Jacqueline M. Barker, PhD - US grants

DESCRIPTION (provided by applicant): The main objective of this proposal is to combine molecular, pharmacological, and behavioral techniques to investigate the role of corticostriatal dopamine signaling in response strategy selection. Using a rodent model of instrumental habit, we will investigate how behavior is influenced by activity at dopamine D1 and D2 receptors in the dorsolateral striatum and infralimbic cortex, brain regions implicated in stimulus- response habit and goal-directed behavior by infusing receptor agonists and antagonists into these regions. Additionally, we will investigate the role of the kappa opioid system, which is known to interact with alcohol to influence dopamine signaling, in habitual and goal-directed responding for alcohol. We hypothesize based on preliminary data that activity at the D1 and D2 receptors will promote differential response strategies, such that D1 will promote habitual responding and D2 activity will promote goal-directed actions. Additionally, we expect that enhanced kappa opioid receptor activity in the dorsolateral striatum will promote goal-directed behavior by decreasing dopamine signaling in this region, while the converse will be true in the infralimbic cortex: kappa opioid receptor activity here will promote habitual responding by decreasing dopamine activity. We hypothesize that blocking kappa opioid receptor activity in these regions will produce the opposite effects. Finally, we propose that the transition from goal-directed actions to habitual responding will be characterized by changes in kappa opioid receptor activity and expression (as measured by Western blot analyses) in the dorsolateral striatum and infralimbic cortex, and that the time course of these changes will be accelerated in animals receiving alcohol reinforcement as compared to those receiving food reinforcers. The findings of the proposed experiments are expected to help inform clinical research move toward developing efficient and successfully therapies for individuals suffering from alcohol use disorders. PUBLIC HEALTH RELEVANCE: Alcohol use disorders are devastating not only to the individuals struggling with alcoholism, but also to society as a whole. As alcoholics transition from casual drug use to compulsive, habitual drug seeking, they show signs of cognitive-motivational dysfunction, resulting in altered reward processing and decision-making that can have highly maladaptive consequences, including heavy drinking, recidivism, risky reward- motivated behaviors, craving and difficultly in terminating consumption. By understanding how the dopamine system interacts with alcohol to influence habitual drug seeking and taking, we can begin to understand neurobiological mechanisms of behavioral flexibility and identify possible therapies for alcohol use disorders that target the restoration of goal-directed behaviors.

DESCRIPTION (provided by applicant): The development of alcohol use disorders involves a transition from casual drinking, to inflexible habitual drinking. Habitual alcohol seeking is no longer mediated by the reinforcing properties of alcohol but rather by drug-paired cues. Our preliminary data suggest that chronic intermittent exposure to ethanol results in rapid formation of alcohol habits - that is, mice chronically exposed to alcohol become unable to flexibly regulate alcohol seeking behavior. Considerable research has highlighted the role of the PFC in regulating the development and expression of stimulus-response habits. In particular, the infralimbic PFC (IL) has been shown to be required for habit formation. Recent data suggest that mGluR2 expression in corticolimbic circuits may be dysregulated after chronic exposure to alcohol. The current proposal is designed to test the novel hypothesis that decreased expression of mGluR2 on IL projection neurons mediates the rapid transition to stimulus-response habits induced by chronic alcohol exposure. We believe that reductions in IL mGluR2 expression result in enhanced IL glutamatergic signaling in subcortical targets, therefore promoting premature expression of habitual reward seeking. Our preliminary data suggest that systemic enhancement of mGluR2 signaling can restore goal-directed alcohol seeking after the transition to habitual alcohol seeking. Aim 1 is designed to confirm the effects of chronic alcohol exposure on habitual ethanol seeking, and to assess the specificity of these effects to alcohol seeking habits. These data are critical for understanding whether chronic alcohol-induced alterations in corticolimbic circuitry promote a general deficit in flexible behavior, or if these effects are specific to alcohol seeking. Aim 2 is designed characterize the full extent of chronic alcohol induced mGluR2 reduction in IL circuitry using cell tracing and immunohistochemical techniques. In Aim 3 we will employ pharmacological and optogenetic techniques using a novel photoswitch to demonstrate a causal role for IL mGluR2 signaling in the restoration of goal-directed ethanol seeking. Finally, we will test the hypothesis that loss of mGluR2 signaling is required for the CIE-induced acceleration of the development of alcohol- seeking habits through the use of viral overexpression of mGluR2 in the IL PFC. The results of these studies are expected to provide considerable information about the effects of chronic alcohol exposure on behavioral flexibility, and further, the mechanisms through which this loss can be restored. We believe that the knowledge gained from these experiments will provide insight into the mechanisms of the development of alcohol use disorders that will ultimately inform the development of novel prevention and therapeutic strategies.

