Harry L. June, PhD - US grants

DESCRIPTION: (Adapted from the APPLICANTS ABSTRACT) The overall goal of this proposal is to identify and systematically examine benzodiazepine (BDZ) receptor ligands which attenuate the reinforcing properties of ethanol (ETOH). To accomplish this goal, the high alcohol drinking (HAD) rat and measures of ETOH reinforcement will be used. Quantitative receptor autoradiography (QAR) will also be used to determine if the binding affinity of effective BDZ ligands at central nervous system (CNS) sites correlates with the magnitude of behavioral effects. The main hypothesis to be tested is whether certain BDZ inverse agonist and antagonist ligands can selectively attenuate measures of ETOH reinforcement; this may be related to their binding at diazepam sensitive (DS), and to a lesser degree at diazepam insensitive (DI) conformations of GABAA-BDZ receptors. Initial dose-effect and time course studies will examine the ability of agents to attenuate ETOH intake using operant methodology. It is hypothesized that agents with high affinity for DS sites will be effective ETOH antagonists; however, agents with high affinity at both DS and DI sites should produce more potent and prolonged antagonism. In a second series of experiments, the threshold for electrical brain stimulation reward (BSR) will be compared in naive HAD and low alcohol drinking (LAD) rats. The role of oral (contingent) ETOH administration will also be tested for comparison with intra gastric (IG) (noncontingent) infusions in HAD rats. In addition, HAD and LAD rats will be compared for sensitivity to BSR following noncontingent ETOH administration. It is hypothesized that HAD rats will evidence a lower threshold and higher rate of responding for BSR compared with LAD rats under naive and following noncontingent ETOH. Contingent ETOH administration is expected to yield a more positive (euphoric) action on BSR compared with the noncontingent route in HAD rats. Studies of effective anti-ETOH agents using the optimal ETOH BSR threshold route will then be conducted in HAD rats. Using QAR, a third series of experiments will examine both inhibition and time course profiles of agents found effective as ETOH antagonists at CNS sites. It is hypothesized that highly effective ETOH antagonists should evidence greater binding at DS and DI sites hypothesized to mediate ETOH reinforcement, and that interactions at these sites may mediate in part (indirectly influence) activation of underlying neuroanatomical substrates contributing to the reinforcing properties of ETOH. It is further hypothesized that the behavioral and binding time course profiles will not be parallel. These studies should advance our understanding of the role the GABAA-BDZ receptor complex plays in mediating ETOH reinforcement, and may lead to the development of treatments for alcohol abuse and alcoholism.

There is increasing evidence that GABAA-benzodiazepine (BDZ) receptors are involved in the regulation of EtOH-self- administration. The goal of this proposal is to identify specific neurobiological substrates of GABAA-BDZ neurotransmission which mediate EtOH reinforcement. To accomplish this goal, the selectively bred high alcohol drinking (HAD) 1 and 2 rat lines will be used. The main hypothesis to be tested is that GABAA-BDZ neuroanatomical circuits in two extended amygdala loci [e.g., central nucleus of the amygdala, bed nucleus of the stria terminalis], mediate in part, activation of underlying neuroanatomical substrates contributing to the reinforcing properties of EtOH. First, dose-effect and time course studies will examine the capacity of site-specific microinjections of high affinity BDZ inverse agonists, a BDZ antagonist and the GABAA antagonist SR 95531 in the central nucleus of the amygdala and bed nucleus of the stria terminalis to attenuate EtOH intake using scheduled controlled responding (i.e., operant methodology). It is hypothesized that inverse agonists and antagonists with similar binding affinity should be equally effective as EtOH antagonists. Effective antagonists (i.e., inverse agonists) of EtOH-maintained responding should be antagonized by competitive BDZ antagonist, since their suppression should be mediated by a direct action at the BDZ component of the GABAA complex. Second, to systematically evaluate the selectivity of the agents to suppress EtOH-motivated responding, a 4-stage behavioral analysis will be employed. It is hypothesized that agents (e.g., inverse agonists, BDZ antagonist, SR 95531) which specifically affect the reinforcing aspects of EtOH should not alter responding of alternative reinforcers with similar reinforcing efficacy (i.e., saccharin), or similar post-ingestional caloric properties (i.e., sucrose). Finally, it is hypothesized that interactions at GABAA-BDZ sites may modulate the function of monoaminergic neurons sustaining EtOH-seeking behaviors. These studies should contribute to a scientific understanding of the role of GABAA-BDZ receptor complex plays in regulating EtOH seeking behavior.

