Presynaptic Mechanisms of Neurotransmission in NTS

描述（由申请人提供）：心血管系统的搏动调节依赖于完整的气压反射。这种反射弧首先在孤束核（NTS）突触，在那里谷氨酸被释放。血压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、可接近的中枢神经系统终末的工作表明，谷氨酸释放的突触前控制涉及离子通道和调节囊泡胞吐的第二信使系统。不同的神经元对谷氨酸的调节是不同的，但人们对其机制知之甚少。调节心血管和呼吸系统的反射通路依赖于脑干神经元，这些反射是由作用于孤束核（NTS）内的颅神经初级传入神经发起的。我们对初级事件的集中行为知之甚少。这些终端的体积小，给直接调查带来了困难。我们已经开发了允许膜片钳记录NTS单个神经末梢的方法。我们的研究计划将使用这些方法来解决我们的驱动假设，即NTS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。我们可以从单个神经末梢直接可视化、识别、刺激和电生理记录2。NTS接受药理学上可区分的传入终末，形成亚类，已知来自分子上不同的周围神经元，3。这些终端的子集可以被标记，以提供功能不同的传入事件的链接。我们的计划包括两个具有适合这一问题的互补专业知识的实验室的努力。我们将采用直接膜片记录和单端刺激，以及成像，来研究频率依赖性突触抑制和谷氨酸释放的肽调节机制。这项工作利用主传入终端的标记来区分各种子类（分别为TRPV1和P2X3； C型和a型传入终端）。具体目的是关注A型和c型颅传入突触末端钠、钾和钙通道的差异表达，以及调节突触间隙钙的新突触前机制的存在。我们还将比较不同亚类的传入神经末梢对谷氨酸释放的控制和肽的调节。

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.