The specific excitatory amino acid agonists, kainate, quisqualate and N-methyl aspartate (NMA) all excite neurons of rat piriform cortex but there is no satisfactory understanding of the mechanisms of the excitation nor of the excitotoxicity to which this brain area is susceptible. We propose to use submerged, perfused rat piriform cortex slices and primary dissociated neuronal cultures to study the mechanisms of excitation and excitotoxicity due to these substances. We will test the hypothesis that there are related but distinct ion channels associated with these three types of receptors, and that permeation through those channels is regulated by divalent cations such as Ca2+ and Mg2+. Extracellular and current and voltage clamp intracellular recordings will be made from identified pyramidal neurons. The ionic bases of the responses to ionophoretic application of the agonists will be studied by variation of the perfusion medium and determination of the conductance changes and voltage dependencies of the responses. The involvement of divalent cations will be studied by varying Ca2+ and Mg2+ concentrations in the medium and by use of a variety of inorganic ions which either substitute for or block Ca2+ currents as well as study of several organic Ca2+ channel blockers. Excitotoxicity will be studied in experiments designed to test two hypotheses. The first is that excitotoxicity is caused by accumulation of intracellular Ca2+ with consequent activation of proteases. This will be tested by study of excitotoxicity under conditions of varying Ca2+ and adding Ca2+ antagonists. The second is that excitotoxicity results from C1- influx and consequent osmotic effects and will be studied by use of C1- substitution. The cell culture system will be developed in parallel to find a preparation where the three agonists, especially kainate, elicit responses with pharmacologic and divalent cation-sensitive properties identical to those in the slice. Investigation of the excitation will be performed as above in order to compare the nature of the responses and the basis of excitotoxicity to that obtained with the brain slice neurons. Using the whole cell patch technique with ion substitutions and pharmacologic manipulations, attempts will be made to isolate currents (Ca2+ and/or C1-) critical to the response and excitoxicity. Finally, the properties of the channels will be studied directly using outside-out patch clamp recordings.
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