Patrick J. Mulholland, Ph.D. - US grants

DESCRIPTION (provided by applicant): Mechanisms underlying ethanol withdrawal-induced neurotoxicity and means to reverse this damage are not completely understood. Research suggests that glucocorticoids and polyamines may contribute to ethanol-associated neurodegeneration. Thus, the broad, long-term objective of this proposal is to establish more precisely the mechanisms involving glucocorticoid-induced exacerbation of ethanol withdrawal (EWD)-mediated damage using organotypic hippocampal slice cultures. To this end, cultures will be exposed chronically to ethanol and withdrawn in the presence of corticosterone. Toxicity will then be assessed using the non-vital fluorescent dye propidium iodide. Some studies suggest that EWDinduced damage may be partially mediated via activation of the polyamine-sensitive portion of NMDA receptors. In addition, corticosterone administration may also influence expression of this polyaminesensitive subunit. These hypotheses will be tested using autoradiographic imaging of spermidine's potentiation of [125l]MK-801 binding. The secondary yet therapeutically relevant objective is to examine the efficacy of novel NMDA polyamine-site and glucocorticoid receptor antagonists against corticosterone's potentiation of EWD-induced toxicity. Regardless of the outcomes of the proposed studies, they may provide considerable insight into some of the mechanisms of EWD-mediated hippocampal degeneration, as well as identify potential therapeutic targets for the treatment of alcohol-associated neurodegeneration.

[unreadable] DESCRIPTION (provided by applicant): Voltage-dependent potassium channels (Kv) dynamically regulate intrinsic neuronal excitability. Increasing evidence suggests that Kv2.1 channels play an especially prominent role in homeostatic plasticity and in modulating excitability and calcium entry during repetitive firing. Thus, the broad, long-term objective of this proposal is to characterize the role that Kv2.1 channels play in controlling hyperexcitability associated with alcohol withdrawal. These studies will employ a well-characterized organotypic hippocampal slice culture model of alcohol withdrawal and techniques including confocal imaging, immunohistochemistry, electrophysiology, and biochemical assays. The specific aims are to: [1] determine if blocking or knocking-down Kv2.1 channels modulate the frequency of action potential firing during ethanol withdrawal, and [2] to test the hypothesis that changes in Kv2.1 modulation of excitability will be reflected as changes in membrane localization and phosphorylation of the channel. Findings from these studies may implicate a novel therapeutic target for possible drug design to treat the severity of alcohol withdrawal and, potentially, relapse. [unreadable] [unreadable] [unreadable]

Recent evidence suggests that ethanol-associated homeostatic plasticity involves compensatory increases in synaptic NMDA receptors that contributes to aberrant hyperexcitability upon cessation of consumption and may underlie craving that leads to the high incidence of relapse in alcohol dependent individuals. Small-conductance calcium-activated potassium (SK) channels regulate NMDA receptor-dependent calcium influx and are critical modulators of hippocampal-dependent synaptic plasticity. This is consistent with the suggestion that SK2 channels and NMDA receptors form a regulatory calcium-mediated feedback loop within individual dendritic spines. Preliminary evidence demonstrates a reduction in surface SK2 channels following chronic ethanol treatment that leads to a disruption of the SK channel-NMDA receptor feedback loop. Moreover, we have demonstrated that modulation of SK channels can influence voluntary drinking behavior. Thus, the overarching hypothesis is that SK2 channels contribute to alcohol-associated plasticity of glutamatergic synapses and that positive modulation of SK channels reduces the severity of withdrawal-related hyperexcitability and decreases alcohol intake. These studies will test the hypotheses that: 1) chronic ethanol exposure produces a homeostatic reduction in SK2 channel expression through PKA signaling, 2) modulation of the SK channel-NMDA receptor feedback loop can reduce ethanol withdrawal , hyperexcitability and neurotoxicity, and 3) modulation of the synaptic feedback loop will reduce voluntary alcohol consumption. Decreases in SK2 channels and increases in NMDA receptors may represent a common homeostatic adaptive response to prolonged reductions in NMDA receptor activity during ethanol exposure. Furthermore, this functional uncoupling of the SK2 channel-NMDA receptor calcium-mediated feedback loop may contribute to tolerance development and to withdrawal hyperexcitability.

