David J. Rossi - US grants

[unreadable] DESCRIPTION (provided by applicant): ' The primary goal of this proposal is to investigate mechanisms that may be involved in cerebellar damage and malfunction during brain ischemia. Brain ischemia, which occurs during cardiac arrest, stroke and perinatal asphyxia, is a leading cause of death and long term disability. The cerebellum is a frequent target of stroke, and cerebellar Purkinje cells are one of the most susceptible brain cells to ischemic damage. However, relatively little is known about how Purkinje cells respond to and are damaged by ischemia. This lack of information is problematic because many of the ischemic mechanisms operating in other brain regions involve molecular processes that are either not expressed or have an unusual configuration in Purkinje cells. For this proposal, a brain slice model will be used to simulate brain ischemia in vitro and patch-clamp recording, confocal fluorescence imaging and pharmacological manipulations will be used to investigate mechanisms of cerebellar ischemic damage. Simulated ischemia induces a severe depolarization of Purkinje cells which is mediated by activation of non-NMDA ionotropic glutamate receptors (and possibly other glutamate receptors/transporters) but its onset is delayed by activation of GABAA receptors. These electrophysiological responses are associated with extensive tissue swelling and the subsequent development of necrotic and apoptotic cell death very similar to that observed with in vivo models. Simulated ischemia also severely disrupts electrical signal transduction through the cerebellum and the disruption persists for long periods after the ischemic episode is terminated. Determining the mechanisms that underlie both cellular damage and disrupted signal processing should provide useful information for the development of therapies. The specific aims of this proposal are: 1) To determine the mechanisms that lead to glutamate accumulation around Purkinje cells, elucidate which receptors mediate the Purkinje cell response and determine their contribution to cell damage; 2) To determine the mechanisms by which GABAA receptor activation delays the onset of Purkinje cell depolarization and to determine if the delay is beneficial or damaging; and 3) To determine where in the cerebellar circuitry and by what mechanism electrical signal transduction is disrupted. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This competing renewal application builds on our studies in the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines and the DBA/2 inbred strain, which suggest that genetic differences in ethanol withdrawal severity are due in part to alterations in the ?-aminobutyric acid (GABA)- modulatory effects of neurosteroids such as allopregnanolone (ALLO), the most potent positive modulator of GABAA receptors. In particular, during ethanol withdrawal, endogenous ALLO levels were reduced, and the anticonvulsant effect of systemically administered ALLO was decreased in WSP and DBA/2 mice, but not in WSR mice. Further, in ethanol withdrawn WSP mice, a reduced sensitivity to ALLO's anticonvulsant effect was also observed when ALLO was infused into specific brain regions within the withdrawal seizure circuit, including the hippocampus. Preliminary electrophysiological experiments in hippocampal slices from ethanol withdrawn WSP mice documented a reduced effect of GABAergic steroids and selective GABAA receptor ligands on GABAA receptor mediated synaptic and tonic currents. Additionally, epileptiform activity in hippocampal slices from ethanol withdrawn WSP mice was reduced by GABAA receptor enhancing ligands. Finally, preliminary experiments with gas chromatography-mass spectrometry (GC-MS) simultaneously quantified hippocampal neurosteroid levels and determined that ethanol injection produced divergent changes in steroids with positive (agonist) and negative (antagonist) effects on GABAA receptor function. Based on these data, the current proposal will adopt a multidisciplinary approach to test the overall hypothesis that a reduction in hippocampal GABAergic function, due to decreased GABAA receptor sensitivity to neurosteroids and a concomitant imbalance in ALLO and related neurosteroids, represents a neurochemical substrate for enhanced susceptibility to ethanol withdrawal severity. Studies will be conducted in WSP and DBA/2 mice, two animal models that are widely used to study genetic liability to ethanol withdrawal in mammals. Aim 1 will use GC-MS to quantify the effect of ethanol withdrawal on multiple endogenous steroid compounds, with agonist and antagonist effects on GABAA receptor function, in the hippocampus at select time points of withdrawal. Aim 2 will use voltage-clamp recording in subregions of hippocampal slices to characterize ethanol withdrawal-induced changes in the pharmacology of synaptic and extrasynaptic GABAA receptors, including alterations in sensitivity to neurosteroids with agonist and antagonist effect and to subunit selective ligands. Aim 3 will integrate information from Aims 1 and 2 and determine if counteracting ethanol withdrawal-induced changes in GABAA receptor properties and in neurosteroid levels rescues the high withdrawal phenotype, measured by epileptiform activity in hippocampal slices and convulsions in vivo. Thus, the proposed experiments will establish the importance of altered hippocampal neurosteroid composition and GABAA receptor pharmacology in mediating ethanol withdrawal severity.

Project Summary/Abstract Alcohol (ethanol; EtOH) abuse is a leading cause of death and disability, making our understanding of the mechanisms that affect transition from healthy use of EtOH to development of an alcohol use disorder (AUD) a high biomedical priority. In this context, the initial neurological responses to socially relevant concentrations of EtOH (5-20mM), and individual differences in those responses are thought to play a critical role in determining vulnerability or resilience to development of AUD. Thus, it is crucial to identify molecular targets and neural circuit responses to low concentrations of EtOH, and to identify the mechanisms by which such responses vary across individuals with high or low risk for developing AUD. In our published and preliminary studies, we have determined that low concentrations of EtOH (10mM, as would occur in the blood of an average adult human after consuming 1-2 standard alcoholic beverages) powerfully affect cerebellar granule cell GABAAR currents, but with opposite polarity in multiple strains of rodents with high and low EtOH consuming phenotypes respectively. Further, we have discovered a previously unknown direct synaptic connection between the cerebellum and the ventral tegmental area (VTA), a brain structure known to influence many aspects of EtOH reward and associated behaviors. These are important discoveries because genetic differences in cerebellar structure, connectivity and sensitivity to EtOH are known to be associated with risk for developing an AUD in humans and with excessive EtOH consumption in rodents, but the underlying mechanisms are unknown. The purpose of this proposal is to advance our understanding of how low concentrations of EtOH affect cerebellar spatiotemporal processing and behaviors, how such actions vary in strains of mice with divergent EtOH related behavioral phenotypes, and to characterize the neural circuitry that translates differential actions at a cellular level into differential behavior, including EtOH reward and excessive EtOH consumption. Success in this endeavor will increase our understanding of how the cerebellum influences predilection to developing an AUD, and will identify novel molecular targets for manipulating responses to low concentrations of EtOH. Collectively, such information should help guide the development of psychological and pharmacological approaches to screening for and deterring development and maintenance of AUD. We will use a combination of patch-clamp recording from cerebellar brain slices, patterned optical light stimulation (to simulate in vivo-like network processing), and behavioral techniques combined with optogenetic manipulations of cerebellar processing to determine how low concentrations of EtOH affect cerebellar processing and output, and EtOH related behaviors. We will then use optogenetic tract tracing techniques to fully characterize cerebellar to VTA circuitry and function, which will provide important insight into the neural circuitry that translates differential actions of EtOH in the cerebellum to divergent EtOH-related behavioral phenotypes. Successful completion of the proposed experiments will improve our understanding of the cellular mechanisms and neural circuitry that influences predilection to AUD.