John Woodward, PhD - US grants

Recent evidence has suggested that membrane ion channels which regulate the flux of important monovalent and divalent ions across the cell membrane may be particularly sensitive to the actions of ethanol and mediate the development of functional tolerance and potent pharmacological actions of ethanol an mediate the development of functional tolerance and physical dependence following chronic exposure. Recent studies have show that there are multiple types (L,N,T) of calcium channels in brain which exhibit different sensitivities to blockade by organic calcium channel blockers. An unequal distribution of multiple calcium channel subtypes throughout the brain may account for brain regional differences in calcium channel blockade observed with ethanol. the specific calcium channel subtype(s) which are blocked by ethanol is not know and attempts to characterize these effects are complicated by the heterogeneous nature of brain tissue. The experiments described in this study are designed to investigate the effects of ethanol on different types of voltage-sensitive calcium channels which can be expressed in cultured rat adrenal medullary chromaffin cells and PC- 12 cells. These cells are good models for the study of calcium-mediated transmitter release processes and appear to possess only the L-type of calcium channel. However, treatment of these cells in culture with nerve growth factor (NGF) results in the expression of a second type of channel which resembles the N-type observed in nerves. Studies will be conducted to characterize the sensitivity of these different channel types to the inhibitory actions of ethanol and will be correlated with changes in the density of calcium channels following NGF treatment. Measurement of fast- phase calcium entry(45Ca 2+), intracellular free calcium (Fura-2 spectroscopy) and endogenous catecholamine release (high performance liquid chromatography) will be made from NGF treated and untreated cells. Changes in calcium channel density following NGF treatment will be measured using radioligand binding techniques. The effects of acute and chronic ethanol exposure on the binding parameters of these cell types will be investigated in order to correlate changes in calcium channel density with functional changes in ion flux, regulation of intracellular free calcium and catecholamine release following NGF treatment. These experiments are designed to study the action of ethanol on multiple types of voltage- sensitive calcium channels which can be expressed in cell culture and should yield valuable insights regarding the molecular mechanisms underlying the complex actions of ethanol on neuronal function.

biomedical equipment purchase;

Ethanol causes widespread and varied actions on the central nervous system that are manifested in alterations in normal neurochemical processes and behavior. The cellular and molecular sites of action of ethanol in the CNS have been the focus of intense research over the past 10 years. Many different neuronal processes have been shown to be sensitive to acute and chronic exposure to ethanol. Of these, the large family of neurotransmitter-gated ion channels has been shown to particularly sensitive to ethanol. It can be hypothesized that many of the actions of ethanol on CNS function and behavior are due to selective effects of ethanol on these ion channel proteins. Research in my laboratory and that of others has shown that a subtype of glutamate receptor, the N-methyl-D-aspartate receptor, is inhibited by ethanol at concentrations at are behaviorally relevant During the course of the current funding period, my laboratory has focused on determining the molecular determinants that confer ethanol sensitivity upon these receptors. Using both native and recombinant NMDA receptors expressed in brain neurons and heterologous expression systems, we have shown that the ethanol inhibition of NMDA receptors is modulate both by subunit composition and intracellular processes that interact with the C-termini of NMDA receptor subunits. Our most recent data show that there are calcium-sensitive and insensitive components to the ethanol sensitivity of NMDA receptors. The calcium-sensitive form involves the C-terminus of the NR1 subunit which possesses distinct binding sites for protein phosphorylation and protein-protein interactions. These processes regulate the activity of the NMDA receptor and appear to be selectively altered by ethanol. The calcium-insensitive component of ethanol's inhibition of NMDA receptors is hypothesized to be mediated via an interaction of ethanol with key residues that make up the transmembrane domains of the receptor. In this continuation of a currently funded R01, we propose to precisely define the molecular sites and mechanisms of action that underlie the calcium-sensitive and insensitive forms of ethanol inhibition of NMDA receptors. This research will utilize two- electrode voltage clamp, single-cell calcium imaging and whole-cell patch clamp to measure the effects of ethanol on NMDA receptors expressed in oocytes, HEK 293 cells, and cultured neurons. Both molecular and pharmacological approaches will be used to unravel the complex interplay between receptors and intracellular signaling processes that ultimately determine the ethanol sensitivity of NMDA receptors.