DESCRIPTION (provided by applicant): Heavy alcohol drinking and repeated withdrawals are associated with increased relapse and allostatic adaptations in the hypothalamic-pituitary-adrenal (HPA) axis. Excessive alcohol intake is also associated with perturbations in cortico-limbic-HPA function that may contribute to alcohol dependence and high rates of relapse. Our preliminary evidence suggests that a critical modulator of high rates of voluntary drinking in alcohol-dependent mice is the small-conductance calcium-activated potassium (SK) channels. SK channels in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) regulate NMDA receptor-dependent calcium influx, intrinsic excitability, and basal firing rates. Results from our preliminary studies demonstrate that SK channel expression is significantly reduced in mPFC and NAc in C57BL/6J mice following chronic intermittent ethanol (CIE) exposure or prolonged stress. Moreover, microinjection studies show that blocking SK channel activity in NAc enhances voluntary consumption in control, but not alcohol-dependent mice. These data suggest that the down-regulation of SK channels observed following CIE is critically involved in the escalation of drinking in CIE exposed mice. Thus, the overarching hypothesis of this proposal is that CIE increases the excitation at glutamatergic synapses through a combination of increased NMDA receptors and a decrease in SK channel activity in key brain regions that control drinking. These studies will test the hypotheses that: 1) chronic ethanol exposure and stress alter glutamatergic synapses, 2) divergent drinking patterns in genetically modified mice are linked to alterations in SK channel expression, and 3) SK channels in mPFC and NAc regulate escalation of drinking in CIE exposed mice. We expect that data collected from these studies will advance our understanding of synaptic plasticity in key brain regions involved in alcohol seeking behaviors and will validate the hypothesis that SK channels are an important new therapeutic target for the treatment of alcohol dependence.

DESCRIPTION (provided by applicant): Alcohol use disorders (AUDs) are a major public health issue and have an enormous societal and economic impact. Current FDA-approved pharmacotherapies for treating AUDs suffer from deleterious side effects and are only effective in a subset of individuals. This signifies an essential need for improved medications. Emerging evidence suggests that anticonvulsants are a promising class of drugs for treating individuals with AUDs. Our preliminary data demonstrates that the anticonvulsant retigabine significantly reduces drinking in two rodent models of voluntary alcohol consumption. Retigabine is a KCNQ (Kv7) voltage-dependent K+ channel positive modulator that is approved by the FDA for treating partial onset seizures. In central neurons, Kv7 channels display activation at voltages close to the resting membrane potential and are the molecular composition of the M-current (IM) in brain. IM activation is important for repolarizing the cell, fine-tuning the resting membrane potential, and controlling action potential generation and frequency. Previous evidence has demonstrated that acute alcohol exposure inhibits IM in ventral tegmental area (VTA) dopamine and CA1 pyramidal neurons. In Drosophila, Kv7 channels have been implicated in acute alcohol-induced memory impairments and tolerance to the sedative effects of acute alcohol exposure. Recent evidence has also demonstrated a role for Kv7 channels in synaptic plasticity and cognition. Chronic alcohol exposure is known to engage neural mechanisms associated with synaptic plasticity. However, it is unknown if chronic alcohol consumption affects Kv7 channel expression or function. Preliminary evidence suggests that prolonged alcohol consumption alters surface trafficking of Kv7.2 channels in the nucleus accumbens (NAc). Interestingly, Kv7.2 channel protein and transcript levels in the NAc negatively correlated with voluntary alcohol intake. Bioinformatics analysis also demonstrated that genes that encode Kv7 channels are included in the support interval for replicated QTLs for alcohol consumption in mice. Thus, our preliminary data have identified Kv7 channels as promising molecular targets that can influence voluntary alcohol consumption. In addition, these results have demonstrated that prolonged alcohol consumption alters Kv7 channel expression. Three specific aims were designed to test the overarching hypothesis of this proposal that Kv7 channels are critical regulators of voluntary alcohol drinking. We have proposed a multifaceted approach using biochemistry, electrophysiology, pharmacology, and mouse transgenic models to determine if: A) positive modulation of Kv7 channels in the NAc, dorsomedial striatum (DMS), or VTA can reduce drinking (Aim 1), B) altered Kv7 channel function in mutant and transgenic mice can influence alcohol consumption (Aim 2), and C) prolonged drinking alters Kv7 channel function and expression in NAc, DMS, and VTA (Aim 3). These studies will advance our knowledge on alcohol-associated neuroadaptations and will in turn help us to understand, at an increasingly sophisticated level, the role of Kv7 channels in alcohol drinking.

