Depolarization-Secretion Coupling in Nerve Terminals

描述（由申请人提供）：虽然有大量证据表明神经元胞体的电活动导致 Ca2 的进入并随后分泌递质（即去极化-分泌耦合），但离子电流如何控制神经末梢释放特定神经活性物质的分子细节仍未确定。加压素 (AVP) 和催产素 (OT) 由下丘脑的大细胞神经元 (MCN) 合成，并从神经垂体 (NH) 末梢分泌。 OT 神经元的特点是在哺乳期间高频放电，导致 OT 的脉动释放。 AVP 神经元的特征在于其在维持 AVP 释放期间的异步阶段性活动（爆发）。在这两种情况下，虽然每种肽的时间进程不同，但尖峰的聚集促进了激素的释放。我们发现 AVP 与 OT 终端存在不同的钙通道亚型，但它们的生物物理特性无法解释这种不同的释放促进作用。因此，我们假设自分泌/旁分泌反馈效应决定了电活动爆发模式的功效，以促进 AVP 与 OT 的释放。 ATP 被认为是与 HNS 肽共同释放的。嘌呤（例如 ATP 和腺苷）与神经元和神经胶质细胞上的特定受体相互作用，从而产生多种效应。然而，尚不清楚这些影响是否存在于中枢神经系统 (CNS) 的体细胞和/或突触。我们已经表征了外源性嘌呤对 HNS 的电和分泌作用，包括对这些 CNS 神经元及其神经末梢的膜离子电导的影响。 HNS 提供了独特的机会，通过比较不同神经元区室的内源性嘌呤对中枢神经系统的复杂影响，揭示这些影响。我们的目标是确定在电刺激的生理模式期间介导内源性嘌呤能诱导的神经激素分泌改变的膜机制。为了实现这些目标，将从已识别的、孤立的 HNS 神经末梢和体细胞中进行 Ca2 和 K 电流的穿孔贴片记录。将通过使用 ELISA 和电容测量来比较完整的 HNS 和 NH 末端对释放的影响。来自完整 HNS 中神经末梢和体细胞的松散膜片钳记录将允许分析爆发活动如何调节整个系统中的肽释放。这些研究将为确定中枢神经系统神经元末端是否发生内源性嘌呤能反馈调节提供独特的机会。

DESCRIPTION (provided by applicant): Although there is considerable evidence that the electrical activity of neuronal somata leads to the entry of Ca2+ and to the subsequent secretion of transmitters (i.e., Depolarization-secretion coupling), the molecular details of how ionic currents control the release of specific neuroactive substances from nerve terminals remain undetermined. Vasopressin (AVP) and oxytocin (OT) are synthesized by magnocellular neurons (MCN) of the hypothalamus and secreted from neurohypophysial (NH) terminals. OT neurons are characterized by a high frequency discharge during suckling which leads to the pulsatile release of OT. AVP neurons are characterized by their asynchronous phasic activity (bursting) during maintained AVP release. In both cases, it is the clustering, albeit with different time courses for each peptide, of spikes, which facilitates hormone release. We have discovered that there are different Calcium-channel subtypes in AVP vs. OT terminals, but that their biophysical properties cannot explain this differential facilitation of release. Therefore, we hypothesize that autocrine/paracrine feedback effects determine efficacy of bursting patterns of electrical activity to facilitate release of AVP vs. OT. ATP is thought to be co-released with the HNS peptides. Purines, such as ATP and adenosine, interact with specific receptors on neurons and glia, leading to a variety of effects. It is not known, however, whether these effects are at somata and/or synapses in the central nervous system (CNS). We have characterized the electrical and secretory effects on the HNS by exogenous purines, including effects on membrane ionic conductances in these CNS neurons vs. their nerve terminals. The HNS affords the unique opportunity of unraveling the complicated effects of endogenous purines in the CNS by comparing such effects on different neuronal compartments. Our goal is to determine membrane mechanisms that mediate endogenous purinergic- induced modifications of neurohormone secretion during physiological patterns of electrical stimulation. To achieve these objectives, perforated-patch recordings of Ca2+ and K+ currents will be made from identified, isolated nerve terminals and somata of the HNS. Effects on release will be compared between the intact HNS and NH terminals by the use of ELISAs and capacitance measurements. Loose patch-clamp recordings from nerve terminals and somata in the intact HNS will allow analysis of how bursting activity regulates peptide release in the complete system. These studies will provide a unique opportunity to determine if endogenous purinergic feedback regulation occurs at the terminals of CNS neurons.