Ethanol has widespread and significant effect on brain function. Acute ethanol causes intoxication with resultant loss of behavioral and motor control that can progress to sedation, coma, and death as lethal blood concentrations are reached. The molecular and cellular alterations induced by ethanol that underlie these effects are beginning to be understood and it is now appreciated that several ligand-gated ion channels activated by brain neurotransmitters are important targets for ethanol's actions in the brain. Of particular interest is the inhibition of the NMDA subtype of the glutamate receptor by ethanol. This receptor gates the flux of calcium ions upon stimulation by the co- agonists glutamate and glycine and is subject to multiple forms of regulation. These include a voltage-sensitive magnesium block, a redox site which can shift the receptor from an agonist to an antagonist preferring state, proton inhibition, and phosphorylation. This complex regulation underscores the fundamental importance of the NMDA receptor in neuronal signaling. The inhibition of the NMDA receptor by ethanol may underlie some of the marked behavioral and cognitive effects associated with intoxication. The goals of this study are to continue our investigation into the mechanisms of action of ethanol on the NMDA receptor at both the biochemical and molecular level. We will continue to use the NMDA-stimulated release of neurotransmitters from adult rat brain slices to examine the multiple regulatory sites of the native NMDA receptor. This assay has been well characterized and is sensitive to all known modulators of the NMD receptor and unlike many test systems it can be used with living adult brain tissue. In separate studies, cDNA clones encoding both rodent and human NMDA recptor subtypes will be used to more carefully determine where ethanol may exert its inhibitory effects on the receptor. For these studies, two approaches will be used. First, experimental cell lines lacking any native ligand-gated or voltage-sensitive calcium channels will be transfected with NMDA receptor subunits to allow for the NMDA-stimulated calcium flux to be monitored by digital calcium imaging and patch-clamp electrophysiology. Secondly, messenger RNA synthesized from the cloned receptors will be expressed in Xenopus oocytes and NMDA-activated currents will be monitored using two-electrode voltage clamp. This system will not only allow for reconstitution of functional multi-subunit NMDA receptor complexes but will also permit an examination on a site by site basis for the molecular site of action of ethanol on the receptor using site- directed mutagenesis. Results from these studies should lead to new insights into the effects of ethanol on neuronal function and may suggest novel approaches in the treatment of disorders resulting from acute and chronic ethanol ingestion.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This application is a competitive renewal of a currently funded K02 award and is focused on advancing the PI's ability to investigate the actions of ethanol on brain ion channels. The PI has conducted research into the effects of alcohol of the NMDA receptor and is currently funded by NIAAA through an R01 to continue this work. The PI has utilized the currently funded K02 award period to obtain training in two-electrode voltage-clamp, whole-cell patch-clamp eletrophysiology and molecular biology. He has used these techniques to make important discoveries with regard to the molecular sites of alcohol action of the NMDA receptor. Results from experiments utilizing transfection of oocytes and HEK293 cells with wild-type and mutant NMDA receptors has led to a better understanding of how subunit assembly, intracellular signaling processes and transmembrane domains modulate the sensitivity of this receptor to alcohol. In this application, the PI will extend the relevance of these findings by learning techniques used to express mutant NMDA receptor subunits with altered ethanol sensitivity in brain neurons and to assess specific areas of molecular biology, electrophysiology, and animal behavior. Specifically, the PI will: 1) learn techniquesneeded to generate and use viral-based gene vectors to deliver NMDA receptor subunits with altered ethanol sensitivity into neurons grown in culture and in vivo; 2) learn methods used to generate genetically modified animals that express mutant NMDA receptor subunits with altered ethanol sensitivity; 3) learn methods to prepare and record ion channel currents from brain slices isolated from animals expressing mutant NMDA receptors with altered ethanol sensitivity and; 4) learn techniques needed to assess the neurobehavioral actions of alcohol in animals expressing mutant NMDA receptors with altered ethanol sensitivity. This training will be obtained during a series of sessions supervised by a variety of consultants with demonstrated expertise in the techniques. The successful completion of this training plan will substantially enhance the PI's research expertise and his ability to fulfill the goals of his research.

