MECHANISMS OF SYNAPTIC MODULATION IN SYMPATHETIC NEURONS

这项建议的主要目标是：(1)描述 交感神经元中神经递质对离子电流的影响 以及(2)确定细胞内生化机制 参与神经递质信号的转导 这些细胞中的膜受体和离子通道。腰椎 将使用牛蛙的椎旁交感神经节 因为它们是离散的、易于访问的，并且在 体外培养数小时。此外，牛蛙的交感神经元表现出 与他们的哺乳动物相同类型的突触反应 对口单位。交感神经节已被用作动物模型 用于研究突触电位，并已被证明是有价值的 在理解脑内突触事件发生的机制方面 神经系统中较难接触的区域，如大脑和 脊髓。 最近的几项技术创新将应用于这项研究 这些神经节。首先，神经节将被酶处理 分离到单个细胞，以避免出现并发症 在研究由于离子堆积而导致的完整神经节中的神经元时 狭窄的细胞间隙和由于释放的 周围细胞的神经递质或代谢物。第二, 整个单元记录配置中的吸入电极将 用于控制膜电压和记录离子电流， 允许在更宽的范围内更精确地控制膜电位 电压范围和比以前更好的时间分辨率 在这些细胞的研究中是可能的。第三，机身内部 记录电极将与放置在附近的细管一起灌流 电极的尖端。通过向电极内部灌流 改变细胞内的成分将是可能的。第四, 将使用单细胞灌流系统来控制 胞外培养基使外部培养基能 变化迅速，并将允许精确应用 神经递质。 神经递质对离子电流的影响将是 量化的。参与生物化学过程的生物化学途径 神经递质的反应将通过操纵 胞内成分。第二条信使通路将被排除- 开采的包括环核苷酸、cAMP和cGMP，以及 G蛋白激活的通路，包括二酰甘油、蛋白质 激酶C和肌醇三磷酸。

The major goals of this proposal are: (1) to characterize the ionic currents affected by neurotransmitters in sympathetic neurons and (2) to determine the intracellular biochemical mechanisms involved in the transduction of neurotransmitter signals between membrane receptors and ion channels in these cells. The lumbar paravertebral sympathetic ganglia from bullfrogs will be used because they are discrete, easily accessible, and remain viable in vitro for hours. Furthermore, bullfrog sympathetic neurons exhibit the same types of synaptic responses as do their mammalian counterparts. The sympathetic ganglia have been used as a model for the study of synaptic potentials and have proven to be valuable in understanding the mechanisms of synaptic events occurring in less accessible areas of the nervous system, such as the brain and spinal cord. Several recent technical innovations will be applied to the study of these ganglia. First, the ganglia will be enzymatically dissociated to single cells to avoid the complications which arise when studying neurons in intact ganglia due to ion accumulation in the narrow intercellular spaces and due to the release of neurotransmitters or metabolites from surrounding cells. Second, a suction electrode in the whole cell recording configuration will be used to control membrane voltage and record ionic currents, allowing a more precise control of membrane potential over a wider voltage range and with better time resolution than has previously been possible in studies of these cells. Third, the inside of the recording electrode will be perfused with a fine tube placed near the tip of the electrode. By perfusing the inside of the electrode it will be possible to change intracellular constituents. Fourth, a single cell perfusion system will be used for controlling the extracellular medium which will allow the external medium to be changed rapidly and will permit precise application of neurotransmitters. The effects of neurotransmitters on ionic currents will be quantified. The biochemical pathways involved in the neurotransmitter responses will be determined by manipulating the intracellular constituents. Second messenger pathways to be exa- mined include the cyclic nucleotides, cAMP and cGMP, and the pathways activated by G proteins, including diacylglycerol, protein kinase C and inositol trisphosphate.