Linda M. Nowak - US grants

The goal of the proposed research is to gain information regarding the functional roles of excitatory amino acid (EAA) receptors in the vertebrate central nervous system (CNS) with the aim of applying it to studies of excitatory synapses in future experiments. Endogenous dicarboxylic amino acids, including L-glutamate, are putative excitatory neurotransmitters in the vertebrate CNS interacting with EAA receptors. There is evidence for at least three classes of EAA receptors; however, they remain poorly defined, largely due the lack of adequate pharmacological tools and the superficial "sameness" of the electrophysiological or biochemical responses generated by applied agonists. Recently, patch clamp recording techniques were applied to the study of EAA pharmacology and with the result of EAA receptors are beginning to be redefined by their associated ion channels (Preliminary Studies). The proposed research is intended to continue some of those studies and to acquire data which will aid in studies of excitatory synapses. It includes patch clamp studies of EAA receptor activated channels and correlational studies with radioligand binding assays of subsynaptic binding sites. The electrophysiological studies feature examination of biophysical properties of channels: 1) activated by application of endogenous EAA ligands in outside-out patches from cortical neurons of 15 day old mouse embryos in dissociated cell culture and patches from acutely dissociated adult murine cortex to see if the channels are similar, 2) in cell attached patches of adult brain neurons to determine if channel properties are altered by forming outside-out patches, 3) that are partially blocked in a voltage dependent way be physiological levels of extracellular MG++ ions, to define kinetic parameters which may related the Mg++ blocking to regenerative responses observed when the channel are activated in non-voltage clamped cells. The importance of these studies is evident when it is considered that EAA receptors are being linked to a variety higher order CNS functions including learning and dysfunctions including seizure disorders and neurodegenerative disease.

This proposal requests support to obtain several pieces of equipment to establish an integrated system for combined quantitative microscopic fluorescence analysis and electrophysiological recording. Six major users have proposed projects that will utilize the instrumentation. Five of these involve Ca2+ measurements by fluorescence coupled to membrane transport or receptor studies (exocytosis in Mast cells, receptor studies in mammalian neurons, intestinal electrolyte transport, nicotinic receptors and Ca2+channel function, Ca2+ movements and hormone production in luteal cells). The sixth project will use Ca2+-mediated fluorescence to study muscle myotubes in culture.

DESCRIPTION: (adapted from Applicant's Abstract) The proposed research involves biophysical and molecular characterization of several ionotropic excitatory amino acid (EAA) receptors, insensitive to N-methyl-D-asparate (NMDA), referred to as nonNMDA receptor-channels. Although nonNMDA EAA channels are likely to be involved in the majority of excitatory synapses in vertebrate brain, detailed functional characterization of this diverse group of channels in neurons is difficult because of their subtype complexity. Nine major subunits of nonNMDA channels have been cloned (GluR1-7, KA1-2), with some of these cDNAs exhibiting additional diversity due to post-transcriptional modifications, including alternative splicing which gives rise to the flip and flop forms of GluR1-4 that differ with respect to desensitization, and RNA editing of GluR2, 5 & 6 which affects channel Ca2+ selectivity. Two projects are proposed that focus on functional aspects of native neuronal channels. The first project involves single channel patch clamp analysis of an unusual ibotenate-activated nonNMDA channel that has two striking features, namely its divalent cation selectivity, and the effects of Ca2+ ions on the gating of this receptor-channel. Molecular analysis of cerebellar Purkinje neurons, and complementary patch clamp studies of candidate subunits expressed in a mammalian cell line are proposed in order to determine which subunits might form this novel channel. The second project is to further characterize two populations of cerebellar granule cells that have biophysically and pharmacologically different responses to kainate, AMPA and L-glutamate. The goal of these experiments is to determine if the functional differences can be explained by differences in the mRNAs expressed in two populations of granule cells. Due to the pharmacological sensitivity of all of the cloned GluR1-4 subunits to kainate, and their pronounced desensitization by AMPA and glutamate, the most likely hypothesis for the differences in the two cell populations is differential expression of the GluR1-4 flip and flop variants which are thought to be developmentally regulated in some brain regions. To test this hypothesis, molecular analysis using a combination of whole cell electrophysiology/pharmacology and single cell molecular biology methods will be conducted on the same cells as the functional studies. The total poly A+ mRNA from single cells will be transcribed using an oligo-dT(24)primer, antisense 32P-RNAs generated and amplified. Expression profiles obtained by hybridizing the single cell 32P-aRNAs with EAA subunit cDNAs bound to filters will be used to estimate the relative abundance of GluR1-7 messages in each cell, and PCR studies of single cell cDNAs will be used to discriminate between flip and flop forms of GluR1-4. Results from the proposed studies will be relevant to understanding the functional capacity of particular nonNMDA channels in excitatory synaptic transmission in normal brain, and in pathological conditions such as seizure disorder and neurotoxin induced cell death where sustained elevation of intracellular Ca2+ is a likely triggering event of the pathology.