DESCRIPTION (provided by applicant): The use of volatile organic solvents as drugs of abuse is an important health problem. These agents, often termed abused inhalants, are voluntarily inhaled or "huffed" for their intoxicating effects. The use of abused inhalants is especially prevalent among children and adolescents since these compounds are legal and are found in a wide variety of household and commercial products including glues, adhesives and paint thinners. Abused inhalants are associated with a variety of adverse effects ranging from reduced academic performance, brain abnormalities and a sudden-death syndrome resulting from solvent-induced cardiac arrhythmia. Although abused inhalants produce ethanol-like signs of intoxication, their sites and mechanisms of action that underlie these effects are largely unknown. Research carried out during the previous funding period of this project used recombinant expression techniques and electrophysiology to study the effects of abused inhalants on ion channels widely expressed by brain neurons. Results from these studies revealed a surprising degree of selectivity for the effects of abused inhalants on both voltage-gated and ligand-gated ion channels that regulate neuronal excitability. In particular, the NR1/2B NMDA receptors and the 1422 nicotinic acetylcholine receptor were among the most sensitive channels identified. In this application, we focus on the role that these channels play in mediating the effects of toluene, the prototype abused inhalant, on the activity of principal neurons within three brain regions known to be important in mediating the effects of drugs of abuse. Whole-cell patch-clamp electrophysiology will be used to study the effects of toluene on dopamine neurons of the ventral tegmental area (VTA), medium spiny neurons of the nucleus accumbens (NAcc) and deep-layer pyramidal neurons of the prefrontal cortex (PFC). Aim 1 will determine the toluene sensitivity of NMDA EPSCs within the addiction neurocircuitry with a special focus on NR1/2B receptors. Aim 2 will determine the effect of acute toluene on VTA DA neuron excitability with a special focus on 1422 nAchRs. Aim 3 will use a novel triple-slice co-culture system to determine the effects of toluene on NR1/2B and 1422 nAchRs that mediate complex firing patterns produced by PFC neurons. Finally, Aim 4 will test the hypothesis that exposure to toluene in vivo induces changes in indicators of plasticity in glutamatergic synapses measured in vitro. Results from these studies will fill an important gap in our knowledge and will define the actions of toluene on key elements of the addiction neurocircuitry. PUBLIC HEALTH RELEVANCE Abused inhalants are an important class of drugs of abuse that have received relatively little attention with respect to their mechanisms of action. Research to be carried out in this proposal will provide a detailed analysis of the effects of abused inhalants on neurons within brain areas involved in addiction.

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse are serious illnesses that have life-threatening consequences if left untreated. Research into understanding the neural substrates that underlie the development of uncontrolled drinking is critical for developing treatments effective at reducing drinking and the incidence of relapse. This application is a renewal of an ongoing NIAAA sponsored training program that is focused on providing cutting-edge training in alcohol research for basic scientists and clinical fellows. The current proposal requests funds to provide alcohol-related research training for 2 pre-doctoral students and 4 post-doctoral fellows. The training will take place within an outstanding academic research environment that is devoted to understanding the neurobiological basis of alcohol and drug addiction. The program will be administered within the Center for Drug and Alcohol Programs (CDAP) that serves as an organizing platform to coordinate research activities focused on drug and alcohol addiction. The training mentors and support faculty are actively involved in NIAAA-sponsored research and are associated with the departments of Physiology and Neuroscience and Psychiatry. The areas of alcohol-related research training available encompass both animal and human studies and include 1) identification of molecular and cellular factors that influence the acute sensitivity of neuronal ion channels to alcohol, 2) characterization of neural adaptations in response to chronic alcohol exposure, 3) use of animal models to investigate novel pharmacological and behavioral treatments aimed at reducing alcohol drinking and the symptoms of withdrawal, 4) elucidation of the importance of classical conditioning in human alcohol drinking, and 5) assessing the efficacy of pharmacological interventions to reduce relapse in alcohol-addicted individuals. The overall goal of this training is to provide basic and clinical scientists with the skills necessary to develop into independent investigators focused on solving the problems associated with alcoholism and alcohol abuse.

alcoholism /alcohol abuse; alcoholism /alcohol abuse chemotherapy; fluorescent dye /probe; mixed tissue /cell culture; prefrontal lobe /cortex; voltage /patch clamp

