This multifaceted project provides new knowledge on the identification and characterization of neurotransmitters, neuromodulators and related enzymes and receptors active at major synapses in brain stem auditory nuclei. Using antibodies against GABA, GAD, and glycine, putative GABAergic and glycinergic neurons in auditory nuclei in guinea pig were identified with immunocytochemistry. GABA-immunoreactive presynaptic terminals in the anteroventral cochlear nucleus of the guinea pig were present on stellate cells and spherical cells, restricted to a subpopulation of terminals containing oval/pleomorphic synaptic vesicles. Using double labeling immunocytochemistry, glycine receptor immunoreactivity was found postsynaptic to terminals containing GABA or GAD immunoreactivities, with oval/pleomorphic synaptic vesicles, at some synapses in the ventral cochlear nucleus. We have continued and expanded a project to characterize excitatory amino acid receptors. Kainic acid receptor was solubilized from frog brain membranes and then purified with DEAE Sepharose and domoic acid affinity chromatography to a purity 481 fold relative to a crude solubilized preparation and 1617 fold relative to the membrane fraction. Polyclonal antibodies were made by injecting rabbits with purified kainic acid receptor. Antibodies were characterized by an Indirect immunoprecipitation assay in which they were bound to Protein A immobilized on Sepharose. Solubilized receptor was then passed through the column and the depletion in receptor binding was determined. Polyclonal antibodies removed about 80% of the receptor binding from a soluble preparation from frog brain, with nearly equal removal of high and low affinity sites. Immunocytochemistry of the kainic acid receptor was done in frog brain with polyclonal antibodies. Immunocytochemical analysis of the kainic acid receptor in frog brain showed a rostral to caudal gradient of immunostaining with highest intensity in the telencephalon. Monoclonal antibodies were made by immunizing Balb C mice with 8-20 Mug of purified frog brain kainic acid receptor in Freund's complete adjuvant. Several monoclonal antibodies which recognize the kainic acid receptor were obtained.
