The overall aim of this study is to characterize the neurotransmitters of the central visual pathways. Four approaches will be pursued: 1) Release of endogenous compounds from tissue slices; including determination of the effect of cortical lesions on release of amino acids, in particular aspartate and glutamate, from tissue slices of lateral geniculate nucleus and superior colliculus; analysis of the release of low level constituents from cortical tissue slices with electron capture and nitrogen specific gas chromatography; use of electrochemical HPLC to monitor release of norepinephrine and serotonin from cortical tissue slices; investigation of neurotransmitter release mechanisms; and effects of putative neurotransmitters on release. 2) Pharmacological and bioassay studies with in vitro recording from tissue slices from visual cortex; including determination of pharmacological responses of different types of identified neurons in tissue slices by puffer micropipette application followed by intracellular marking with fluorescent dyes or horseradish peroxidase; and use of intracellular recording and puffer micropipette application of extracts from cortex onto tissue slices as a bioassay system for possible neurotransmitters. 3) Immunohistochemical and autoradiographic localization of neurotransmitter systems; including preparation of monoclonal or serum antibodies to rat brain choline acetyltransferase; use of new or existing antibodies to choline acetyltransferase to more precisely localize cholinergic neurons in retina and in visual cortex; preparation of monoclonal or serum antibodies to glutaminase from calf brain to attempt to achieve immunohistochemical localization of glutaminergic pathways; and use of neurotransmitter-specific autoradiographic tracing with radioactively labelled transmitters or precursors. 4) Fluorescent neuronal labelling, enzymatic dissociation and fluorescence-activated cell sorting; including optimizing dye labelling and cell sorting conditions; measurement of levels of endogenous compounds in sorted cells; and analysis of proteins present in sorted cells. Identification of the neurotransmitters used in the central visual pathway is important for understanding and treating clinical disorders of this and possibly other sensory systems.
