Depolarization-secretion coupling

 描述（由申请人提供）：去极化-分泌偶联被认为通过导致钙进入和随后分泌递质的电活动发生。然而，在完整的中枢神经系统（CNS）中，这种活动模式如何控制神经末梢释放神经肽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合成，并从神经垂体（NH）终末分泌，它们共同组成下丘脑-神经垂体系统（HNS）。OT神经元的特征在于在哺乳或分娩期间的高频放电，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。在这两种情况下，都是尖峰的聚集（爆发）促进了神经肽的释放。我们发现AVP和OT终末有不同的钙通道亚型，但它们的生物物理特性本身不能解释这种爆发模式对释放的差异促进作用。相反，我们已经证明，自分泌/旁分泌嘌呤能反馈效应有助于确定不同的爆发模式的electrica活动，促进释放AVP与OT的功效。沿着ATP，锌也与HNS肽共同释放。Zn ~（2+）与神经元上的许多效应物相互作用，导致多种效应。然而，尚不清楚这些效应发生在中枢神经系统突触的哪些特定部位。HNS现在提供了一个独特的机会，通过比较孤立的终端与完整的，整个系统的内源性锌在中枢神经系统中的复杂作用解开。本研究的目的是确定在电刺激的生理模式期间介导内源性锌诱导的神经肽分泌促进的分子机制。为了实现这些目标，将从成年大鼠和小鼠的HNS的鉴定的（AVP与OT）、分离的神经末梢与完整的制备物中记录ATP电流和钙电流。选择性荧光指示剂染料将监测NH末端的细胞内Ca 2+和Zn 2+变化。通过使用明确定义的体外灌注试验和电容测量，将在完整的HNS和分离的NH末端之间比较对神经肽释放的影响。该建议利用了新的可用的遗传工具，这些工具有助于阐明新型锌受体的功能，其特异性比传统拮抗剂药物更高。这些受体敲除研究将提供一个独特的机会，以确定内源性锌反馈调节是否发生在中枢神经系统神经元的终端。此外，由于突触囊泡/神经分泌颗粒似乎含有ATP和Zn 2+，这些反馈机制可能在CNS中的许多其他突触中具有生理学重要性。

 DESCRIPTION (provided by applicant): Depolarization-secretion coupling is thought to occur via electrical activity leading to the entry of calcium and the subsequent secretion of transmitters. The molecular details of how such patterns of activity control the release of neuropeptides from nerve terminals in the intact central nervous system (CNS), however, remain undetermined. Vasopressin (AVP) and oxytocin (OT) are synthesized by magnocellular neurons of the hypothalamus and secreted from neurohypophysial (NH) terminals; together they comprise the Hypothalamic-Neurohypophysial System (HNS). OT neurons are characterized by a high frequency discharge during suckling or parturition which leads to the pulsatile release of OT. AVP neurons are characterized by their asynchronous phasic activity during maintained AVP release. In both cases, it is the clustering of spikes (bursting) which facilitates neuropeptid release. We have discovered that there are different calcium-channel subtypes in AVP vs. OT terminals, but that their biophysical properties alone cannot explain the differential facilitationof release by such burst patterns. Conversely, we have demonstrated that autocrine/paracrine purinergic feedback effects help determine the efficacy of different bursting patterns of electrica activity in facilitating release of AVP vs. OT. Along with ATP, Zinc is also co-released with the HNS peptides. Zn2+ interacts with many effectors on neurons, leading to a variety of effects. It is not known, however, at what specific sites these effects occur at synapses in the CNS. The HNS now affords the unique opportunity of unraveling the complicated effects of endogenous Zinc in the CNS by comparing such effects on isolated terminals vs. the intact, whole system. The goal of the research proposed here is to determine molecular mechanisms that mediate endogenous Zinc-induced facilitation of neuropeptide secretion during physiological patterns of electrical stimulation. To achieve these objectives, recordings of ATP- and calcium-currents will be made from identified (as AVP vs. OT), isolated nerve terminals vs. intact preparations of the HNS of adult rats and mice. Selective fluorescent-indicator dyes will monitor intracellular Ca2+ and Zn2+ changes in NH terminals. Effects on neuropeptide release will be compared between the intact HNS and isolated NH terminals by the use of well-defined in vitro perfusion assays and capacitance measurements. This proposal takes advantages of newly available genetic tools that facilitate the elucidation of the function of novel Zinc receptors with greater specificty than is possible with traditional antagonist drugs. These receptor knockout studies will provide a unique opportunity to determine if endogenous Zinc feedback regulation occurs at the terminals of CNS neurons. Furthermore, since synaptic vesicles/neurosecretory granules appear to contain ATP and Zn2+, these feedback mechanisms could be physiologically important at many other synapses in the CNS.