The major theme of our Alcohol Research Center is treatment through research. By enhancing our basic knowledge of alcohol's action on the brain and of individual risk factors for the development of alcohol abuse and dependence, better treatments can be developed. One important way to facilitate this translation approach to alcohol research is to encourage the development of new tools and ideas. Recent advances in molecular biology and genetics, neuroscience, psychology, psychiatry, and related fields offer great promise for better defining key mechanisms and pathways that underlie excessive drinking. In turn, these targets can be used to develop effective and personalized treatment strategies. The major goal of the ARC Pilot Project Component is to identify and recruit individuals to use these unique tools and skills in order to generate novel and interesting data of relevance to alcohol treatment. This goal will be accomplished by carrying out three Specific Aims: 1) Provide a mechanism to recruit and mentor basic science and clinical investigators into the alcohol research field and to promote their ability to generate publications and independent grant funding;2) Increase efforts in promoting and developing translational research approaches in the alcohol research field by identifying critical areas where basic science and clinical practice overlap;and 3) Identify gaps in our knowledge regarding the effects of alcohol on brain and behavior, and apply specific technologies and approaches to solving these problems. Each year, we will invite researchers across the MUSC campus to submit a 5-page proposal describing a research plan that specifically addresses novel alcohol treatments or has implications for treatment. A system is in place to review and select pilot projects for funding. Projects supported by this Component will be rigorously monitored for progress, and mentoring will be offered to junior investigators and to those new to the alcohol field. By actively seeking out pilot projects from MUSC researchers across the diverse set of disciplines within the university, we will not only expand our research capabilities, but will provide training and support to investigators who seek to join us in solving the problems associated with alcoholism and excessive drinking.

Alcoholism is characterized by a loss of control over drinking, suggesting that there are long-lasting changes in higher cortical brain areas that normally control compulsive behaviors. Despite this general understanding, there is little known about the specific actions of alcohol on neurons within these cortical circuits. During the last funding cycle, we addressed this shortcoming and completed a series of electrophysiological studies that examined the effects of ethanol on "persistent" activity in the medial prefrontal cortex (PFC). This activity is characterized by spontaneous and rhythmic transitions between quiescent down-states and depolarized up-states that generate relevant patterns of firing. Persistent activity may allow PFC neurons to integrate and process sensory information derived from internal and external cues and to use this information to control sub-cortical circuits. The results from these pioneering studies showed that prefrontal up-states and associated firing are inhibited by concentrations of ethanol associated with mild to moderate intoxication. They also demonstrated that this effect resulted from inhibition of synaptic NMDA receptors and that PFC AMPA receptors and GABA{A} receptors are largely insensitive to behaviorally relevant concentrations of ethanol. In this application, we extend these studies and will investigate the effects of chronic ethanol on prefrontal cortex function. These studies use a well-established mouse model of chronic intermittent ethanol (CIE) exposure that increases levels of drinking as compared to non-dependent animals. We hypothesize that repeated cycles of CIE exposure will produce long-lasting changes in the excitability and plasticity of neurons within the orbitofrontal region (OFC) of the prefrontal cortex, an area known to be dysfunctional in human alcoholics. This hypothesis will be tested using four specific aims that will i) Assess the effect of chronic ethanol exposure on behaviors that require a functional OFC network;ii) Determine the acute ethanol sensitivity of glutamatergic and GABAergic transmission in OFC neurons;iii) Monitor changes in glutamatergic and GABAergic synaptic transmission in OFC neurons from control and ethanol dependent mice and iv) Determine the effects of chronic ethanol exposure on plasticity mechanisms of OFC neurons. The results from these studies will be critical in advancing our understanding of the effects of chronic ethanol on higher cortical function.

