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某些物质，如氧气和葡萄糖，但阻止了包括激素在内的大多数其他物质的交换。然而，有一些区域被称为感觉周脑室器官，没有血脑屏障，包含专门的神经元来检测循环激素的水平(反映生理状态)以及循环的属性，如渗透压(反映动物脱水的程度)和钠浓度。这些神经元检测到的信息然后被传输到中枢神经系统的其他调节稳态的中心。穹隆下器(SFO)是脑室周围感觉器官之一，在调节摄食、水盐平衡和心血管输出等方面起着重要作用。我提出的研究重点是了解作用于SFO的一种名为神经肽Y(NPY)的信号蛋白的生物学效应。在中枢神经系统中，NPY是一种神经递质，在调节摄食行为和其他几个过程中发挥关键作用。神经肽Y也是一种多肽荷尔蒙，可以释放到血液中，导致心率和血压升高。我们的初步证据(由我们之前发表的数据支持)表明，SFO神经元表达几种类型的NPY受体。此外，我们观察到，当应用NPY时，分离的SFO神经元变得活跃，显然是通过激活一种非常丰富的离子通道，称为“Na+泄漏通道，非选择性”(NALCN)，它是神经元电活动的强大调节器。因此，本研究有三个目的：(1)研究SFO神经元对NPY的电反应，包括确定哪种受体亚型(S)参与NPY介导的激活以及信号通路中分子的识别；(2)利用基因工具上调和下调NALCN，以确认其在NPY介导的信号转导中的作用，并进一步研究NALCN在SFO神经元中的其他生物学功能；(3)直接向麻醉大鼠SFO微量注射NPY，研究其在心血管输出调节等生物学过程中的作用。因此，这项研究将通过跨越分子和细胞生物学到整个动物生理学的方法来深入了解SFO和NPY的生物学作用。意义：作为中枢神经系统中的中枢，SFO一直是密集调查的对象，它有助于调节摄食行为、水盐平衡和心血管输出等功能。同样，NPY是一种神经肽激素，与SFO一样，在许多相同的动态平衡过程中汇聚。有人提出，SFO信息处理的错误或NPY信号的异常可能导致摄食和/或心血管输出的异常调节。我们提案中的实验将探索我们最近在SFO是NPY的作用地点这一新颖观察方面的进展。这些数据将提供SFO神经元中NPY的详细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被归因于它对周围组织的作用，但这项研究将通过揭示中枢神经系统中的作用部位来提供证据，帮助改变这一范式。这项工作将对那些对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具有重要意义。

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.