DESCRIPTION (provided by applicant): The goal of this proposal is to evaluate the role of the alphal and alpha5 GABAA receptor subunits in EtOH reinforcement. To accomplish this, the high alcohol drinking (HAD) rat lines, the alphal knock out (KO) mice and their wild type counter part (WT) will be used. The first hypothesis to be tested is whether postsynaptic alpha1 receptors in the ventral pallidum (VP) of HAD rats contribute to the reinforcing properties of EtOH. Site-specific microinjection of selective alphal antagonists in the VP will be evaluated for their capacity to attenuate EtOH-maintained responding. It is hypothesized that the alphal antagonist will selectively decrease EtOH responding since the alphal subunit is present in very high levels throughout the VP. The second hypothesis will evaluate the degree to which complete deletion of the alphal subtype modulates acquisition of alcohol-seeking behaviors using the KO and WT mice. It is hypothesized that the KO mice will initiate alcohol-maintained responding because alcohol reward has been shown to be regulated by multiple neurotransmitter systems; however, the drinking in the KO mice is predicted to be significantly lower than the WT. Moreover, we hypothesize that the magnitude of reduction with alphal antagonists in the KOs will be less than that seen in the WT because the KOs will be devoid of a functional alphal subunit. The third specific aim will test the hypothesis that enhanced alphal binding selectivity, longer lived in vivo and more water soluble ligands will result in a more optimal alcohol antagonist. Finally, the fourth hypothesis will assess whether alpha5 receptors in the hippocampus (CA1 and CA3) modulate putative GABAergic EtOH reward substrates (e.g., nucleus accumbens, basal amygdala, bed nucleus of the stria terminalis). To accomplish this, microinjection of selective alpha5 inverse agonists in the hippocampus to attenuate EtOH-maintained responding will be evaluated. We hypothesized that the alpha5 ligands will selectively decrease EtOH responding in the hippocampus since the alpha5 containing receptors are primarily localized in the hippocampus. In contrast, the selective alpha5 ligands infused in the VP will not alter EtOH responding, since this locus is completely devoid of alpha5 subunits, and the ligands have a very low affinity for the alphal receptor subtype. These studies should further our understanding of the GABAA receptor mechanisms in EtOH-seeking behavior and may possibly identify agents which may have potential in reducing alcohol drinking in humans.

[unreadable] DESCRIPTION (provided by applicant): It is well established that the rewarding properties of ethanol [EtOH] are mediated in part by GABAA-receptor mechanisms; however, only recently has research demonstrated that the a1 receptor subunit, particularly those within the ventral pallidum [VP], may be the critical GABAergic receptor regulating EtOH reinforcement. The primary objective of this proposal will be to further evaluate the role of the GABAA a1-containing receptor subunit in regulating EtOH-seeking behaviors by employing a combination of neuropsychopharmacological and molecular biology techniques. To accomplish this goal, the selectively-bred high alcohol-drinking [HAD-1] rats will be used. The primary hypothesis to be tested is that a selective inhibition of the GABAA a1 receptor containing subunit within the VP of HAD-1 rats will lead to selective reductions in EtOH-motivated behavior, with little or no effect on sucrose-motivated behaviors. The inhibition of the GABAA a1 subunit will be accomplished by constructing a herpes simplex virus [HSV] vector, which will utilize the siRNA sequence specific, posttranscriptional gene silencing mechanism. This vector will then be infused directly into the VP of HAD-1 rats via bilateral guide cannulae. It is hypothesized that a selective reduction in EtOH-maintained responding will be observed following infusion of the vector-mediated siRNA amplicon into the VP. Specifically, reinforcer and neuroanatomical specificity will be expected, as neither suppression on sucrose maintained responding following infusion of the active virus in the VP, nor suppression on alcohol responding infusion of the active virus into the neuroanatomical control locus [e.g., caudate putamen] will be expected. In subsequent studies, a combination of in situ reverse transcriptase-PCR, immunohistochemistry, and Western analyses [immunoblotting] will be used to determine the success of the HSV-siRNA viral vector manipulations. These studies should extend our understanding of the role of the GABAA a1 receptor subtype in the regulation of alcohol-drinking behaviors. [unreadable] [unreadable] [unreadable]

