFIC AIM II by directly targeting the signaling systems about which specific hypotheses have been formulated based on preliminary data. Specifically, levels of Orx/Hcrt and CART immunoreactivity as well as mGlu2 and mGlu3 receptor expression will be determined to test whether a differential role of these signaling systems in seeking behavior induced by the EtOH vs. PNR cue can be traced to differential alterations within distinct brain sites. Additionally, both in the case of Orx/Hcrt and CART, and novel systems be identified, dual-labeling with one of the ITF markers showing activation will provide direct information on the phenotype of neurons showing activation in response to the EtOH vs. PNR cues. A role of signaling systems within specific brain sites implicated in the differential persistence of EtOH vs. PNR cue-induced reinstatement by results of Specific Aims I and II, then will be verified by site-specific pharmacological manipulations in SPECIFIC AIM III. The research plan has been developed with the objective of advancing understanding the biological basis of alcohol dependence, as well as of the neuroscience of motivated behavior, in general. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Alcohol addiction is a chronically relapsing disorder characterized by compulsive drug-seeking and use. The objective of this proposal is to identify neuroanatomical and neuropharmacological substrates responsible for the distinctly compulsive nature of alcohol-seeking as opposed to those mediating behavior motivated by natural reward essential for survival, well being and "healthy" hedonic pursuits. This research is expected to (a) advance understanding the biological basis of alcohol dependence, (b) reveal novel treatment targets for alcohol abuse and alcoholism, and (c) advance basic science understanding of normal and pathological goal- directed behavior, in general [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The conditioning of ethanol's (EtOH) reinforcing effects with environmental stimuli is a major factor in the abuse potential of this drug. EtOH-related stimuli elicit strong EtOH seeking in animal models of relapse and these models are widely employed to study the neurobiological basis of EtOH craving and relapse. However, behavioral and neurobiological information on conditioning factors in EtOH seeking from animal models is limited largely to that from EtOH nondependent subjects. In alcoholics, a significant positive correlation exists between the history of dependence and the severity of cue-induced EtOH craving. EtOH consumption during withdrawal modifies subjects' EtOH reinforcement history to include learning about amelioration or avoidance of adverse withdrawal states as a novel and essential aspect of EtOH's reinforcing actions, rendering the drug a qualitatively different, more potent reinforcer. Thus, understanding the control of behavior by stimuli conditioned to EtOH under conditions that encompass the reinforcing dimension of this drug that emerges with the experience of withdrawal states will be essential for advancing the understanding and treatment EtOH addiction. Preliminary data that stimuli conditioned to EtOH reinforcement during withdrawal elicit significant reinstatement confirm the need for this understanding. The purpose of this exploratory and developmental project is to investigate the significance of withdrawal-related conditioning in EtOH seeking and to gain understanding of the neurocircuitry regulating this behavior implementing novel methodology. Behavioral hypotheses to be tested are (a) that withdrawal-related conditioning overrides the control of EtOH seeking by cues conditioned to EtOH earlier in the nondependent state, (b) that, compared to cues conditioned to EtOH in the nondependent state, stimuli conditioned to EtOH reinforcement during withdrawal produce greater EtOH seeking following a stress challenge, and (c) greater resistance to conditioned suppression of EtOH seeking. Parallel studies will establish neural activation patterns associated with EtOH seeking in rats with and without histories of withdrawal-related learning using quantitative Fos immunohistochemistry. These studies are guided by the hypothesis that cues conditioned to EtOH during withdrawal produce a different pattern of neural activation, with a stronger engagement of stress-regulatory regions, compared to cues conditioned to EtOH in the nondependent state. Finally, to confirm a role of brain regions in EtOH seeking linked to withdrawal-related conditioning, Daun 02 pharmacogenetic inactivation in cfos-lacZ transgenic rats will be employed to selectively lesion neuronal ensembles in these sites. These developmental studies will test the hypothesis that neuronal ensembles can be identified within brain reward and stress circuitry that preferentially mediate the effects of stimuli conditioned to EtOH reinforcement during withdrawal. This research will provide the groundwork for future in-depth investigations of the significance of withdrawal-related conditioning in the abuse potential of EtOH to advance understanding of the neurobiology and of treatment strategies for alcoholism.

