DEVELOPMENT AND MODULATION OF PRESYNAPTIC ION CHANNELS

该提案要求为我正在进行的研究项目提供工资支持 发展中国家因此，它侧重于两个相关但具体的领域， 过去两年在实验室进行的研究：（1） 突触前离子通道的发育及其在胆碱能神经系统中的作用 体外静脉曲张;（2）生长抑素对神经元 钙（Ca++）通道。两者都是我长期目标的组成部分 制定一个强有力的研究计划，在发展，监管， 神经元突触前功能的调节。 第一个具体的目标是集中在突触前离子的发展 通道及其在体外胆碱能静脉曲张中的作用。这些 本实验旨在研究突触前神经元的诱导， 表达的特异性（Ca++和钙激活的K+通道）， 在培养物中沿着沿着形成的释放递质的静脉曲张 非洲爪蟾脊髓神经元和肌细胞。使用膜片钳 技术，我建议（1）直接表征电流的类型 存在于新形成的突触前结构，（2）确定它们的作用 在发射机释放调节中，以及（3）识别小区-小区 调节这些特化表达的相互作用。从 这些研究应该能更全面地了解运动神经 末端Ca++和钙激活的K+通道，它们在 递质释放和突触前特化的诱导。 第二个具体目标是集中在调制的机制， 副交感神经元的钙通道。生长抑素对 将在睫状神经节神经元中研究Ca++电流，作为 在脉络膜神经末梢发生的调制。很难 直接研究突触前离子电流的调制， 准备.这在很大程度上是由于规模小， 发射器释放区域的不可接近性。急性分离 睫状神经节神经元将在体外用作模型，并且使用 膜片钳技术，我建议阐明的信号转导 将生长抑制素受体与Ca++抑制偶联的级联反应 渠道拟议的实验将提供有价值的见解， 钙离子通道的调控机制。 独立科学家奖（NINDS - RCDA）将提供自由 为了实现这些研究目标，我不会花费太多时间， 教学和行政职责。神经科学系 匹兹堡大学是一个理想的环境，在那里我可以 促进和利用科学交流的气氛。那里 这个系里的很多研究人员 突触生理学某些方面（LTP、NMDA受体研究、运动 控制，离子通道调节和突触的生物化学研究 传输）。从很多方面来说，这是一个让我成长的理想环境 专业，并制定了强大的研究计划，在突触 physiology.

The proposal requests salary support for the research program that I am developing. As such, it is focused on two related, but specific areas of research developed in the laboratory over the past two years: (1) development of presynaptic ion channels and their role at cholinergic varicosities in vitro, and (2) somatostatin modulation of neuronal calcium (Ca++ ) channels. Both are integral parts in my long-term goal to develop a strong research program in the development, regulation, and modulation of neuronal presynaptic function. The first specific aim is focused on the development of presynaptic ion channels and their role at cholinergic varicosities in vitro. These experiments are designed to study the induction of presynaptic specialization (Ca++ and calcium-activated K+ channels) expressed at transmitter-releasing varicosities that form along neurites in cultures of Xenopus spinal cord neurons and myocytes. Using patch clamp techniques, I propose to (1) characterize directly the types of currents present at newly formed presynaptic structures, (2) determine their role in transmitter release regulation, and (3) identify cell-cell interactions that regulate the expression of these specializations. From these studies should come a more thorough understanding of motor nerve terminal Ca++ and calcium-activated K+ channels, their role in transmitter release and the induction of presynaptic specialization. The second specific aim is focused on the mechanisms of modulation of Ca++ channels in parasympathetic neurons. The effects of somatostatin on Ca++ currents will be studied in ciliary ganglion neurons as a model for modulation that occurs at choroid nerve terminals. It is very difficult to study directly the modulation of presynaptic ionic currents in this preparation. This is due, in large part, to the small size and inaccessibility of the transmitter releasing regions. Acutely dissociated ciliary ganglion neurons will be used in vitro as a model, and using patch clamp techniques, I propose to elucidate the signal transduction cascade that couples somatostatin receptors to inhibition of Ca++ channels. The proposed experiments will provide valuable insights into the mechanisms of modulation of Ca++ channels. The Independent Scientist Award (NINDS - RCDA) would provide the freedom to pursue these research goals without large demands on my time for teaching and administrative duties. The Department of Neuroscience at the University of Pittsburgh is an ideal environment in which I can both contribute to, and draw from, an atmosphere of scientific exchange. There are many researchers in this department engaged in research addressing some aspect of synaptic physiology (LTP, NMDA receptor studies, motor control, ion channel regulation, and biochemical studies of synaptic transmission). In many ways, this is an ideal environment for me to grow professionally, and develop a strong research program in synaptic physiology.