The goal of this project is to use in vivo two-photon laser scanning microscopy (2PLSM) to examine changes in neuronal activity in ethanol-dependent mice. Chronic drinking that leads to dependence is associated with marked changes in brain function including impairments in cortical processing and cognition. These impairments likely underlie the transition to heavy drinking and elucidating the mechanisms that underlie these changes is critical towards developing treatments and interventions that restore control over behavior. A key limitation in the study of the mechanistic actions on alcohol on brain function is the reliance upon in vitro techniques to monitor neuronal function. These approaches, while extremely valuable, are hindered by the loss of relevant sensory input and disruption of normal circuitry during preparation of the in vitro tissue sample. While brain imaging approaches can avoid this complication, they are compromised by a lack of specificity with regard to spatial resolution and neuronal sub-type. In this application, we will use in vivo 2PLSM to image neuronal activity and neurovascular function in alcohol-dependent mice. In Aim 1, we will utilize a recently developed line of mice that, when crossed with a specific Cre-driver mouse line, expresses the calcium-sensor protein GCaMP3 in a defined sub-population of neurons. Neuronal calcium dynamics will be monitored in glutamatergic pyramidal neurons and fast-spiking interneurons by crossing the GCaMP3 mice with the Wfs1-Tg2-CreERT2 line and Pvalb-2A-Cre line; respectively. Activity will be monitored both during rest and during presentation of visual cues that reliably induce activity in visual cortex neurons. In Aim 2, neurovascular coupling (e.g., activity-dependent changes in local brain blood flow) will be monitored by the use of a dye recently shown by the Co-I to effectively signal changes in arteriolar blood flow. Together, results from these two complementary aims will establish the technique of in vivo 2PLSM in the study of alcohol action and will support future studies designed to analyze the effects of alcohol on cortical function.

PROJECT SUMMARY Alcoholism is characterized by a loss of control over drinking and increased risk of adverse events including traumatic injury, organ damage and loss of normal social interactions. The neural substrates that underlie the transition from controlled social drinking to uncontrolled alcohol abuse are not fully understood, but they likely involve disruption of brain areas responsible for assessing risk versus reward and in inhibiting maladaptive behaviors. During the current funding cycle, we have focused on defining the actions of acute and chronic ethanol on neurons within the orbitofrontal cortex (OFC), a part of the prefrontal cortex that is critical for choice and decision-making. Results from these studies show that acute ethanol inhibits OFC neuron activity via effects on processes that regulate intrinsic excitability and synaptic glutamatergic transmission. Further, chronic ethanol exposure disrupts OFC-dependent behaviors and results in marked enhancement of OFC excitability and glutamate synaptic plasticity, which may contribute to escalation in drinking associated with ethanol dependence. In this continued Center Project, we propose three major aims designed to expand on our findings by addressing the selectivity of these changes with respect to connections between OFC neurons and brain areas involved in goal-directed and habit-based behaviors. These studies will use our well-characterized mouse model of chronic intermittent ethanol (CIE) exposure and will: (1) use retrograde labeling, slice electrophysiology and optogenetic stimulation to interrogate the input and output function of OFC neurons projecting to areas involved in reward, action and habit (e.g., ventral tegmental area and dorsal and ventral striatum). Alterations in dendritic complexity and spine morphology of OFC neurons that project to these areas will be examined using a novel AAV/Rabies transynaptic labeling technique and Cre-dependent lines (e.g., TH- Cre; D1-Cre; D2-Cre) that provide synapse specific control of connectivity; (2) test how CIE treatment alters the intrinsic excitability of OFC neurons and alters the ability of local (glycine) and long-distance (monoamines) modulators to regulate OFC neuron excitability and synaptic function. These studies will also use retrograde labeling and slice electrophysiology to identify projection-specific changes in the modulation of neuronal function of OFC neurons in control and CIE exposed animals; and (3) test how controlling OFC output via excitotoxic lesions and inhibitory and excitatory DREADDs impacts drinking before and following repeated cycles of CIE exposure. Results from these studies will yield important new insights into the role that OFC neurons play in the escalation of drinking observed during the development of ethanol dependence.  