DESCRIPTION (provided by applicant): A major challenge for the successful treatment of alcoholism is long-lasting susceptibility to relapse. Several processes have been implicated in the compulsion to resume drinking during abstinence. These include drinking urges produced by ethanol (EtOH)-related environmental cues or contexts, EtOH-induced neuroadaptation resulting in anxiety and hypersensitivity to stress, as well as cognitive deficits associated with EtOH-induced neurodegeneration that can lead to impaired impulse control. Thus, considering that various risk factors exist that elicit vulnerability states in alcoholics, approaches to treatent drug discovery aimed at providing protection for multiple precipitating factors are likely to be more effective than approaches targeting only a single factor. An agent with an emerging profile of actions relevant for multiple relapse vulnerability states is cannabidiol (CBD), the main non-psychoactive and non-addictive component of the cannabis sativa plant. A factor limiting CBD's therapeutic potential in man has been the drug's low oral bioavailability paired with lack of a readily available and suitable drug delivery method. However, evidence has become available that the transdermal route of administration provides an effective delivery method for CBD. Therefore, preclinical evaluation of the profile of actions of transdermal CBD (tCBD) is timely and will close a major gap in knowledge on CBD's clinical potential. Preliminary studies confirmed that tCBD ameliorates several vulnerability states associated with relapse risk as measured by attenuation of cue- and stress-induced reinstatement of EtOH seeking, anxiety-like behavior, and reversal of impulsive behavior following EtOH intoxication. Of particular significance was the finding that the reduction of EtOH seeking remained unabated at the end of a nearly five-month post-treatment test period. This observation, paired with the attenuation of EtOH-induced impulsivity, is of substantial interest from both a medication development and neurobiological perspective in that it is suggestive of neuroregulatory actions of CBD that restore normal function to circuitries regulating reward, incentive motivation, impulsivity, stress and anxiety. The purpose of this project is to confirm the hypothesis that tCBD has therapeutic potential for multiple vulnerability states associated with relapse risk. This will be accomplished using rats with a history of EtOH dependence, a status essential for providing translational relevance, as follows: By establishing the short- and long-term profile of tCBD actions (1) on compulsive EtOH seeking and relapse, (2) on post-withdrawal manifestations of negative affect as measured by anxiety-like behavior and sensitivity to stress challenges, and (3) on impaired impulse control produced by EtOH intoxication. A parallel objective is to identify neuropharmacological systems mediating the diverse behavioral effects of tCBD and to examine whether tCBD has neuroprotective or proneurogenic actions relevant for the prevention or reversal of impaired impulse control. The results are likely to have significant implications fo treatment drug development and understanding of the neural basis of relapse.

? DESCRIPTION (provided by applicant): This exploratory/developmental project is designed is to establish novel methodology as a research tool in the PI's laboratory to explore the neurobiological basis of intriguing preliminary findings that the phytocannabinoid cannabidiol (CBD) attenuates cocaine seeking with effects that significantly outlast treatment. A major factor contributing to the compulsive and chronically relapsing nature of cocaine addiction is drug desire elicited by environmental stimuli that have become conditioned to cocaine's subjective effects. In animals, the efficacy of these stimuli to elicit cocaine seeking perseverates over long periods of abstinence despite frequent exposure under non-reinforced conditions, reflective of the compulsive nature of cocaine addiction. In preliminary studies, CBD (the main non-psychoactive and non-addictive component of the cannabis sativa plant) significantly attenuated reinstatement in an animal model of perseverating, compulsive- like cocaine seeking, with effects that were still unabated six weeks after treatment termination. These findings suggest that CBD reverses neuroplasticity underlying cocaine craving and relapse beyond mere transient pharmacological amelioration of vulnerability to relapse. Insight into the mechanisms underlying these effects may therefore have major implications for treatment drug development and understanding of the neural and molecular basis of compulsive cocaine seeking. The objective of this proposal is to explore the neurobiological basis of CBD's lasting anti-reinstatement actions by exploiting recent advances in fluorescence-activated cell sorting (FACS) and associated methodologies. These advances, spearheaded by the Co-I, Dr. Hope, permit rapid high-throughput regionally specific identification (neural mapping) of Fos-expressing neurons that encode specific behaviors while at the same time providing RNA in a rapid and quantitative manner to permit characterization of molecular alterations in behaviorally activated neurons from single rats. The research plan is to extend the exploration of CBD's long-lasting effects on responsiveness to cocaine cues and ensuing cocaine seeking at the behavioral level, and to establish FACS and associated methodologies in the lab to identify brain sites and gene expression linked to the lasting attenuation of cocaine seeking by CBD. The results are expected to provide essential insight into neural and molecular targets through which CBD exerts its actions and to lay the foundations for subsequent full-scale projects ranging from systematic investigation of causal roles of identified neural targets and gene expression changes in mediating CBD's interference with cocaine seeking, to utilization of FACS for the identification of key genes and their epigenetic regulatory mechanisms responsible for the persistence of CBD's actions, and thereby to reveal novel therapeutic targets for relapse prevention.

