Neurotransmitter function of identified cortical neurons will be studied in tissue culture. Cortical neurons, e.g. layer 5 corticocollicular cells, will be labeled by retrograde transport of fluorescent latex microspheres (beads) and put into monolayer tissue culture. The synaptic physiology and pharmacology of these identified neurons will be studied by simultaneously recording intracellularly from a labelled cell and a nearby cell to create a """"""""driver/follower"""""""" pair in which driven postsynaptic potentials can be recorded. The effects of various excitatory amino acid agonists and antagonists on these psp's will be determined. Immunocytochemistry and autoradiography will be used to provide neurotransmitter-specific labeling of the cells in culture. Identified cortical neurons labeled with fluorescent beads will be sorted on an EPICS-V cell sorter. The endogenous pattern of amino acids and related compounds and of proteins will be determined in the sorted neurons. This may identify transmitter or other molecules related to specialized neuronal function. Homogeneous sorted cells will be placed in culture to study intrinsic cellular properties of identified cells and the effects of interactions between different cell types on neuronal function. The nature of geniculate afferent and layer 6 recurrent input to cells in layer 4 will be investigated in an in vitro tissue slice preparation. Neurotransmitter systems will be characterized with histochemical staining. Combined retrograde HRP and ChAT immunocytochemical staining will be used to attempt to localize the source of cholinergic input to the LGN, superior colliculus, and vestibulo-ocular cerebellum (mossy fiber inputs). In the long term, our goal is to determine what the connections and roles are for the various neurochemically or anatomically identifiable cell types in the visual pathways. In addition to being important for understanding normal function, these studies are important for understanding abnormal function. Many neurological deficits appear to be related to failures in specific types of molecular signaling, and many neurological diseases appear to be associated with the degeneration of specific neurotransmitter pathways. Our results may have importance not only for learning more about normal visual processing, but also for learning more about neuropathological mechanisms.
