Depolarization-Secretion Coupling in Nerve Terminals

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。钙和其他离子电流如何控制神经肽从完整的中枢神经系统（CNS）的神经末梢释放的分子细节，然而，仍然没有确定。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，正是尖峰的聚集促进了神经肽的释放。我们已经发现，有不同的钙通道亚型AVP与OT终端，但它们的生物物理特性本身不能解释这种突发模式的差异促进释放。因此，我们假设自分泌/旁分泌反馈效应可能有助于确定不同的电活动爆发模式的功效，以促进AVP与OT的释放。ATP与HNS肽共同释放。嘌呤，如ATP和腺苷，与神经元上的特定受体相互作用，导致各种效应。目前尚不清楚;然而，在什么特定的受体，这些影响发生在中枢神经系统中的突触。我们已经表征了嘌呤通过P2 X2、P2 X3和A1受体对HNS终端的电和分泌作用，包括对这些CNS终端中的离子电导的影响。HNS提供了一个独特的机会，通过比较孤立的终端与完整的，整个系统的内源性嘌呤在中枢神经系统中的复杂作用解开。本文提出的研究目标是确定介导内源性嘌呤能诱导的电刺激生理模式期间神经肽分泌功效的膜机制。为了实现这些目标，将从成年大鼠和小鼠的HNS的已鉴定的、分离的神经末梢与完整制备物中进行静息电位、钙电流和动作电位电流的穿孔和松散膜片钳记录。将通过使用ELISA和电容测量比较完整HNS和分离NH末端之间对释放的影响。该建议现在利用了新的遗传工具，这些工具有助于阐明这些嘌呤能受体的功能，具有比传统拮抗剂药物更高的特异性。此外，由于所有的突触囊泡/神经分泌颗粒似乎含有ATP，这些嘌呤能反馈机制可能是在CNS中的许多其他突触生理上重要的。这些受体敲除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。公共卫生相关性：嘌呤、ATP和腺苷介导的作用包括伤害感受和机械感觉转导、神经传递抑制、睡眠诱导、抗缺血、乙醇诱导的运动不协调、心脏功能的自主控制和肾钠潴留。药理学抑制剂的使用表明嘌呤能受体在突触可塑性中的关键作用以及在帕金森病和亨廷顿病中的可能作用;然而，这些拮抗剂对于嘌呤能受体家族的个体成员不够特异。该建议利用了新的可用的遗传工具（基因敲除），这些基因敲除有助于阐明这些嘌呤能受体的功能，其特异性比传统拮抗剂药物通常可能的更高，并为确定CNS突触的反馈效应带来了希望。只有这样，治疗药物才能靶向生理相关的嘌呤受体，以减轻此类疾病。

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 calcium and other ionic currents 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 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 of spikes which facilitates neuropeptide 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 facilitation of release by such burst patterns. Therefore, we hypothesize that autocrine/paracrine feedback effects might help determine the efficacy of different bursting patterns of electrical activity to facilitate release of AVP vs. OT. ATP is co-released with the HNS peptides. Purines, such as ATP and adenosine, interact with specific receptors on neurons, leading to a variety of effects. It is not known; however, at what specific receptors these effects occur at synapses in the CNS. We have characterized the electrical and secretory effects on the HNS terminals by purines via P2X2, P2X3 and A1 receptors, including effects on ionic conductances in these CNS terminals. The HNS affords the unique opportunity of unraveling the complicated effects of endogenous purines in the CNS by comparing such effects on isolated terminals vs. on the intact, whole system. The goal of the research proposed here is to determine membrane mechanisms that mediate endogenous purinergic-induced efficacy of neuropeptide secretion during physiological patterns of electrical stimulation. To achieve these objectives, perforated and loose patch-clamp recordings of resting potential, calcium- and action potential-currents will be made from identified, isolated nerve terminals vs. intact preparations of the HNS of adult rats and mice. Effects on release will be compared between the intact HNS and isolated NH terminals by the use of ELISAs and capacitance measurements. This proposal now takes advantages of newly available genetic tools that facilitate the elucidation of the function of these purinergic receptors with greater specificity than is possible with traditional antagonist drugs. Furthermore, since all synaptic vesicles/neurosecretory granules appear to contain ATP, these purinergic feedback mechanisms could be physiologically important at many other synapses in the CNS. These receptor knockout studies will provide a unique opportunity to determine if endogenous purinergic feedback regulation occurs at the terminals of CNS neurons. PUBLIC HEALTH RELEVANCE: The purines, ATP and Adenosine, mediate effects that include nociception and mechanosensory transduction, depression of neurotransmission, sleep induction, anti-ischemia, ethanol-induced motor incoordination, autonomic control of cardiac function, and renal sodium retention. The use of pharmacological inhibitors has suggested a key role for purinergic receptors in synaptic plasticity and possible roles in Parkinson's and Huntington's diseases; however, these antagonists are not specific enough for individual members of the purinergic receptor family. This proposal takes advantages of newly available genetic tools (knockouts) that facilitate the elucidation of the function of these purinergic receptors with greater specificity than is generally possible with traditional antagonist drugs and gives hope for the determination of such feedback effects at CNS synapses. Only then can therapeutic drugs be targeted to physiologically relevant purinergic receptors to alleviate such diseases.