Richard A. Morrisett - US grants

Neuronal hyperexcitability and epileptiform activity due to withdrawal from chronic ethanol exposure encompasses a continuing problem for the modem clinician. The N-methyl-D-aspartate receptor/channel complex potently regulates the development of epileptiform activity in both in vitro and in vivo neural systems. Ethanol effects upon NMDA systems may be of primary importance for the expression of epileptiform activity especially in relation to the development of, and our ability to treat, alcohol withdrawal seizures. We propose to test the hypothesis that chronic ethanol exposure and subsequent withdrawal modifies the functioning of the NMDA complex resulting in changes in the induction and expression of epileptiform activity. The primary aim of this proposal is to increase our understanding and treatment of epileptiform activity which accompanies chronic ethanol exposure and withdrawal (alcohol withdrawal seizures). This proposal utilizes the electrographic seizure model (EGSs) elicited in the in vitro rat hippocampal slice. A comprehensive analysis of the effects of acute and chronic ethanol exposure and withdrawal on the processes responsible for induction and expression of hippocampal electrographic seizures is proposed. Extracellular recording techniques win be used to identify changes in hippocampal EGSs. Tight seal whole-cell voltage clamp recording techniques (in vitro slice patch) will be used to directly identify and measure cellular changes due to ethanol exposure. Initial evidence shows that ethanol markedly elevates the threshold for the expression of established hippocampal EGSs. This action of ethanol occurs at concentrations known to inhibit NMDA processes. The inhibitory effect of in vitro ethanol exposure is similar to the effect of the NMDA receptor antagonist, D-APV, which also raises the EGS threshold. These data indicate that the NMDA complex may be an important site of ethanol action resulting in modification of seizure generation. Analysis of ethanol effects on seizure processes should increase our understanding and aid in the development of novel therapies for alcohol withdrawal seizures.

DESCRIPTION (provided by applicant): Evidence suggests that glutamate receptor trafficking induces long-term changes in the nucleus accumbens (NAc) which may constitute an important neuroadaptation contributing to the development of ethanol dependence. We have observed that low frequency conditioning stimulation (LFS) of excitatory afferents to shell NAc medium spiny neurons (msns) normally induces NMDA-receptor dependent synaptic depression in slices from ethanol na?ve mice. However, a single 4 day bout of passive chronic intermittent ethanol (CIE) in vivo exposure induces a polarity shift in plasticity to LFS conditioning and induces synaptic potentiation (LTP) rather than LTD (metaplasticity). In addition, the shell and core subregions of the NAc likely encode different aspects of drug responding with the shell responding more to novelty and the core encoding more established learned behaviors. These findings promote our overarching hypothesis that D1-dopamine receptor-expressing medium spiny neurons of the shell and core of the nucleus accumbens differentially encode passive and operant ethanol experience through selective neuroadaptations in glutamatergic synaptic transmission and plasticity We propose in aim 1 to measure glutamatergic transmission and metaplasticity in shell/core NAc medium spiny neurons from transgenic mice expressing eGFP under the control of the dopamine D1- receptor promoter (drd1-eGFP mice) (1) after passive exposure to chronic intermittent ethanol vapor or (2) trained for volitional, operant ethanol self-administration. All experiments in aim 1 will use whole-cell voltage clamp recordings from identified drd1-eGFP shell/core msns. Alterations in glutamatergic transmission will be assessed by pharmacological isolation of NMDA and AMPA-subcomponents and AMPA:NMDA ratios. Metaplasticity will be assessed by determining the expression of LTD or LTP in response to conditioning stimulation in shell/core drd1-eGFP msns from the different ethanol experience groups. In aim 2, we will determine whether interference with GluR2 internalization alters operant self-administration behavior in mice. Injection of a peptide fragment which disrupts GluR2- mediated AMPA channel internalization will be made into the NAc to determine whether interruption of AMPAR trafficking can modulate or occlude operant responding for ethanol in the same mouse model used in aim 1. Injections will also be made into other mesocorticolimbic structures (VTA, PFC) to determine whether the behavioral effects of occlusion of LTD are limited to the NAc. These aims will advance our understanding of neuroadaptive alterations in an important mesocorticolimbic structure which may underlie the development of ethanol dependence.