Project Summary The legal status and widespread use of alcohol among the US population is associated with adverse health outcomes in both adolescents and adults. Nearly 88% of the US population have used alcohol at least once during their lifetime and rates of binge drinking and heavy alcohol use continue to be of concern. Pharmacological treatments for alcohol abuse show limited effectiveness and only three medications are FDA approved for treating alcohol dependence. One factor that underlies this problem is a lack of understanding of the mechanisms that contribute to problem drinking. While many studies have revealed that alcohol excites midbrain dopamine neurons involved in reward-based behaviors, agents targeting dopamine have not been shown to be effective in reducing drinking. More recent studies have begun to investigate connections between higher brain areas such as the medial prefrontal cortex and orbitofrontal cortex and down-stream targets such as dorsal and ventral striatum, amygdala and hippocampus that are involved in the control of motivated behaviors. One key brain region that has largely been ignored in studies of alcohol consumption is the cerebellum that is known to communicate with cortical and reward-sensitive brain areas and is sensitive to both acute and chronic exposure to alcohol. In this proposal, we address this gap in our knowledge with experiments outlined under two Aims that explore how specific lobules of the cerebellum contribute to the regulation of ethanol drinking. Aim 1 uses models of homecage drinking and operant ethanol self-administration combined with lickometer circuitry to measure the amount, motivation and micro-structure of ethanol drinking in mice expressing DREADDs in selective cerebellar lobules. Control studies will examine whether DREADD-induced alterations in alcohol consumption are accompanied by motor deficits and concomitant changes in consumption of alternative rewards and tastants. Aim 2 uses slice electrophysiology to determine how DREADDs that affect ethanol drinking alter the firing of cerebellar Purkinje neurons and those in the deep cerebellar nuclei that are the sole output of the cerebellum. Neurons and regions outside the cerebellum affected by DREADD manipulation of ethanol drinking will be identified using the newly developed Fos-TRAP2 mice that permanently labels activated neurons during a brief window opened by tamoxifen injection. Results from these studies will provide the first comprehensive assessment of how cerebellar sub-regions contribute to alcohol drinking and will support the submission of a full- length R01 to continue this work.

SUMMARY A cardinal feature of alcohol use disorder (AUD) is loss of control over drinking. The neural substrates that underlie transition from controlled social drinking to uncontrolled alcohol abuse are not fully understood but likely involve disruption of brain areas involved in assessing risk versus reward and in inhibiting maladaptive behaviors. During the current funding cycle, we focused on defining the actions of alcohol on neurons within the orbitofrontal cortex (OFC), a part of the prefrontal cortex that is critical for choice and decision making. Studies also examined how manipulating the activity OFC neurons affects drinking. Results show that acute alcohol inhibits OFC neuron activity via a glycine receptor and dopamine D1 receptor-dependent mechanism, while monoamine inhibition of OFC spiking requires D2, a2 and 5HT-1a receptors that are coupled to inhibitory GIRK channels. Following repeated cycles of chronic intermittent ethanol (CIE) exposure or voluntary drinking, OFC neurons were hyper- excitable and no longer sensitive to alcohol or monoamines. Blocking OFC output via excitotoxic lesions or inhibitory DREADDs had little effect on alcohol drinking in non-dependent animals but further enhanced elevated consumption in CIE-treated mice. In this renewal application, we propose three aims that complement and extend findings from the current project. These studies focus on defining how neurons in the OFC and basolateral amygdala (BLA) converge on and influence activity and functional output of medium spiny neurons (MSNs) in the dorsal striatum (DS). Studies will use our well-characterized mouse model of CIE exposure combined with operant alcohol self-administration and in vitro and in vivo measures of neural activity. Aim 1 studies use ex vivo slice electrophysiology and retrograde labeling to examine the excitability and acute alcohol sensitivity of OFC and BLA neurons that project to the DS in non-dependent and alcohol-dependent male and female C57BL/6J mice. Dual color optogenetic constructs and transgenic reporter mice will be used to evoke OFC and BLA synaptic currents onto DS D1 and D2 MSNs in slices from Air and CIE treated mice. Aim 2 studies use in vivo calcium fiber photometry to measure CIE-induced changes in the activity and alcohol sensitivity of DS projecting OFC and BLA neurons during operant self-administration of alcohol and alcohol with tastants that reduce (quinine) or enhance (sucrose) drinking in non-dependent mice. Complementary fiber photometry studies will monitor the activity of DS MSNs innervated by OFC and BLA inputs. Studies in Aim 3 will use in vivo optogenetics (e.g. Chronus, Chrimson, ArchT) to bi-directionally manipulate DS projecting OFC and BLA inputs to assess how these pathways regulate drinking and how this is altered by alcohol dependence. Overall, the results of these studies will advance our understanding of mechanisms that contribute to the escalation of drinking in alcohol- dependent subjects and provide key data needed to develop better treatments for individuals with AUD.