Microanalytical Methods in Neurochemistry

本研究关注的是用于细胞内易氧化神经递质测定的超小伏安电极的开发和应用。这项研究的目标之一是构建比目前使用的伏安电极更小的伏安电极（尖端直径1-2微米）。尖端直径小于100纳米的电极应该是可以制造出来的，并且可以用于刺穿非常小的神经元。利用石英微血管内烃类热解制备电极，对池螺（Planorbis corneus）巨多巴胺神经元细胞体进行伏安测定。转运到细胞的速率，多巴胺的代谢和泡化将在单个细胞的水平上进行评估。此外，还将开发一种新的工具来研究多巴胺总储存的药理作用，并研究多巴胺摄取到突触后细胞体的可能性。进行大鼠黑质、蓝斑、下丘脑和纹状体的原代培养。培养的游离神经元可作为研究神经递质在细胞体中的功能的模型系统。此外，超小型伏安电极和可见神经元过程的结合将允许在终端纤维束附近放置传感器，并对单个轴突进行局部刺激。因此，可以从单个神经元观察到神经递质的释放。虽然计划中的大多数实验都涉及多巴胺，但去甲肾上腺素和血清素也将研究它们在影响多巴胺神经化学中的作用。由于神经递质在神经元细胞体中的功能尚未得到深入研究，因此本实验为研究神经元通信的这一维度提供了一种新的途径。

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.