Depolarization-Secretion Coupling in Nerve Terminals

描述（由申请人提供）：去极化-分泌偶联被认为是通过导致钙进入和随后的递质分泌的电活动发生的。然而，在完整的中枢神经系统（CNS）中，钙和其他离子电流如何控制神经末梢神经肽释放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合成，并从神经垂体（NH）末端分泌；它们共同构成下丘脑-神经下丘脑系统（HNS）。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，是尖峰的聚集促进了神经肽的释放。我们发现在AVP和OT终末中存在不同的钙通道亚型，但它们的生物物理特性不能单独解释这种突发模式对释放的不同促进作用。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对促进AVP和OT释放的功效。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，导致各种各样的影响。这是未知的；然而，在中枢神经系统的突触上，这些效应发生在哪些特定的受体上。我们通过P2X2， P2X3和A1受体表征了嘌呤对HNS末端的电和分泌作用，包括对这些中枢神经系统末端离子电导率的影响。HNS提供了独特的机会，通过比较内源性嘌呤在孤立末端和完整的整个系统中的作用，揭示了内源性嘌呤在中枢神经系统中的复杂作用。本研究的目的是确定在电刺激的生理模式下，内源性嘌呤能诱导神经肽分泌的膜机制。为了实现这些目标，将对成年大鼠和小鼠经鉴定的分离神经末梢与完整的HNS制剂进行静息电位、钙电位和动作电位电流的穿孔和松散膜片钳记录。通过使用elisa和电容测量来比较完整的HNS和分离的NH末端对释放的影响。这一建议现在利用了新的遗传工具的优势，这些工具有助于阐明这些嘌呤能受体的功能，比传统的拮抗剂具有更大的特异性。此外，由于所有突触囊泡/神经分泌颗粒似乎都含有ATP，这些嘌呤能反馈机制可能在中枢神经系统的许多其他突触中具有重要的生理意义。这些受体敲除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。公共卫生相关性：嘌呤、ATP和腺苷介导的影响包括伤害感觉和机械感觉转导、神经传递抑制、睡眠诱导、抗缺血、乙醇诱导的运动不协调、心功能的自主控制和肾钠潴留。药物抑制剂的使用表明嘌呤能受体在突触可塑性中起关键作用，并可能在帕金森病和亨廷顿病中起作用；然而，这些拮抗剂对嘌呤能受体家族的个体成员没有足够的特异性。该建议利用了新获得的遗传工具（基因敲除）的优势，这些基因工具有助于阐明这些嘌呤能受体的功能，比传统拮抗剂具有更大的特异性，并为确定中枢神经系统突触的反馈效应提供了希望。只有这样，治疗药物才能针对生理上相关的嘌呤能受体来缓解这类疾病。

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.