Microanalytical Methods in Neurochemistry

This research concerns the development and application of ultra-small voltammetric electrodes for intracellular determination of easily oxidized neurotransmitters. One goal of this research is to construct voltammetric electrodes smaller than those presently used (1-2 um tip diameters). Electrodes with tip diameters less than 100 nm should be possible to fabricate and can be used for impaling very small neurons. Electrodes fabricated by hydrocarbon pyrolysis inside quartz microcapillaries will be used for voltammetry inside the cell body of the Giant dopamine neuron of Planorbis corneus (pond snail). The rate of transport into the cell, metabolism and vesicularization of dopamine will be evaluated on the level of a single cell. In addition, a novel tool for study of pharmacological effects on total dopamine stores will be developed and the possibility of dopamine uptake into post-synaptic cell bodies will be investigated. Primary cultures of rat substantia nigra, locus coeruleus, hypothalamus and striatum will be carried out. Dissociated neurons in culture will serve as a model system to investigate the function of neurotransmitter in the cell body. In addition, the combination of ultra-small voltammetric electrodes and visible neuronal processes will permit placement of a sensor near a bundle of terminal fibers with local stimulation of a single axon. Hence, release of neurotransmitter might be observed from a single neuron. Although most of the experiments planned concern dopamine, norepinephrine and serotonin will also be examined with respect to their roles in influencing dopamine neurochemistry. Since the functions of neurotransmitters in neuronal cell bodies have not been heavily investigated, the experiments here represent a novel approach to the study of this dimension of neuronal communication.