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.
