Hay M, Hasser E M
Dalton Cardiovascular Research Center, Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA.
Am J Physiol. 1998 Aug;275(2):H710-6. doi: 10.1152/ajpheart.1998.275.2.H710.
The purpose of this study was to evaluate the use of the fluorescent membrane label FM1-43 as a measure of synaptic terminal exocytosis during stimulation of labeled aortic baroreceptor and unlabeled nodose ganglia neurons. Activation of the nerve terminals with electrical stimulation or depolarization with 90 mM KCl in the presence of 2.0 microM FM1-43 resulted in bright, punctate staining of synaptic boutons. Additional depolarization in the absence of dye resulted in destaining with a time course that was consistent and repeatable in multiple boutons within a given terminal. Destaining was dependent on calcium influx and was blocked by bath application of 100 microM CdCl2. Whole cell patch-clamp studies have reported that depolarization-induced calcium influx in aortic baroreceptor cell bodies is predominantly caused by the activation of omega-conotoxin GVIA (omega-CgTx)-sensitive N-type calcium channels. In addition, these N-type channels have been shown to be inhibited by activation of metabotropic glutamate receptors. In the present study, exocytosis in aortic baroreceptor terminals was not affected by bath application of 5 microM nifedipine and only partially inhibited by bath application of 2.0 microM omega-CgTx. However, depolarization-induced exocytosis was significantly inhibited by bath application of 200 microM L-AP4, a type III metabotropic glutamate receptor agonist. Results from this study suggest that 1) FM1-43 can be used to measure synaptic vesicle exocytosis in baroreceptor neurons; 2) the N-type calcium channel may not be involved in the initial phase of vesicle exocytosis; and 3) activation of L-AP4-sensitive metabotropic glutamate receptors inhibits 90 mM KCl-induced vesicle release.
本研究的目的是评估荧光膜标记物FM1-43在刺激标记的主动脉压力感受器和未标记的结状神经节神经元时作为突触终末胞吐作用指标的用途。在存在2.0微摩尔FM1-43的情况下，用电刺激或用90毫摩尔氯化钾进行去极化激活神经终末，会导致突触小体出现明亮的点状染色。在没有染料的情况下进行额外的去极化会导致褪色，其时间进程在给定终末内的多个小体中是一致且可重复的。褪色依赖于钙内流，并可被浴槽施加100微摩尔氯化镉阻断。全细胞膜片钳研究报告称，主动脉压力感受器细胞体中去极化诱导的钙内流主要是由ω-芋螺毒素GVIA（ω-CgTx）敏感的N型钙通道激活引起的。此外，这些N型通道已被证明可被代谢型谷氨酸受体的激活所抑制。在本研究中，主动脉压力感受器终末的胞吐作用不受浴槽施加5微摩尔硝苯地平的影响，仅被浴槽施加2.0微摩尔ω-CgTx部分抑制。然而，浴槽施加200微摩尔L-AP4（一种III型代谢型谷氨酸受体激动剂）可显著抑制去极化诱导的胞吐作用。本研究结果表明：1）FM1-43可用于测量压力感受器神经元中的突触小泡胞吐作用；2）N型钙通道可能不参与小泡胞吐作用的初始阶段；3）L-AP4敏感的代谢型谷氨酸受体的激活会抑制90毫摩尔氯化钾诱导的小泡释放。