PROJECT SUMMARY The development of alcohol use disorders involves a transition to inflexible habitual drug-seeking behavior, resulting in difficulty regulating or terminating drinking behavior. Ethanol exposure is thought to facilitate the expression of habitual ethanol seeking, and indeed our own data indicate that chronic intermittent ethanol exposure (CIE) promotes the expression of this behavior. A growing body of literature suggests that glutamatergic signaling within corticostriatal circuits is dysregulated across chronic ethanol exposure. In particular, data suggest that CIE increases extracellular glutamate in the nucleus accumbens shell (NAcS), and that this effect may be mediated by the loss of presynaptic mGluR2. Despite strong evidence that glutamate signaling within these circuits mediates behavioral flexibility, little is known regarding how NAcS glutamate signaling changes across the development of inflexible reward seeking, and further, how alterations in NAcS glutamate signaling can regulate the expression of actions versus habits. This K99/R00 proposal contains a comprehensive training and research plan based on the applicant?s preliminary findings that regulation of glutamate signaling within the NAcS can reverse CIE-induced deficits in goal-directed behavior, directly implicating NAcS glutamate signaling in response strategy selection. During the mentored portion of the award, the applicant will receive training in cutting-edge laboratory techniques including multielectrode array (MEA) recording in awake behaving mice, pharmacogenetics and optogenetics. The candidate will use this training to test the novel hypothesis that alterations in NAcS glutamate signaling resulting from CIE exposure impair behavioral flexibility. Aim 1 is designed to confirm the hypothesis that CIE-induced deficits in goal-directed ethanol seeking can be reversed by mGluR2/3 agonism and to confirm the neuroanatomical locus of this effect. In Aim 2 we will use MEA recordings to test the hypothesis that acquisition of habitual behavior is accompanied by changes in neuronal activity in the NAcS, as well as synchrony between the NAcS and structures that send glutamatergic projections to this brain region, including the infralimbic prefrontal cortex (IfL), the basolateral amygdala (BLA) and the ventral hippocampus (VH). Aim 3 is designed to test the hypothesis that pharmacogenetic and optogenetic manipulations of activity within distinct glutamatergic projections to the NAcS ? from the IfL, BLA and VH - alter the expression of habitual ethanol seeking. The results of these experiments are expected to provide considerable information on the neurobiology of habitual ethanol seeking and to identify mechanisms through which behavioral flexibility can be restored. We expect that the knowledge gained from these studies will provide significant insight into the development of alcohol use disorders that will ultimately inform the generation of novel prevention and treatment strategies. The training that the candidate receives during the mentored portion of this award is expected to facilitate her career development and transition toward becoming an independent neuroscientist.

PROJECT SUMMARY Substance use disorders (SUDs) occur in the HIV-infected population at nearly twice the rate of the uninfected population. Nearly 50% of HIV+ individuals will develop HIV-associated neurocognitive disorders (HAND) characterized by impairments in memory and attention, deficits in executive control over behavior, and reductions in cognitive flexibility. Deficits in cognitive control over behavior may maintain addiction by promoting compulsive drug seeking and poor combined antiretroviral therapy (cART) adherence which further exacerbates disease progression. Despite the potential contribution of HIV infection to compulsive drug seeking, little research has investigated the neural mechanisms underlying this relationship as animal models of progressive HIV infection are limited. Many neurocognitive impairments observed in HAND are likely mediated by impairments in corticostriatal structure and function. Individuals with HIV show structural and functional impairments in the prefrontal cortex (PFC). Animal models of HIV infection exhibit spine loss and reductions in dendritic branching in prefrontal subregions. SUDs are associated with similar changes to the PFC, including cortical atrophy and aberrant dendritic morphology. The dopaminergic system appears to be particularly vulnerable to dysregulation by HIV infection. Alterations in dopamine (DA) signaling in corticostriatal circuits likely contribute to impairments in behavioral and cognitive flexibility. Indeed, PFC DA mediates the ability to flexibly regulate behavior, including compulsive-like reward seeking that persists despite adverse consequences. This proposal will determine whether progressive HIV-infection promotes compulsive-like cocaine seeking and alters corticostriatal regulation of drug seeking. To do this, humanized mice infected with HIV will be trained in a modified conditioned place preference task which allows assessment of reward-related learning and compulsive-like behavior. We will further determine whether HIV infection alters the ability of DA D2/D3 receptor agonists to reduce compulsive- like behaviors. Additionally, we will determine whether HIV interacts with exposure to a reward-paired context to drive changes in corticostriatal activity, as measured by expression of a putative marker of neural activity and of PSA-NCAM, a modified form of a cell adhesion molecule critically involved in DA-induced plasticity. This will enable direct investigation into the relationship between neuronal consequences of HIV infection with changes in reward learning and compulsive cocaine seeking. The development of this novel mouse model of HIV infection- induced changes in neurocognitive function and drug seeking will enable further elucidation of the precise neural circuits and substrates that are altered following HIV, and how these changes contribute to drug seeking. Future research using this model will investigate the impact of cART and existing treatment strategies on neurocognitive function as well as the neural mechanisms by which infection promotes addictive behavior. We expect that the knowledge gained from these studies will contribute to the development of treatment and prevention strategies for HIV-induced neurocognitive impairments.