? DESCRIPTION (provided by applicant): Alcohol use disorders are a major public health issue and emerging evidence suggests that high-risk drinking during adolescence has long-term consequences on behavior and brain development. The brain undergoes profound structural and functional adaptations throughout adolescence, and some critical brain regions even continue to mature into early adulthood. Over the last few years, studies published from the NADIA Consortium and other laboratories showed that adolescent intermittent alcohol (AIE) exposure produces profound behavioral, cognitive, electrophysiological, and neuroanatomical impairments that persist in adult rodents. The underlying neuroadaptations that contribute to the persistent consequences of high-risk adolescent drinking remain largely unknown. Three components of the NADIA Consortium found that AIE exposure alters dendritic spine density and morphology in the adult amygdala (Pandey component), prefrontal cortex (Chandler component), and hippocampus (Swartzwelder component). An analysis of the morphological characteristics of spines revealed that AIE exposure selectively increased the prevalence of `immature' long, thin dendritic spines in the adult prefrontal cortex and hippocampus. Previous findings suggest that maturation of asymmetric synapses during the natural process of developmental pruning involves replacing immature synapses associated with long, thin spines with mature synapses associated with mushroom-shaped dendritic spines. Thus, AIE exposure appears to impair the normal maturation of synaptic pruning in the adult brain. Because dendritic spine morphology influences synaptic physiology and behavior, aberrant structural plasticity of neurons in the adult brain is a likely neural mechanism underlying deficits in cognition and behavior associated with AIE exposure. The convergence of these findings prompted the formation of a NADIA Dendritic Spine Core that will integrate dendritic spines changes in multiple brain regions induced by AIE exposure. The overarching hypothesis of this NADIA Dendritic Spine Core is that AIE exposure locks-in an adolescent morphological phenotype in the adult brain that diverges from the normal pruning process. The purpose of this Core is to provide a detailed analysis of dendritic spine density and spine morphology in brain regions relevant to the behavioral, electrophysiological, and epigenetic studies of the NADIA Consortium. The Dendritic Spine Core will provide analysis of dendritic spine changes in a primary and secondary brain region for each NADIA component. This Core will also characterize developmental changes in dendritic spine density and morphology in brain regions related to the NADIA Consortium components. The data provided to the NADIA Consortium by this Core on the impact of AIE exposure on dendritic spine adaptations and the developmental trajectory of spine morphology will influence the neuroscience field and inform public health.

