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钙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特定神经活性物质从神经末梢释放的分子细节仍未确定。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。催产素神经元在哺乳过程中以高频放电为特征，导致催产素的搏动性释放。AVP神经元的特征是在维持AVP释放的过程中其异步期活动(爆发)。在这两种情况下，促进激素释放的是尖峰的聚集，尽管每个多肽的时间进程不同。我们发现，AVP和OT终末存在不同的钙通道亚型，但它们的生物物理性质不能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有效性，以促进AVP与OT的释放。ATP被认为是与HNS多肽共同释放的。嘌呤，如三磷酸腺苷和腺苷，与神经元和神经胶质细胞上的特定受体相互作用，导致各种效应。然而，目前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。我们已经确定了外源性嘌呤对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。HNS提供了独特的机会，通过比较内源性嘌呤对不同神经元的影响，来揭示内源性嘌呤在中枢神经系统中的复杂影响。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。为了达到这些目的，将从识别的、分离的HNS的神经末梢和胞体中记录钙电流和钾电流的穿孔贴片记录。使用ELISA和电容测量来比较完整的HNS和NH端子对释放的影响。完整的HNS中神经末梢和胞体的松散膜片钳记录将使我们能够分析爆发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终末。

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.