Evidence from our laboratory indicates that ethanol has a regionally specific action in brain to antagonize NMDA-induced excitation of neural activity. The NMDA receptor is composed of multiple protein subunits which form a functional ion channel. These NMDA receptor subunits have differing neuroanatomical distributions, thereby providing evidence for heterogeneity of NMDA receptors in mammalian brain. The purpose of this grant is to test the hypothesis that the regionally specific effect of ethanol on responses to NMDA in brain is linked to the expression of different combination of NMDA receptor subunits. Based upon our present observation of ethanol- induced regional inhibition of NMDA (i.e., ethanol-sensitive and ethanol- insensitive brain regions), the first Specific Aim of this proposal will determine the regional localization of the mRNA for the subunits cloned for the NMDA receptor using PCR to identify NMDAR-1 splice variants and the NMDAR-2a,b,c,&d subunits found in micropunches from brain regions shown electrophysiologically to be either insensitive or sensitive to the action of ethanol to antagonize NMDA-induced excitation.
In Specific Aim II, we will determine the sensitivity of dissociated neurons to ethanol's antagonism of NMDA using fluorescent measures of increased intracellular calcium flux induced by NMDA. Subsequently, the subunit mRNA composition of the NMDA receptors in dissociated cells with proven sensitivity to ethanol will be determined with PCR amplification.
In Specific Aim III, patch-clamp electrophysiology as well as changes in fluorescence will be used to compare the structural components of NMDA isoreceptors within individual neurons with their differing sensitivities to ethanol. Additionally, receptor components will be related to phosphorylation drug actions and to ethanol's inhibition of NMDA. Once the subunit composition for an NMDA receptor in individual neurons sensitive to ethanol has been documented in Specific Aim III, Specific Aim IV will map the NMDA subunits important for ethanol's action throughout brain using in situ hybridization. Following this, extracellular electrophysiological studies will be performed to test additional brain sites predicted from the mapping study to be sensitive to ethanol. Thus, with this multidisciplinary effort, it will be determined whether specific structural components making up NMDA isoreceptors predict ethanol's action on NMDA responses and will delineate 'new"""""""" sites where ethanol does or does not affect responses to NMDA. Such data will define the neurobiological and molecular basis of ethanol's regional action on this ligand-gated ion channel.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA010025-03
Application #
2682977
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1996-04-01
Project End
2001-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Psychology
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Criswell, Hugh E; Ming, Zhen; Kelm, M Katherine et al. (2008) Brain regional differences in the effect of ethanol on GABA release from presynaptic terminals. J Pharmacol Exp Ther 326:596-603
Knapp, Darin J; Overstreet, David H; Moy, Sheryl S et al. (2004) SB242084, flumazenil, and CRA1000 block ethanol withdrawal-induced anxiety in rats. Alcohol 32:101-11
Criswell, Hugh E; Ming, Zhen; Griffith, Benjamin L et al. (2003) Comparison of effect of ethanol on N-methyl-D-aspartate- and GABA-gated currents from acutely dissociated neurons: absence of regional differences in sensitivity to ethanol. J Pharmacol Exp Ther 304:192-9
Ming, Zhen; Griffith, Benjamin L; Breese, George R et al. (2002) Changes in the effect of isoflurane on N-methyl-D-aspartic acid-gated currents in cultured cerebral cortical neurons with time in culture: evidence for subunit specificity. Anesthesiology 97:856-67
Haberman, Rebecca; Criswell, Hugh; Snowdy, Stephen et al. (2002) Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity. Mol Ther 6:495-500
Ming, Z; Knapp, D J; Mueller, R A et al. (2001) Differential modulation of GABA- and NMDA-gated currents by ethanol and isoflurane in cultured rat cerebral cortical neurons. Brain Res 920:117-24
Yang, X; Criswell, H E; Breese, G R (2000) Ethanol modulation of gamma-aminobutyric acid (GABA)-mediated inhibition of cerebellar Purkinje neurons: relationship to GABAb receptor input. Alcohol Clin Exp Res 24:682-90
Criswell, H E; McCown, T J; Ming, Z et al. (1999) Interactive role for neurosteroids in ethanol enhancement of gamma-aminobutyric acid-gated currents from dissociated substantia nigra reticulata neurons. J Pharmacol Exp Ther 291:1054-9
Yang, X; Knapp, D J; Criswell, H E et al. (1998) Action of ethanol and zolpidem on gamma-aminobutyric acid responses from cerebellar Purkinje neurons: relationship to beta-adrenergic receptor input. Alcohol Clin Exp Res 22:1655-61
Duncan, G E; Moy, S S; Knapp, D J et al. (1998) Metabolic mapping of the rat brain after subanesthetic doses of ketamine: potential relevance to schizophrenia. Brain Res 787:181-90

Showing the most recent 10 out of 11 publications