DESCRIPTION (provided by applicant): A major goal of INIA-West is to identify novel therapeutics for excessive alcohol consumption and prior INIA-West efforts have identified and characterized rodent animal models of excessive intake, and candidate genes and associated targets that are altered in these models. The next iteration of INIA-West is directed at identifying which of these gene targets should be targeted for further analysis. Therefore, a simple yet functionally meaningful assay to test whether a target may exhibit efficacy for excessive ethanol intake would be extremely valuable. We and our co-investigators have developed substantial evidence that changes in glutamatergic synaptic plasticity in the nucleus accumbens constitute a critical neuroadaptive response to repeated exposure to positive and negative reinforcers. Furthermore, disruption of the processes underlying the changes in glutamate receptor location and function (GluR2 subunit internalization) modulates behavioral responses to such reinforcers. Therefore, we propose that accumbal plasticity constitutes exactly such a functionally meaningful assay. We propose two main hypotheses to investigate the utility of NAc plasticity as a functional screen for novel targets for ethanol dependence. In Specific Aim 1 we propose to identify how prolonged disruption of GluR2 subunit internalization in vivo modulates excessive ethanol intake using two models: CIE with two-bottle choice and operant ethanol self-administration. In Specific Aim 2 we propose to screen novel gene targets identified by INIA-West components for modulation of accumbal plasticity under baseline conditions and following CIE exposure. This aim will use the INIA-West RNA interference (RNAi) core and we will work in close coordination with our INIA-West colleagues who are focused upon detailed analyses of these individual INIA-West targets. Thus, in aim 2, we will determine whether interference with INIA-West target expression modulates NAc LTD. Finally, as we show in our preliminary data, NAc LTD is predominant in NAc MSNs ofthe direct pathway that express Dl-dopamine receptors, we will ensure that plasticity will be screened for all experiments in DI-NAc MSNs using mice derived from MIVIRRC GENSAT drdl-eGFP Swiss Webster mice back-crossed to the C57BI6/J background.

PROJECT SUMMARY/ABSTRACT This Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune [INIA-N]) consortium application is for a U24 Research Resource that will act as a service core to provide electrophysiological evaluation of targets identified or developed by INIA-N investigators. The guiding hypothesis of INIA-N is that genetic differences and neuroadaptations in immune-related pathways contribute to the vulnerability to excessive alcohol consumption. In addition to validating neuroimmune signaling pathways already identified during the prior funding period, this consortium will use genomic datasets and computational approaches to predict novel compounds that may be efficacious in reducing excessive alcohol consumption. This core resource will provide essential services to INIA-N investigators by performing studies that examine the cellular electrophysiological functions of brain immune signaling systems and their causal or therapeutic role(s) in excessive alcohol consumption. These studies will be divided among three major subgroups of INIA-N target identification and investigation. In Specific Aim 1, novel INIA-N compounds that are efficacious in reducing excessive alcohol consumption will be evaluated. In Specific Aim 2, the focus will be on genetic targets, of which the majority will be long non-coding RNAs shown to influence drinking. Specific Aim 3 will focus on studies in animal models of hereditary excessive drinking. Electrophysiological experiments, designed in collaboration with the relevant INIA-N investigators, will use new technologies to study neural circuits involved in excessive drinking, and, in cases of wholly novel targets, will employ an unbiased approach of measuring basic membrane properties along with excitatory and/or inhibitory synaptic transmission to screen for mechanisms of action in mesolimbic brain structures. The results of these studies will provide basic information on the role of brain neuroimmune systems in excessive ethanol consumption and help prioritize novel targets for further development.