Evidence from several independent laboratories using different experimental approaches indicates that acute ethanol selectively inhibits N-methyl-D-aspartate receptor (NR) function at pharmacologically relevant concentrations, whereas chronic ethanol produces NR supersensitivity due, at least in part, to receptor upregulation. As a consequence, excessive activation of NRs during alcohol withdrawal is thought to contribute to the associated seizures, autonomic instability and neurotoxicity. Pharmacologic treatment with available NR antagonists is undesirable due to generalized inhibition of these ubiquitous receptors. However, NRs are functionally diverse due to differences in subunit composition and associated signal transduction systems. This diversity may account for the selective effects of ethanol on NRs in different brain regions, and in different neuronal populations within a particular region. By exploring the mechanistic basis for this regional selectivity, it should be possible to design and utilize in the alcoholic patient more selective antagonists that are targeted to vulnerable neuronal populations. To further define the role of NRs in chronic ethanol-induced neurotoxicity in hippocampus, the following hypotheses will be tested in adult rats: (I) increased levels of specific NR subunits, and possibly mRNAs, will be observed in hippocampal subfields (CA1, CA3, DG) of ethanol-treated rats, relative to pair-fed control rats, in a manner that corresponds to development of ethanol dependence. Moreover, increased levels of NR subunits will be positively correlated with increased expression or activity of a type II Ca2+/calmodulin-dependent protein kinase (CaM KII) that is co-localized with NRs at excitatory synapses and has been implicated in other forms of excitotoxicity; (ii) chronic ethanol ingestion will differentially sensitize CA1, CA3 and DG neurons to NR-mediated excitotoxicity, relative to pair-fed controls, in a reversible manner. This effect will be mediated by CaM KII. To test these hypotheses, ethanol will be administered to rats for 1-12 weeks and, in some cases, will include a withdrawal period. In ai-m 1, hippocampal subfields from ethanol-treated and control rats will be compared using NRI, NR2 and CaM KII immunoblotting and RT-PCR assays.
In aim 2, hippocampal slices will be compared for regional differences in vulnerability to glutamate- or NMDA-mediated excitotoxic damage using a """"""""live-dead"""""""" assay in conjunction with confocal imaging analysis.
