Depolarization-Secretion Coupling in Nerve Terminals

描述（由申请人提供）：尽管有大量证据表明神经元体的电活动导致Ca2+的进入和随后的递质分泌（即去极化-分泌偶联），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。抗利尿激素（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。在哺乳期间，OT神经元的特点是高频放电，导致OT脉冲释放。AVP神经元在持续的AVP释放过程中具有非同步相活动（爆发）的特征。在这两种情况下，虽然每个肽的时间过程不同，但正是聚类促进了激素的释放。我们发现在AVP和OT末端存在不同的钙通道亚型，但它们的生物物理特性不能解释这种促进释放的差异。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促进AVP与OT释放的有效性。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上的特定受体相互作用，导致各种各样的影响。然而，尚不清楚这些影响是否发生在中枢神经系统（CNS）的躯体和/或突触上。我们描述了外源性嘌呤对中枢神经系统的电和分泌作用，包括对这些中枢神经系统神经元及其神经末梢的膜离子电导率的影响。通过比较内源性嘌呤对不同神经元室的影响，HNS提供了独特的机会来揭示内源性嘌呤在中枢神经系统中的复杂作用。我们的目标是确定在电刺激的生理模式中，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，Ca2+和K+电流的穿孔贴片记录将从鉴定的、分离的神经末梢和HNS的躯体中进行。通过使用elisa和电容测量来比较完整的HNS和NH端对释放的影响。从完整HNS的神经末梢和躯体中松散的膜片钳记录将允许分析破裂活动如何调节整个系统中的肽释放。这些研究将提供一个独特的机会来确定内源性嘌呤能反馈调节是否发生在中枢神经系统神经元的末端。

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.