This application for a First Award seeks research support for five years to study the afferent control of substantia nigra dopaminergic neuron activity. Many lines of evidence point towards a dopaminergic component in schizophrenia, but there is actually no evidence that the dopaminergic neurons themselves are dysfunctional, and the possibility exists that the problem is one of altered afferent modulation of dopaminergic neuron activity. Previous attempts to study the anatomy and physiology of afferents nigral dopaminergic neurons have been hindered by the complex cytoarchitectonic organization of the substantia nigra, the lack of identification of the neuron being studied as dopaminergic, and uncertainty as to the site of origin of evoked electrophysiological responses or synapses contacting nigral neurons. This application proposes to study dopaminergic afferents by electrophysiological- neuroanatomical techniques in which dopaminergic neurons will be electrophysiologically identified in vivo and recorded intracellularly with horseradish peroxidase (HRP)-containing electrodes. Intrinsic properties and responses to stimulation of suspected afferents will be observed and measured, and then the neuron will be intracellularly injected with HRP and processed for sequential light and electron microscopic analyses. HRP-stained neurons will be examined, drawn and photographed with a light microscope, and representative regions of the entire dendritic extent of identified and individually labelled dopaminergic neuron will be trimmed out, re-sectioned on an ultramicrotome, and examined in the electron microscope, where a qualitative and quantitative analyses of afferent synapses will be conducted. Immunocytochemical labelling of synaptic terminals made onto these neurons will be used to correlate morphologies of synaptic endings with specific neurotransmitters and neurotransmitter-related substances known to exist in substantia nigra, for example, glutamic acid decarboxylase,'substance P, choline acetyltransferase, enkephalin, dopamine beta-hydroxylase and serotonin. The sites of origin of afferents to elec- trophysiologically identified, HRP-filled nigral dopaminergic neurons will be studied by electron microscopic anterograde tracing experiments using Phaseolus vulgaris leucoagglutinin. Lesion degeneration studies will be performed as well in order to identify the sources of both physiologically observed responses to afferent stimulation as well as to correlate physiological responses with the ultrastructural morphology of synapses onto identified dopaminergic neurons in substantia nigra.
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