PROJECT SUMMARY Alcohol use disorder is a major public health issue associated with altered activation of brain stress systems, cognitive deficits, and escalated alcohol drinking. The prefrontal cortex (PFC) is a key structure involved in executive cognitive function and imposing inhibitory control over reward-motivated behaviors. Altered stress responsivity is implicated in the development and maintenance of excessive alcohol drinking, and both chronic ethanol and stress negatively impact PFC function. Cognitive deficits in individuals with alcohol use disorder are thought to hinder successful treatment and contribute to increased risk for relapse. Preclinical studies show that chronic ethanol exposure produces an exaggerated stress response in the PFC that mediates cognitive impairments and escalated ethanol drinking. In the current funding period, we identified chronic ethanol- sensitive proteins and K+ channel genes in the PFC and nucleus accumbens (NAc) of mice and monkeys. We also identified predictive relationships between candidate K+ channel genes and drinking in ethanol-dependent BXD recombinant inbred strains of mice, and pharmacological validation showed that positive modulation of a KCa2 channels significantly reduces escalated drinking in stressed, dependent mice. Moreover, we demonstrated that chronic stress elevates drinking only in ethanol dependent mice and enhances the magnitude of the early component of long-term potentiation (LTP) in the mouse PFC. In addition to the enhanced LTP we observed in the stressed mice, our preliminary data shows that glutamatergic signaling in the PFC is enhanced in macaques following chronic ethanol self-administration. While it is known that chronic ethanol and stress exposure elicit maladaptive plasticity in the PFC, the underlying neural mechanisms and the adaptations in the PFC circuitry induced by ethanol-stress interactions remain poorly understood. To address this gap in our knowledge, we propose three specific aims that will test the overarching hypothesis that disruption of PFC circuitry and plasticity underlies the excessive drinking and cognitive impairments produced by chronic stress and ethanol dependence. Studies in Aim 1 will test the hypothesis that chronic ethanol self- administration and interactions between ethanol dependence and stress produce functional adaptations in the PFC of monkeys and mice. Aim 2 will test the efficacy of drugable proteogenetic targets to reduce escalated drinking in stressed dependent male and female mice. Finally, studies in Aim 3 will test the hypothesis that ethanol dependence and chronic stress produce aberrant signaling in PFC circuitry that contributes to escalated drinking and cognitive impairments. In addition to identifying drugable targets, the findings from these studies will provide data on the temporal aspects of circuit-specific, functional, and morphological adaptations produced by chronic stress and ethanol in the mouse and monkey PFC.

ABSTRACT Confocal microscopy has allowed a large number of biomedical researchers in the Departments of Neuroscience and Psychiatry & Behavioral Sciences at the Medical University of South Carolina (MUSC) to elucidate morphological and protein neuroadaptations in rodent models of neuropsychiatric diseases. A number of these investigators are recognized leaders in the field of addiction neurosciences, and a major focus of the faculty in these departments is to understand human disease using innovative approaches. The faculty identified in this application are currently using a 15-year-old and technologically obsolete confocal microscope. There are recent advances in confocal imaging technology that provide substantial improvements in speed, sensitivity, and resolution over the current capabilities of the existing confocal microscope. Thus, the overall goal of this application is to upgrade the obsolete confocal and create a Shared Confocal Facility equipped with a new versatile and high-resolution confocal microscope system that will serve the faculty in the Departments of Neuroscience, Psychiatry & Behavioral Sciences, and other departments at MUSC. We propose to replace the older system with a Zeiss LSM 880 that has an Airyscan detector capable of superresolution imaging. Unlike our current system, the versatile Zeiss LSM 880 has an open interface and modular architecture that will allow reconfiguration of the system as our future needs change to meet the rigorous demands of biomedical research. Moreover, our preliminary data show that images acquired with a superresolution detector are far superior in quality and resolution, and quantitation of superresolution images revealed an increase in the density of fine dendritic protrusions (i.e., dendritic spines) compared with the same images acquired with a GaAsP detector. These qualitative and quantitative findings strongly demonstrate the necessity of this state-of-the-art technology to allow the investigators to examine cellular structures and relationships of proteins with enhanced detail. There is substantial institutional support provided by the Departments of Neuroscience and Psychiatry & Behavioral Sciences and Office of the Provost at MUSC to cover the instrument for its lifetime and to expand image analysis capabilities in an effort to facilitate superresolution technology on MUSC's campus. In addition to supporting individual R01 awards, two NIH-funded P50 Center grants, and two NIH-funded T32 Training grants at MUSC, the superresolution confocal will provide data to two national consortia (Integrative Neuroscience Initiative on Alcoholism and Neurobiology of Adolescent Drinking in Adulthood). Thus, this upgrade will immediately influence the current research programs at MUSC and across the US, and will provide users with equipment that can grow and change in parallel with cutting-edge neuroscience research. A technological upgrade of this magnitude will be critical in furthering the long-range biomedical research goals at the Medical University of South Carolina.

