Ronald L. Alkana - US grants

Past studies demonstrate that one effect of ethanol (body temperature change during intoxication) can strongly influence sensitivity to other effects of ethanol and can alter genetically determined differences in brain sensitivity to ethanol. However, little is known about the cellular mechanisms by which temperature affects ethanol sensitivity and the role temperature plays in mediating differences in ethanol sensitivity established by selective breeding. In addition, recent studies indicate that temperature can significantly influence the sensitivity of in vitro systems to ethanol, but systematic information is not available. These gaps in our knowledge cloud interpretation of many ethanol studies. The present proposal addresses these issues. Specific Aim 1 tests the hypothesis that ethanol-induced changes in body temperature during intoxication mediate genetically determined differences in ethanol sensitivity established by selective breeding. We will accomplish this goal by determining ethanol sensitivity in the presence and absence of ethanol-induced hypothermia in selectively bred animals commonly used in ethanol research [Withdrawal Seizure Prone (WSP) and Resistant (WSR) mice and High Alcohol Sensitive (HAS) and Low Alcohol Sensitive (LAS) rats]. We will measure ethanol sensitivity using the respective measure upon which selection pressure was placed to establish the line (withdrawal seizures and loss of righting reflex.) Body temperatures and blood and brain ethanol concentrations will be determined. These studies, in conjunction with our previous work, will provide a definitive test of the hypothesis. Specific Aim 2 investigates the mechanisms of temperature's effects on brain sensitivity to ethanol by testing hypothesis that specific cellular systems are involved. In addition, Aim 2 will systematically test the hypothesis that temperature is a critical variable in vitro studies of ethanol's cellular actions and effects. We will Aim 2's overlapping goals by studying the effects of temperature (23-38 degrees C) and ethanol (12.5- 400 Mm) in vitro on cellular systems implicated in mediating or modulating ethanol's behavioral effects [membrane order (polarization of fluorescent probes), GABA-A stimulated 36 C1- uptake, NMDA-stimulated dopamine release and NMDA mediated dendritic EPSPs]. Aim 2's experiments will test four mouse genotypes with demonstrated differences in the qualitative effects of temperature on their behavioral sensitivities to ethanol (C57BL,129,LS and SS mice) in order to determine whether genotypic differences in the effects of temperature on ethanol sensitivity in specific cellular systems exist and may underlie their differences in temperature's effects on ethanol sensitivity in vivo. Overall, the proposed research will provide essential information which will enhance the use of pharmacogenetic tools and in vitro techniques in ethanol research and will also contribute to our long term objectives which are to understand the importance and mechanisms of temperature's effects on ethanol sensitivity.

Knowledge regarding the mechanisms by which ethanol initiates, and the subsequent events that mediate, its behavioral effects has increased markedly over the past 5-10 years. These findings have fundamentally changed the working hypotheses regarding ethanol's initial mechanism(s). They have also identified several cellular systems which may represent the initial sites upon which ethanol acts to begin the cascades resulting in behavioral alterations. The goal of the Fifth Gordon Conference on Alcohol is to use this new information as a springboard to re-assess important questions in our field, to identify mechanisms and sites of ethanol's action and to link these sites to specific behavioral effects of ethanol. The long-term goal is to use knowledge regarding the biological bases of ethanol's behavioral effects to improve and develop new alcoholism risk assessment, prevention, diagnosis and treatment strategies. The proposed Conference has two Specific Aims. Aim 1 is to address key questions regarding the biological bases of ethanol's behavioral effects. These include: a) What are the initial biological events (mechanisms) underlying ethanol's behavioral effects? b) What are the critical sites of action initiating, mediating or controlling specific behavioral effects of ethanol? c) What role do different brain regions play in mediating specific behavioral effects of ethanol? and d) How can we begin to address systems interactions in assessing the biological bases of ethanol's behavioral effects? Aim 2 is to present and discuss new discoveries in Neuroscience that could lead to new research approaches and questions in ethanol research over the next five years. The goal is to present information that will stimulate thoughts about new approaches and questions. Nine scientific sessions will be presented to achieve these aims. Session I will concentrate on new issues and developments regarding the mechanisms by which ethanol acts. Session II will be devoted to posters. Sessions III-VII will be devoted to linking individual putative sites upon which ethanol acts to the behavioral alterations they induce. The banquet speaker will continue the conference theme via the topic "Biological Bases of Ethanol's Behavioral Effects: Speculative flow chart from initial sites through cascades, interactions (chemical/regional), final common pathways to behavioral change." Session VIII will be devoted to recent developments in Neuroscience that may enhance our ability to establish links between cellular and behavioral effects of ethanol. Session IX will switch to a more systems oriented approach by investigating new roles steroids may play in mediating ethanol's behavioral effects. This meeting will fill a unique niche for alcohol researchers due to: 1) the Gordon Conference format, with its emphasis on bringing together leading researchers from different disciplines for informal discussion and the generation of new ideas, and 2) the specific focus of linking molecular and cellular changes induced by ethanol to their resultant behaviors.

