This proposal concerns the use of capillary electrophoresis as an ultrasmall volume analysis method to investigate specific issues in neurobiology requiring quantitative single cell analysis. We have pioneered the use of capillary electrophoresis for single cell and subcellular analysis and propose to apply several new strategies to investigate these issues. First, the two compartment model of neurotransmitter storage and the effect of psychostimulants on autoreceptors and catecholamine release will be studied by capillary electrophoresis with electrochemical detection. Pharmacological and temperature manipulation will be used to examine the kinetics of neurotransmitter transfer between compartments and the generality of this model to other cell systems. Experiments are proposed to identify and quantitate substances released from cells immobilized in micro-vials having volumes in the 8 to 95 picoliter range. Second, capillary electrophoresis with fluorescence detection will be used to determine the levels of transferrin in single myelinating and nonmyelinating oligodendrocytes in culture. Combined with assays of proteins and peptides in myelin-producing vs myelin-inhibiting culture systems, these measurements should provide important information concerning oligodendrocyte growth and proliferation. Third, noncompetitive, heterogeneous, enzyme-amplified immunoassays are proposed in picoliter microvials, or conversely, to carry out detection by capillary electrochemical immunoassay. The goal is to determine the peptides, beta- endorphin and corticotropin releasing hormone, at zeptomole levels in individual cerebrospinal fluid lymphocytes involved in the etiology of the experimental disease autoimmune encephalomyelitis - a model for multiple sclerosis. Finally, it is proposed to determine these target peptides in lymphocytes of schizophrenic and normal patients.
