Presynaptic Mechanisms of Neurotransmission in NTS

描述（由申请人提供）：心血管系统的逐搏调节取决于完整的压力反射。这种反射弧首先在孤束核（NTS）中形成突触，在那里释放谷氨酸。压力反射功能在常见的危及生命的疾病状态中受损：高血压、休克和心力衰竭。在大的、可接近的CNS末梢中的工作表明，谷氨酸释放的突触前控制涉及调节囊泡胞吐的离子通道和第二信使系统。谷氨酸调节在神经元之间存在差异，但其机制知之甚少。调节心血管和呼吸系统的反射通路依赖于脑干神经元，并且这些反射由作用于孤束核（NTS）内的颅神经初级传入发起。人们对初级传入神经如何集中行为知之甚少。这些码头的面积很小，直接调查很困难。我们已经开发了方法，允许膜片钳记录从单个神经末梢在NTS。我们的研究计划将使用这些方法来解决我们的驱动假说，即NTS中的重要调节机制取决于传入神经元的身份。我们的初步工作表明，NTS神经元提供了一个独特的机会，因为：1。可以直接从单个神经末梢进行可视化、识别、刺激和电生理记录; NTS接收可区分的传入终末，这些终末形成已知从分子上不同的外周神经元产生的亚类。这些终端的子集可以被标记以提供与功能不同的传入的链接。我们的计划包括两个实验室的努力，它们具有适合这个问题的互补专业知识。我们将采用直接膜片钳记录和刺激单个终末，以及成像，研究频率依赖性突触抑制和谷氨酸释放的肽调节机制。这项工作利用初级传入末梢的标记来区分各种亚类（TRPV 1和P2 X3;分别为C型和A型传入末梢）。具体目标涉及跨A型和C型颅传入突触末梢的差异钠、钾和钙通道表达，以及调节突触间隙钙的新突触前机制的存在。传入神经末梢的不同亚类也将在它们对谷氨酸释放的控制和肽的调节方面进行比较。

DESCRIPTION (provided by applicant): Beat to beat regulation of the cardiovascular system depends on an intact baroreflex. This reflex arc first synapses in the nucleus tractus solitarius (NTS) where glutamate is released. Baroreflex function is compromised in common life threatening disease states: hypertension, shock, and heart failure. Work in large, accessible CNS terminals suggests that the presynaptic control of glutamate release involves ion channels and 2nd messenger systems that regulate vesicle exocytosis. Glutamate regulation differs across neurons, but the mechanisms are poorly understood. Reflex pathways regulating the cardiovascular and respiratory systems depend on brainstem neurons and these reflexes are initiated by cranial nerve primary afferents acting within the nucleus tractus solitarius (NTS). Little is known about how primary afferents behave centrally. The small size of these terminals has made direct investigation difficult. We have developed methods to permit patch clamp recording from single nerve terminals in NTS. Our Research Plan will use these methods to address our driving hypothesis that important mechanisms of regulation in NTS depend on the identity of the afferent neuron. Our Preliminary Work demonstrates that NTS neurons offer a unique opportunity because: 1. we can directly visualize, identify, stimulate and electrophysiologically record from single nerve terminals, 2. NTS receives pharmacologically distinguishable afferent terminals that form subclasses known to arise from molecularly distinct peripheral neurons, 3. Subsets of these terminals can be labeled to provide links to functionally distinct afferents. Our Plan encompasses the efforts of two labs with complementary expertise suited to this problem. We will use direct patch recording and stimulation of single terminals, as well as imaging, to study the mechanism of frequency dependent synaptic depression and peptide modulation of glutamate release. The work capitalizes on using markers of primary afferent terminals to distinguish the various sub classes (TRPV1 and P2X3; C- and A-type afferent terminals, respectively). Specific Aims concern differential sodium, potassium and calcium channel expression across A- and C-type cranial afferent synaptic terminals plus the presence of new presynaptic mechanisms regulating synaptic cleft calcium. The different sub classes of afferent nerve terminal will be also be compared in terms of their control of glutamate release and modulation by peptides.