Regulation of Subfornical Organ Neurons by the Novel Na+ leakage channel NALCN: Interactions with Neuropeptide Y

脊椎动物的中枢神经系统受到血脑屏障的保护。这种屏障允许血液和神经元周围的液体之间交换一些物质，如氧气和葡萄糖，但阻止大多数其他物质的交换，包括激素。然而，存在称为感觉室周器官的区域，其没有血脑屏障，并且含有专门的神经元以检测循环激素的水平（反映生理状态）以及循环的性质，例如渗透压（反映动物可能脱水的程度）和钠浓度。这些神经元检测到的信息然后被传递到中枢神经系统的其他中心，这些中心调节体内平衡。穹窿下器（subfornicalorgan，SFO）是室周感觉器官之一，在摄食、水盐平衡和心血管输出等方面起重要作用。** 我的研究重点是了解一种名为神经肽Y（NPY）的信号蛋白在SFO中的生物学效应。在中枢神经系统中，NPY是一种神经递质，在调节进食行为和其他几个过程中起着关键作用。NPY也是一种肽激素，可以释放到血液中，导致心率和血压升高。我们的初步证据（由我们以前发表的数据支持）表明，SFO神经元表达几种类型的NPY受体。此外，我们观察到，当施加NPY时，分离的SFO神经元变得活跃，显然是通过激活称为“Na+泄漏通道，非选择性”（NALCN）的非常丰富的离子通道，这是神经元电活动的强大调节剂。因此，本研究有三个目的：（1）表征SFO神经元对NPY的电反应，包括确定哪些受体亚型参与NPY介导的激活以及信号通路中分子的身份;（二）使用遗传工具上调和下调NALCN，以证实其在NPY介导的信号传导中的作用，并进一步研究NALCN的其他生物学功能（3）将神经肽Y（NPY）直接微量注射到麻醉大鼠的SFO内，探讨其在心血管输出调节等生物学过程中的作用。因此，这项研究将提供深入了解SFO和NPY的生物学作用的方法，从分子和细胞生物学到整个动物生理学。** 重要性：SFO作为中枢神经系统的一个中心，一直是深入研究的对象，它有助于调节摄食行为、水盐平衡和心血管输出等功能。类似地，NPY是一种神经肽激素，其与SFO会聚在许多相同的稳态过程中。已经提出，在SFO处的信息处理中的错误或在异常的NPY信号传导中的错误可能导致进食和/或心血管输出的异常调节。在我们的提案中的实验将探索我们最近的进展到新的观察，SFO是一个网站的行动NPY。这些数据将提供详细的神经肽Y在SFO神经元中的细胞和分子作用机制。此外，虽然NPY调节心血管输出的能力历来归因于其对外周组织的作用，但这项研究将提供证据，通过揭示中枢神经系统中的作用部位来帮助改变这种范式。这项工作对于那些对SFO或NPY调节基本稳态功能感兴趣的人以及对人类疾病中心血管调节生物学感兴趣的人来说非常重要。

The central nervous system of vertebrates is protected by a blood brain barrier. This barrier allows the exchange of some materials such as oxygen and glucose between the blood and the fluid surrounding neurons, but prevents the exchange of most other materials, including hormones. There are areas however called sensory circumventricular organs which have no blood brain barrier, and contain specialized neurons to detect levels of circulating hormones (reflecting physiological status) as well as properties of the circulation such as osmolarity (reflecting how dehydrated an animal may be) and sodium concentration. The information detected by these neurons is then transmitted to other centres of the central nervous system which regulate homeostasis. The subfornical organ (SFO) is one of the sensory circumventricular organs, and plays critical roles in regulating feeding, water and salt balance and cardiovascular output. **My proposed research is focused on understanding biological effects of a signaling protein called neuropeptide Y (NPY) acting at the SFO. Within the central nervous system, NPY is a neurotransmitter that plays a key role in regulating feeding behavior and several other processes. NPY is also a peptide hormone which can be released into the bloodstream causing elevated heart rate and blood pressure. Our preliminary evidence (supported by our previously published data) indicates that SFO neurons express several types of receptor for NPY. Moreover, we observed that isolated SFO neurons become active when NPY is applied, apparently by activation of a very abundant ion channel called "Na+ leakage channel, non-selective" (NALCN), which is a powerful regulator of electrical activity of neurons. Therefore this proposed research has three objectives: (1) to characterize the electrical response of SFO neurons to NPY, including determining which subtype(s) of receptor are involved in the NPY-mediated activation and the identity of the molecules in the signaling pathway; (2) to use genetic tools to upregulate and downregulate NALCN to confirm its role in NPY mediated signaling and further investigate other biological functions of NALCN in SFO neurons and (3) to microinject NPY directly into the SFO of anesthetized rats to investigate its role in biological processes such as regulation of cardiovascular output. Therefore this research will provide insight into the biological roles of SFO and NPY using approaches that span molecular and cellular biology to whole animal physiology. **Significance: The SFO has been the subject of intense investigation as a centre in the central nervous system that contributes to regulation of functions such as feeding behavior, water and salt balance and cardiovascular output. Similarly, NPY is a neuropeptide hormone that converges on many of the same homeostatic process as SFO. It has been proposed that errors in processing of information at the SFO, or in aberrant NPY signaling may result in abnormal regulation of feeding and/or cardiovascular output. Experiments in our proposal will explore our recent progress into the novel observation that SFO is a site of action for NPY. The data will provide detailed cellular and molecular mechanisms of action for NPY in SFO neurons. Moreover, while NPY's ability to regulate cardiovascular output has historically been attributed to its action on peripheral tissue, this research will provide evidence to help shift this paradigm by revealing a site of action in the central nervous system. The work will be of great importance to those who are interested in regulation of basic homeostatic functions by SFO or NPY, as well as those interested in the biology of cardiovascular regulation in human disease.