7. SUMMARY/ABSTRACT Excessive alcohol (ethanol) consumption is a hallmark characteristic of individuals with alcohol use disorder (AUD) and a risk factor for developing ethanol dependence. Currently, there is a substantial gap in our understanding of the neural mechanisms and circuits that drive initiation of excessive drinking. Gaining insight into the neurobiological factors that facilitate the transition from moderate to excessive ethanol intake may lead to the development of new treatment strategies for reducing relapse rates. The prefrontal cortex (PFC) is a crucial neural substrate for executive cognitive function and appetitive responding, and its ability to impose inhibitory control over reward-motivated behaviors is disrupted following excessive drinking. While the heterogeneous architecture and function of principal PFC neurons has limited the understanding of drinking-induced adaptations in behaving animals, there are newly developed and powerful tools that allow for genetic access to unique subpopulations of neurons that drive behaviors. The Targeted Recombination in Active Populations (TRAP) mouse line (FosTRAP) is one such technology that allows for identification, measurement, and manipulation of neural ensembles activated in response to ethanol drinking behavior. Using this novel technology, our preliminary results show that intermittent access to ethanol activated (or `TRAPed') subpopulations of neurons in subregions of the PFC, including the infralimbic (IL), orbitofrontal, insular, and anterior cingulate cortices. Importantly, the TRAPed pyramidal neurons in the IL-PFC of ethanol drinking mice fired more evoked action potentials in comparison with adjacent non-activated neurons, suggesting that enhanced intrinsic excitability in activated IL-PFC neurons is a functional signature of ethanol consumption. Thus, the overarching hypothesis of the present proposal is that TRAPed neurons that are activated by initial ethanol drinking display functional plasticity and control future excessive drinking. To test this hypothesis, studies in Aim 1 will use electrophysiological, immunofluorescent, and single-cell calcium imaging approaches in ethanol-drinking FosTRAP double transgenic mice. In Aim 2, we will combine FosTRAP technology with chemogenetics to test the hypothesis that neurons activated by initial drinking drive subsequent excessive consumption of ethanol. With the emergence of novel techniques, we can now study the function of a subpopulation of cortical neurons and control their activity during development of excessive drinking in the behaving mouse. The findings from these studies using a combination of newly developed technology will identify stable and specific subsets of neural populations that are activated by the initiation of ethanol consumption that drive subsequent drinking behaviors. Collectively, the proposed research will characterize the functional adaptations in PFC engrams that contribute to excessive ethanol intake.

