Calcium-dependent autoregulation of vasopressin neurons in a rodent model of heart failure.

项目摘要 这个K99/R 00的提议将测试一个关于加压素（VP）神经元如何自我调节其神经元的关键假设。 兴奋性，其局部旁分泌血管加压素的释放，以及这些机制如何在啮齿动物模型中改变， 心力衰竭，同时为PI提供额外的科学和专业技能，使其成功过渡到 作为研究教授的独立职业。 心力衰竭（HF）是全球范围内的主要死亡原因。HF的标志是交感神经过度活跃， 神经激素如VP的循环水平。慢后超极化（sAHP）被认为是一种重要的机制 影响VP放电，在形成VP神经元的定型阶段性爆发模式中起关键作用。的 VP HF神经元sAHP受损。独特的是，VP的Ca 2+依赖性胞吐作用发生在体树突（VP-SDR）， 以旁分泌的方式作为一种自我抑制反馈机制。目前尚不清楚VP-SDR是否是 VP HF神经元受损。本文提供的数据显示sAHP和VP-SDR共享许多共同的信号传导途径 这两种现象被单独观察到，但从未同时观察到。此外，我们有令人兴奋的初步数据表明， VP本身可以增强分离的sAHPs，表明内源性VP-SDR可以调节sAHPs的时间进程，从而 射击活动。了解这些机制如何导致异常的VP神经元活动在HF是至关重要的。 因此，我提出了一个独特的假设，即sAHP和VP-SDR激活不仅是由相同的激活。 信号和途径，但VP-SDR可以直接调节sAHPs。我假设观察到的损伤 HF神经元中sAHP的降低反映了受损的细胞内Ca 2+信号传导和VP-SDR，导致 在这种情况下观察到过度兴奋和过量外周VP释放。目标1将检验假设， VP激活模式（例如，连续相对于阶段性刺激）有利于sAHP或VP-SDR的激活。目的2 将检验内源性VP-SDR本身调节sAHP的假设。目的3将检验细胞内 HF大鼠VP神经元Ca ~（2+）敏感性/有效性和VP SDR受损。这项提案将大大提高 主要研究者的职业发展，并推动他朝着成为一名独立研究者的目标前进。拟议 该项目提供技术技能和专业发展方面的培训，重点是独立性和获得 管理实验室所需的技能。我们将通过独特地将以前掌握的技巧整合到一部小说中来实现这一点 通过膜片钳和利用VP检测“嗅探”细胞同时测量sAHP和VP-SDR的方法， 分别来自健康和HF大鼠。格鲁吉亚州立大学还提供了一个特殊的环境， 进行研究所需的设施和设备，研讨会和讲习班的知识环境， 机构内外的持续合作，最重要的是指导高级PI， 为独立职业做准备的博士后经验。的确，导师已经提供了一个明确的计划，积极准备 独立派总之，本建议书将为申请人提供必要的培训， 独立性，同时为我们理解流行疾病中的VP神经元做出了重大贡献。

PROJECT SUMMARY This K99/R00 proposal will test a critical hypothesis on how vasopressin (VP) neurons self-regulate their neuronal excitability, their local paracrine release of vasopressin, and how these mechanisms are altered in a rodent model of heart failure while providing the PI with additional scientific and professional skills to transition him to a successful independent career as a research professor. Heart failure (HF) is the leading cause of death worldwide. A hallmark of HF is sympathetic hyperactivity and increased circulating levels of neurohormones such as VP. The slow afterhyperpolarization (sAHP) is recognized as a key mechanism that influences VP firing, playing a critical role in shaping the stereotyped phasic bursting patterns in VP neurons. The sAHP is impaired in VP HF neurons. Uniquely, Ca2+-dependent exocytosis of VP occurs somatodendritically (VP-SDR) as well, acting in a paracrine manner as an auto-inhibitory feedback mechanism. It is currently unknown whether VP-SDR is impaired in VP HF neurons. Data presented herein shows sAHPs and VP-SDR share many common signaling pathways which have been observed separately but never in tandem. Furthermore, we have exciting preliminary data that demonstrates VP itself can enhance isolated sAHPs, suggesting that endogenous VP-SDR may modulate sAHP time course, and thus firing activity. Understanding how these mechanisms lead to aberrant VP neuronal activity in HF is of critical importance. I therefore present the unique hypothesis that sAHP and VP-SDR activation are not only activated by the same signaling and pathways, but that VP-SDR can directly modulate sAHPs. I hypothesize that the observed impairment of the sAHP in HF neurons reflects impaired intracellular Ca2+ signaling and VP-SDR, leading to the hyperexcitability and excess peripheral VP release observed in this condition. Aim 1 will test the hypothesis that certain modes of VP activation (ex. continuous vs. phasic stimulation) favors the activation of either sAHPs or VP-SDR. Aim 2 will test the hypothesis that endogenous VP-SDR itself modulates sAHPs. Aim 3 will test the hypothesis that intracellular Ca2+ sensitivity/availability and VP-SDR are impaired in VP neurons of HF rats. This proposal will significantly enhance the PI’s career development and advance him towards his goal of becoming an independent investigator. The proposed project provides training in technical skills and professional development with an emphasis on independence and acquiring skills needed to run a laboratory. We will accomplish this by uniquely integrating previously mastered techniques in a novel way to measure sAHPs and VP-SDR simultaneously via patch clamp and utilization of VP detecting “sniffer” cells, respectively from healthy and HF rats. Georgia State University also provides an exceptional environment for training with the facilities and equipment needed to perform the research, an intellectual environment of seminars and workshops, ongoing collaborations both within and outside the institution, and most critically a mentoring senior PI with a wealth of experience preparing postdocs for an independent career. Indeed, the mentor has provided an explicit plan to actively prepare the PI for independence. In summary, this proposal will provide the training necessary to prepare the applicant for independence while making a significant contribution to our understanding of VP neurons in a prevalent disease.