? DESCRIPTION (provided by applicant): This exploratory/developmental project is designed is to establish novel methodology as a research tool in the PI's laboratory to explore the neurobiological basis of intriguing preliminary findings that brief treatment with the phytocannabinoid cannabidiol significantly reduces ethanol (EtOH) seeking with effects that outlast treatment by several months. A major factor contributing to the compulsive and chronically relapsing nature of alcoholism is EtOH desire elicited by environmental stimuli that have become conditioned to the drug's subjective effects. Indeed, the efficacy of these stimuli to elicit EtOH seeking perseverates over long periods of abstinence despite frequent exposure under non-reinforced conditions, reflective of the compulsive nature of alcoholism. In preliminary studies, cannabidiol (CBD), the main non-psychoactive and non-addictive component of the cannabis sativa plant significantly attenuated reinstatement in an animal model of compulsive-like perseverating EtOH seeking, with effects that were still unabated five months after treatment termination. These findings suggest that CBD reverses neuroplasticity linked to maladaptive learning underlying EtOH craving and relapse, beyond mere transient pharmacological amelioration of vulnerability to relapse. Insight into the mechanisms underlying these effects may therefore have major implications for treatment drug development and understanding of the neural and molecular basis of compulsive EtOH seeking. The objective of this proposal is to explore the neurobiological basis of CBD's lasting anti- reinstatement actions by exploiting recent advances in fluorescence-activated cell sorting (FACS) and associated methodologies. These advances, spearheaded by the Co-I, Dr. Hope, permit rapid high-throughput regionally specific identification (neural mapping) of Fos-expressing neurons that encode specific behaviors while at the same time providing RNA in a rapid and quantitative manner to permit characterization of molecular alterations in behaviorally activated neurons from single rats. The research plan is to extend the exploration of CBD's long-lasting effects on responsiveness to EtOH cues and ensuing EtOH seeking at the behavioral level, and to establish FACS and associated methodologies in the lab to identify brain sites and gene expression linked to the lasting attenuation of EtOH seeking by CBD. The results are expected to provide essential insight into neural and molecular targets through which CBD exerts its actions and to lay the foundations for subsequent full-scale projects ranging from systematic investigation of causal roles of identified neural targets and gene expression changes in mediating CBD's interference with EtOH seeking, to utilization of FACS for the identification of key genes and their epigenetic regulatory mechanisms responsible for the persistence of CBD's actions, and thereby to reveal novel therapeutic targets for relapse prevention.

The conditioning of ethanol?s (EtOH) reinforcing effects with environmental stimuli is a major factor in the abuse potential of this drug. EtOH-related stimuli elicit strong EtOH seeking in animal models of relapse and these models are widely employed to study the neurobiological basis of EtOH craving and relapse. However, behavioral and neurobiological information on conditioning factors in EtOH seeking derived from animal models is limited to that from EtOH nondependent animals or, in animals with a dependence history, use of stimuli conditioned to the reinforcing effects of EtOH before dependence induction without consideration of conditioning factors related specifically to EtOH consumption in the dependent state. In alcoholics, a significant positive correlation exists between the history and degree of dependence and the severity of drinking urges induced by alcohol-related environmental stimuli. One process to explain this observation is that repeated consumption of EtOH during withdrawal states allows for learning of the negative contingency between EtOH consumption and adverse withdrawal symptoms, modifying an individual?s reinforcement history to include learning about amelioration/avoidance of adverse states as a novel and essential aspect of EtOH?s reinforcing actions. This proposal is designed to address the implications of this process with regard to EtOH craving and relapse, with the major hypothesis that a consequence of withdrawal-related learning (WDL) the conditioned effects of EtOH- associated environmental stimuli come to exert more powerful control over EtOH-directed behavior than stimuli conditioned to the positive reinforcing effects alone, and thereby play a dominant role in eliciting and maintaining compulsive EtOH seeking. In support of this hypothesis, preliminary data that provide the basis for this proposal show that stimuli conditioned to EtOH availability during withdrawal (WDL) elicit significant reinstatement in a manner that is punishment- and effort- resistant, whereas EtOH seeking induced by stimuli conditioned to EtOH in the nondependent state in rats without a withdrawal-related learning (N-WDL) history is not. The purpose of the proposed project is to solidify and extend the preliminary findings across three Specific Aims: (1) to establish the significance of environmental conditioning to withdrawal relief by EtOH (WDL) in initiating and maintaining compulsive EtOH seeking, (2) to identify neuronal ensembles mediating compulsive ethanol seeking linked to WDL and to confirm their role in this behavior via activity-dependent pharmacogenetic neural inactivation, and (3) to establish the neurochemical/phenotypic profile of behaviorally critical neuronal ensembles in WDL- vs. N- WDL-motivated EtOH seeking utilizing RNAscope®. Successful completion of this project is expected to establish WDL as a major previously not well recognized factor in relapse vulnerability and provide the necessary foundations for future studies to uncover the learning and neuroadaptive and mechanisms responsible for the control of EtOH seeking by stimuli linked to WDL.