7. SUMMARY/ABSTRACT Excessive alcohol (ethanol) consumption is a hallmark characteristic of individuals with alcohol use disorder (AUD) and a risk factor for developing alcohol dependence. Mood and anxiety disorders that are often comorbid with AUD can hinder psychosocial treatment interventions and increase the risk of relapse. While current FDA approved medications are not effective in the general population, they also do not target comorbid conditions. This represents a considerable gap in our understanding of the neural mechanisms driving excessive drinking and its comorbid neuropsychiatric disorders. Gaining insight into the neurobiological factors that facilitate excessive ethanol intake and negative affective disturbances may lead to the development of new treatment strategies for reducing relapse rates. In the previous funding period, our studies demonstrated that KV7 channels are a target for reducing alcohol drinking, especially in rodents with a high-drinking phenotype. There is emerging evidence implicating KV7 channels as a mediator of negative affective behaviors in humans and rodents. In agreement with these results, our preliminary data provide additional evidence for KV7 channel regulation of behaviors related to negative affective states. Because of the overlapping role for KV7 channels in regulating intrinsic excitability, alcohol intake, and negative affective behaviors, the long-term goal of our studies is to understand circuit- and cell-specific adaptations in KV7 channels that are caused by and drive excessive alcohol drinking and affective disturbances. Our overarching hypothesis of this grant is that down-regulation of KV7 channels drives plasticity of intrinsic excitability, excessive alcohol drinking, and maladaptive behaviors that contribute to the maintenance of alcohol use disorder. To test this hypothesis, studies in Aims 1 and 2 will use emerging technology, electrophysiological, and immunofluorescent approaches to characterize KV7 channel- dependent adaptations in specific circuits and subpopulations of prefrontal cortex, nucleus accumbens, and ventral tegmental area projection neurons during development and maintenance of and abstinence from excessive alcohol intake in mice. In addition, we will determine the ability of the KV7 channel activator retigabine to reverse these adaptations. These studies will explore morphological adaptations in KV7 channels located in the axon initial segment produced by excessive alcohol intake. Studies in Aim 3 are designed to determine the role that adaptations in KV7 channels contribute to the development of negative affective disturbances during abstinence from excessive alcohol drinking. The proposed research will characterize cell- and circuit-specific adaptations in projection neurons that contribute to excessive ethanol intake and negative affective behaviors. Collectively, the findings from these preclinical studies will provide evidence that KV7 channels in specific neural circuits are a target for reducing alcohol consumption and symptoms of neuropsychiatric conditions that are comorbid with AUD.

PROJECT SUMMARY Excessive, uncontrolled alcohol consumption is a hallmark characteristic of individuals with alcohol use disorder (AUD). Chronic alcohol drinking produces neuroadaptations in corticothalamic and corticostriatal circuits that may reduce behavioral flexibility and diminish engagement in behaviors for non-alcohol rewards. While deficits in executive cognitive functioning in individuals with AUD hinder treatment and lead to relapse, the mechanisms and neural circuits driving excessive alcohol drinking and alcohol-biased choice behaviors represent a substantial gap in our understanding of factors that lead to the development and maintenance of AUD. The mediodorsal thalamus (MDT) is a higher-order thalamic nucleus that integrates cortical and subcortical signaling via its reciprocal glutamatergic projections with the prefrontal and orbitofrontal cortex. The MDT is innervated by reward-processing mesolimbic structures and contributes to adaptive goal-directed choice behavior and higher- order cognitive flexibility. However, it is unknown if the MDT is a critical region in addiction-related circuitry involving PFC dysfunction and alcohol-biased choice behaviors. In alcoholics, the pathway between the MDT and medial PFC is degenerated, and our preliminary data demonstrates that chronic intermittent ethanol (CIE) exposure increases intrinsic excitability of MDT projection neurons. Moreover, we provide evidence that voluntary alcohol drinking in FosTRAP2 mice activates cells in the MDT and chemogenetic activation of the MDT reduces alcohol intake. Thus, the overarching hypothesis of this research project in the Charleston Alcohol Research Center (ARC) is that cortical-projecting MDT neurons drive excessive alcohol drinking and alcohol- biased choice behaviors. In Aim 1, we will identify neural ensembles and characterize functional adaptations in MDT neurons that are activated by alcohol drinking and CIE exposure, and project to the infralimbic (IfL) cortex, a region that controls excessive alcohol intake. Studies in Aim 2 will use fiber photometry to test the hypothesis that activity of MDT?IfL neurons will be modulated by alcohol drinking and this pattern of activity will be altered in alcohol-dependent mice. In Aim 3, studies will test the hypothesis that MDT?IfL projecting neurons drive excessive drinking and alcohol-biased choice behaviors in dependent mice. The results from this project using emerging technology and circuit-based approaches will identify specific subsets of neural populations in the MDT that are activated by excessive alcohol drinking in dependent mice. Collectively, the proposed research will identify unique functional signatures and novel neurocircuits that control heavy drinking and contribute to loss of reward-based flexible behaviors. The focus of this new Charleston ARC project on thalamocortical projections and alcohol-biased choice behaviors complements the overarching theme and overlaps extensively on conceptual, technical, and circuitry levels with the other basic science and clinical projects in this Center renewal.