Project Abstract Alcoholism and anxiety frequently co-occur in humans;however, it has been difficult to find a single treatment which is effective against both conditions. Substantial evidence suggests that the motivational aspects of alcohol withdrawal [e.g., increased anxiety and anhedonia] referred to as negative affective states play an important role in the maintenance of excessive alcohol drinking, and may also be associated with relapse. Evidence also suggests a salient role for GABAergic mechanisms in regulating excessive alcohol drinking and the negative affective states associated with abstinence. The initial objective of the present proposal is to identify novel [unreadable]1 GABAA subtype-preferring ligands at the preclinical level that may serve as prototypes for further evaluation of clinical efficacy in treating both excessive alcohol drinking and the negative affective states associated with abstinence. To accomplish this, Aim 1A will employ our established pharmacophore/receptor model of BDZ binding sites to synthesize novel [unreadable]1 subtype-preferring ligands with reduced efficacies at diazepam sensitive [DS] subtypes [e.g., [unreadable]1-3]. Once the three agents [e.g., [unreadable]CCt, 3-PBC, WYS8] have been synthesized, Aim 1B will test the hypothesis that their chronic oral administration for 30 consecutive days can effectively attenuate excessive binge alcohol drinking in high alcohol drinking [HAD] rats. Aim 1C will test the hypothesis that a similar treatment will attenuate the negative affective states associated with abstinence. We hypothesize that chronic BDZ treatments will attenuate both binge dependence drinking and the negative affective states associated with abstinence. The second objective will be to identify select GABAA receptor subunits which may play a role in the regulation of excessive alcohol drinking and the negative affective states associated with abstinence. Aim 2A will test the hypothesis that inhibition of the [unreadable]1 receptor subunits within the ventral pallidum [VP] will lead to selective time-dependent reductions in binge alcohol responding, while Aim 2B will evaluate the hypothesis that their inhibition within the lateral/basolateral amygdala [BLA] will lead to time-dependent reductions in negative affective states. To downregulate the [unreadable]1 subunit, a novel siRNA sequence will be delivered into the VP and BLA by bilateral microinfusion using a herpes simplex virus-1 [HSV-1] amplicon vector. These studies should identify novel pharmacotherapies for further evaluation of clinical efficacy in treating comorbid alcoholism and anxiety at the preclinical level. In addition, they should shed light on the salient neuronal mechanisms in the regulation of the comorbid condition, which could be important in ultimately leading to a successful treatment for the comorbid condition.