Substance use disorders are highly comorbid with HIV infection, with nearly 25% of individuals with HIV needing treatment. With the success of combinatorial antiretroviral therapies (cART), HIV-associated mortality has substantially declined. This has resulted in a growing population of chronically HIV-infected and cART treated adults in the United States. Both HIV infection and cART may interact with drug exposure to alter neurobiology, thus creating a behaviorally and biologically distinct condition and requiring novel, targeted pharmacotherapeutic strategies to reduce the compulsive drug use and high relapse propensity that characterize substance use disorders. The corticostriatal glutamate system is a key regulator of these forms of behavioral inflexibility. Projections from the medial prefrontal cortex to the nucleus accumbens are critical mediators of reinstatement of drug seeking, a model of relapse, and the inability to terminate drug seeking despite adverse consequences, a model of compulsivity. A growing literature in both HIV patient populations and preclinical models has identified infection-induced alterations in the corticostriatal glutamate system as a contributor to HIV-associated neurocognitive disorders. Despite this, a robust characterization of corticostriatal glutamate system alterations in HIV infection and cART treatment, with circuit- and cell-type specificity, is lacking. We will capitalize on the advent of new mouse models of HIV infection and perform studies in parallel in (1) a humanized mouse model that successfully develops human-like microglia, thus enabling progressive central nervous system infection with HIV, and (2) wild-type mice infected with a chimeric virus ? EcoHIV. To determine whether current industry standards of treatment impact behavior or glutamate system biology independently or in combination, a subset of mice will receive chronic bictegravir, emtricitabine, and tenofovir alafenamide (B/F/TAF) treatment. This research will use multiplexed in situ hybridization to test the hypothesis that gene and protein expression are altered in glutamate neurons and astrocyte populations in mouse models of HIV infection and cART. We will also test the hypothesis that HIV infection and B/F/TAF interact to regulate astrocyte and neuron calcium signaling during cocaine reward learning, cocaine reinstatement, and compulsive-like cocaine seeking via simultaneous fiber photometric assessment in corticostriatal astrocytes and neurons. To demonstrate a role for gliotransmission and glutamatergic corticostriatal neuron activity in reducing compulsive-like or relapse-related cocaine seeking in mice, we will use cell- and circuit-specific chemogenetic manipulations. Finally, we will use novel and preclinically-validated pharmacological tools for regulating glutamate release and uptake in humanized, HIV-infected and EcoHIV-infected mice to determine whether pharmacological strategies to reduce compulsivity and relapse are similarly effective in infected and B/F/TAF-treated states. My lab will address this unmet need through collaboration with leaders in the immunology, HIV, and drug discovery fields, enabling us to provide thorough, convergent data on corticostriatal glutamate regulation of drug seeking in HIV infection.

PROJECT SUMMARY Historically men have been diagnosed with alcohol use disorders (AUDs) at higher rates than women, but greater risk for adverse health consequences is observed in women who are problem drinkers. In recent years, the gap in diagnosis of AUDs between women and men has been closing. Despite this, minimal research has investigated factors mediating sex differences in relapse-related behavior. Men and women appear to be differentially sensitive to the ability of alcohol-paired cues or stress to drive alcohol craving or relapse-related behaviors, such that men are particularly sensitive to reward-paired cues, while women show escalated stress- induced craving. Animal models of relapse to alcohol seeking indicate that male rodents are similarly susceptible to cue-induced reinstatement, while females show elevations in stress-facilitation of reinstatement. The neural circuits and signaling systems that mediate these sex differences are only beginning to be understood. Nucleus accumbens (NAc) glutamate signaling is a critical regulator of reinstatement to ethanol seeking, and discrete glutamatergic projections play separable roles in stress- and cue-induced reinstatement. Further, pharmacological regulation of glutamate signaling can reduce reinstatement to ethanol seeking. Despite strong evidence that glutamate signaling within the NAc is critical for the regulation of reinstatement in males, sex differences in engagement of glutamatergic projections to the NAc or in expression of glutamate receptors and transporters following chronic alcohol exposure has not been extensively investigated. This R21 proposal will test the overarching hypothesis that sex differences in accumbens glutamate circuit function result in differential propensity toward stress- and cue-induced reinstatement. Specifically, we will assess innate and ethanol dependence-driven sex differences in neural function during cue- or stress-induced reinstatement. Aim 1 is designed to test the hypothesis that chronic alcohol exposure promotes reinstatement with sex-specific patterns. We expect that CIE facilitates cue-induced reinstatement in males, but stress-induced reinstatement in females. We will further use photometric assessment of calcium signaling in infralimbic, ventral hippocampus, and basolateral amygdalar glutamatergic projections to the NAc to determine if these circuits are engaged during cue- and stress-induced reinstatement in a sex- and ethanol dependence-driven manner. Aim 2 will investigate sex-specific effects of CIE on the expression of glutamate receptors and transporters in the NAc through the use of immunofluorescence and RNAscope in situ hybridization. The results of these experiments are expected to provide considerable information on the sex differences in neural circuits and environmental factors regulating reinstatement of ethanol seeking. We will also gain significant knowledge of sex differences in the impact of chronic alcohol exposure on glutamate signaling and relapse-related behavior. These findings are expected to enable development of novel drug targets for the reduction of relapse behavior as well as the establishment of targeted treatments strategies for men and women.