MECHANISMS OF SYNAPTIC MODULATION IN SYMPATHETIC NEURONS

本建议的主要目标是：（1）描述 交感神经元中神经递质对离子电流的影响 和（2）确定细胞内生化机制 参与神经递质信号的传导， 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，并且在 几个小时 此外，牛蛙交感神经元表现出 与哺乳动物一样的突触反应 同行 交感神经节已经被用作模型 用于突触电位的研究，并已被证明是有价值的 在理解突触事件发生的机制， 神经系统中不易进入的区域，如大脑， 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，神经节将被酶促地 分离成单个细胞，以避免出现并发症 当研究完整神经节中的神经元时， 狭窄的细胞间隙， 神经递质或周围细胞的代谢物。 第二、 整个细胞记录结构中的抽吸电极将 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， 电压范围和更好的时间分辨率比以前 在这些细胞的研究中是可能的。 第三，内部 记录电极将被灌注一个细管放置在附近 电极的尖端。 通过灌注电极内部 将有可能改变细胞内成分。 第四， 单细胞灌注系统将用于控制 细胞外介质，这将允许外部介质 变化迅速，将允许精确应用 神经传递素 神经递质对离子电流的影响将是 量化。 参与的生化途径 神经递质反应将通过操纵 细胞内成分 第二信使途径是exa- 包括环核苷酸，cAMP和cGMP，以及 G蛋白激活的途径，包括甘油二酯，蛋白质 激酶C和三磷酸肌醇。

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.