Depolarization-Secretion Coupling in Nerve Terminals

描述（由申请人提供）：尽管有相当多的证据表明神经元胞体的电活动导致Ca 2+的进入和随后的递质分泌（即，去极化-分泌耦合），离子电流如何控制特定神经活性物质从神经末梢释放的分子细节仍然没有确定。加压素（AVP）和催产素（OT）由下丘脑大细胞神经元（MCN）合成，由神经垂体（NH）终末分泌。OT神经元的特征在于在哺乳期间的高频放电，其导致OT的脉冲式释放。AVP神经元的特征在于它们在维持AVP释放期间的异步阶段性活动（爆发）。在这两种情况下，尽管每种肽具有不同的时间过程，但尖峰的聚集促进了激素的释放。我们已经发现，在AVP和OT终末中存在不同的钙通道亚型，但是它们的生物物理特性不能解释这种差异的释放促进作用。因此，我们假设自分泌/旁分泌反馈效应决定了电活动的爆发模式的功效，以促进AVP与OT的释放。ATP被认为与HNS肽共同释放。嘌呤，如ATP和腺苷，与神经元和神经胶质细胞上的特异性受体相互作用，导致各种效应。然而，尚不清楚这些影响是否发生在中枢神经系统（CNS）的胞体和/或突触上。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神经元与其神经末梢的膜离子电导的影响。通过比较内源性嘌呤对不同神经元隔室的影响，HNS提供了解开内源性嘌呤在CNS中的复杂作用的独特机会。我们的目标是确定介导内源性嘌呤能诱导的神经激素分泌在电刺激的生理模式的修改的膜机制。为了实现这些目标，穿孔补丁记录的钙离子和K+电流将从确定的，孤立的神经末梢和胞体的HNS。通过使用ELISA和电容测量，比较完整HNS和NH末端对释放的影响。松散的膜片钳记录神经末梢和胞体在完整的HNS将允许分析如何突发活动调节肽释放在整个系统。这些研究将提供一个独特的机会，以确定是否内源性嘌呤反馈调节发生在中枢神经系统神经元的终端。

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.