Project Summary/Abstract Alcoholism and depression often co-occur in humans, but it has been difficult to find a single treatment which is effective against both conditions. This comorbid condition is frequently observed following impulsive binge alcohol consumption, as well as in compulsive drinking in humans. The primary objective of the present proposal is to identify effective compounds at the preclinical level that may serve as prototypes for further evaluation of clinical efficacy in treating the comorbid condition. To accomplish this, a series of triple monoamine uptake inhibitors [TUIs] [e.g., DOV 216,303, DOV 21,947, and DOV 102,677], which have been successfully tested in Phase I studies for depression with published preclinical-antidepressant [AD] efficacy, will be evaluated. The first aim will test the hypothesis that orally-administered TUIs can effectively attenuate excessive alcohol drinking in the binge and prolonged repeated alcohol deprivation [PRAD] models using the alcohol-preferring [P] rat. Initial studies will employ DOV 102,677 [our lead compound], recently shown to reduce limited alcohol responding for six days after a single administration. It is hypothesized that acute treatment for binge drinking, and chronic treatment for PRAD drinking, will selectively reduce intake in both models. The second aim will test the hypothesis that TUIs will effectively attenuate the negative affective states [e.g., withdrawal symptomatology], characterized by reductions in pleasure [i.e., anhedonia] and increased immobility [i.e., indicative of depressive-like behaviors] following alcohol-induced abstinence from binge and PRAD drinking. Negative affective states will be inferred using the intracranial self-stimulation [ICSS] and forced swim test [FST] models. We hypothesize that both acute and chronic DOV treatments will attenuate the negative affective states associated with alcohol-induced abstinence from the two heavy drinking models. Aim 3 will test the hypothesis that similar neurobiological substrates mediate alcohol dependence and the negative affective states associated with binge drinking within the extended amygdala [EA] [i.e., bed nucleus of the stria terminalis (BST); central nucleus of the amygdala (CeA); shell of the nucleus accumbens (nAcc)]; and medial prefrontal cortex (mPfc). To evaluate this hypothesis, site-specific microinjection of our lead TUI [DOV 102, 677] will be given in the EA loci and mPfc. However, because little if any data are available on the precise brain substrates which regulate the actions of TUIs, we will initially employ the c-fos technology in na¿ve P rats to delineate multiple CNS loci which may mediate the actions of DOV 102, 677. These studies should identify effective compounds at the preclinical level which may serve as prototypes for further evaluation of clinical efficacy in treating comorbid alcoholism and depression, as well as shed light on the neurobiological commonalities which regulate the two conditions.

DESCRIPTION (provided by applicant): Excessive alcohol drinking is an enormous public health burden in need of more radical therapy. Most health problems associated with excessive drinking are due to two types of drinking: (i) binge (BALs > 0.08 grams % in a 2-hour period) and (ii) relapse (sustained heavy drinking for at least 2 days following a single or multiple abstinence periods). Previously, we showed that alcohol- preferring (P) rats express elevated levels of the GABAA subunits for alpha1, alpha2 and toll-like receptor 4 (TLR4) innate immunity receptors, and using specific siRNA vectors infused into the central amygdala (CeA), demonstrated that regulation of binge drinking at this site is mediated by GABAA a2 regulated TLR4 (a2/TLR4 axis). While the alpha1 subunit was also shown to regulate binge drinking, it was associated with the ventral pallidum (VP) and was independent of TLR4 (Liu et al., PNAS, 2011). In the current grant, we propose to better elucidate the mechanism responsible for the TLR4 effect, focusing on the chemokine monocyte chemotactic protein-1 (MCP-1) and the dopamine rate- limiting enzyme tyrosine hydroxylase (TH)--implicated by data obtained after the application was last submitted--and on brain sites that regulate different domains of the alcohol addiction cycle. The working hypothesis is that neuronal TLR4 induces MCP-1 expression via activation of cell type- specific transcription factors and it, in turn, functions as a neurotransmitter to stimulate dopamine release and excitability (inferred by TH) in select reward loci. The specific aims are: Aim I. Define the expression of GABAA a2/a1, TLR4, MCP-1 and/or TH at alcohol reward loci from P vs NP rats. Aim II. Define the role played by the a2/TLR4 axis that encompasses MCP-1 and/TH at alcohol reward loci in impulsive binge drinking. Aim III. Define the contribution of a1, a2, TLR4 and its downstream targets (MCP-1 and/or TH) at alcohol reward loci in compulsive relapse drinking. Aim IV. Define the mechanism of TLR4-mediated regulation of binge drinking by focusing on downstream signals that upregulate MCP-1 and TH expression, through the use of the pHSVsiMCP-1 amplicon. Better understanding of the role of chemokines in neurotransmission and the regulation of distinct (viz. dopaminergic) neurons will help broaden current concepts of neuroimmune communication and their roles in excessive drinking. This highly innovative proposal will create a paradigm shift in understanding the relationship between innate immunity signals and neuronal responses that impact alcohol addiction. Use of non-toxic herpes simplex virus (HSV)- siRNA constructs to inhibit relevant genes at specific brain loci will define therapeutic gene targets and test the potential of gene therapy approaches for binge and relapse drinking.