Ronald L. Alkana

ABSTRACT

The overall goal of the proposed work is to increase understanding of the basic mechanisms involved in communication between nerve cells in the brain. The proposal builds on previous findings demonstrating that increased atmospheric pressure (hyperbaric exposure) is a direct, highly selective blocker of a little explored, poorly defined process-allostenc coupling-that modulates the effectiveness of a family of neurotransmitters receptors in the brain, called ligand gated ion channels (LOICs), that play major roles in nerve-nerve communication. In particular, the proposed work focuses on one of these channels. This channel responds to gamma-aminobutyric acid (GABA) and is called the GABAA channel. When GABA binds to its receptor on the GABAA the channel opens a channel and allows chloride ions to enter the nerve cell. The resultant build-up of chloride ions in the nerve cell inhibits the cells action and decreases its excitablity. The GABAA system is the major inhibitory system in the mammalian brain.

The effectiveness of GABA' 5 action in opening chloride ion channels can be modulated by several classes of compounds that act at distinct but interacting sites on the GABAA receptor. These sites include those for the benzodiazepines, barbiturates and neuroactive steroids. When one of these compounds (ligands) bind to their respective sites on the GABAA receptor, it causes a conformation change in the receptor that affects the ability of other ligands to bind or affect the receptor. This process, referred to as allosteric coupling between sites, modulates the effectiveness of the primary agonist-GAB A-as well as the effectiveness of other allosteric modulators.

The molecular structures and functions of the portions of the GAB AA receptor that bind ligands has been extensively studied. In contrast, little attention has been devoted to understanding the elements that underlie allosteric coupling. The elements mediating coupling are difficult to study directly due to a lack of tools. Recent behavioral and biochemical findings in our laboratory suggest that hyperbaric exposure offers a new approach that can help in studying allosteric coupling. Moreover, this work with hyperbaric exposure suggests that there are fundamental, previously unrecognized, differences in the manner in which binding sites on the GABAA receptor are coupled and that hyperbaric exposure can be used to study these differences.

The specific objective of the research to be undertaken is to test two hypotheses: Hypothesis 1:
The different pattern of sensitivity to pressure antagonism among allosteric modulators of GABAA receptor function will provide new insights into the structural and functional determinants of coupling. The logic for this hypothesis is based on the assumption that the selectivity of pressure antagonism results from pressure's ability to block common physico-chemical changes underlying coupling and that the sensitivity of these physico-chemical changes to pressure reflect similarities in their underlying molecular structures. The hypothesis will be tested by systematically investigating four predictions regarding the selectivity of pressure antagonism based on known functional distinctions in coupling within and between different sites on the GABAA receptor using biochemical measures of GABAA receptor function in mouse brain cell membranes. Hypothesis 2:
Differences in the structural and functional determinants of GABAA receptor coupling identified in
ABSTRACT DRAAT 12/4/98

testing Hypothesis 1 reflect differences in the protein subunits that comprise the receptor. This hypothesis will be tested by determining the sensitivity to pressure antagonism of allosterically modulated events using biochemical and molecular biological techniques.

The proposed work will increase knowledge regarding the manner in which the effectiveness of nerve cell transmission is controlled. The proposed work will lay the foundation for future studies that will use hyperbaric exposure in combination with molecular manipulations in recombinant cells to identify molecular components that mediate allosteric coupling. This information in turn will facilitate the development of molecular models of these structures. The proposed work will also lay the foundation for future investigations that will investigate coupling in other allosterically modulated channels (e.g., NMDA, 5HT3...). These studies could support known and/or could reveal previously unrecognized similarities between allosterically modulated ion channels. Finally, future studies will investigate whether differences in coupling mechanisms have currently unrecognized physiological and behavioral significance. Therefore, the proposed and future work should lead to important new insights regarding the role LGICs play in mediating and modulating brain function and behavior.