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

新型钠渗漏通道 NALCN 对穹窿下器官神经元的调节：与神经肽 Y 的相互作用

• 批准号: RGPIN-2014-05230
• 负责人: Fry, William
• 金额: $ 2.55万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=651235
• 关键词: Regulation; Subfornical; Organ; Neurons; Novel

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.

脊椎动物的中枢神经系统受到血脑屏障的保护。这个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，有一些区域被称为感觉心室周围器官，它们没有血脑屏障，含有专门的神经元，用于检测循环激素水平（反映生理状态）以及循环特性，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随后被传送到中枢神经系统的其他中枢，这些中枢神经系统负责调节体内平衡。皮层下器官（SFO）是感觉心室周围器官之一，在调节摄食、水盐平衡和心血管输出量等方面起着重要作用。**我提出的研究重点是了解一种名为神经肽Y （NPY）的信号蛋白作用于SFO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起关键作用。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，当施加NPY时，分离的SFO神经元变得活跃，显然是通过激活一个非常丰富的离子通道，称为“Na+泄漏通道，非选择性”（NALCN），这是神经元电活动的强大调节剂。因此，本研究有三个目标：(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了NPY介导的激活以及信号通路中分子的身份；(2)利用遗传工具上调和下调NALCN，确认其在NPY介导的信号通路中的作用，并进一步研究NALCN在SFO神经元中的其他生物学功能；(3)将NPY直接微注射到麻醉大鼠SFO，研究其在调节心血管输出量等生物学过程中的作用。因此，本研究将利用跨越分子和细胞生物学到整个动物生理学的方法，深入了解SFO和NPY的生物学作用。**意义：SFO作为中枢神经系统中的一个中枢，参与调节摄食行为、水盐平衡和心血管输出量等功能，一直是人们关注的焦点。同样，NPY是一种神经肽激素，它与SFO在许多相同的稳态过程中聚集。有人提出，SFO信息处理错误或NPY信号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的新进展，即SFO是NPY的作用位点。这些数据将为NPY在SFO神经元中的作用提供详细的细胞和分子机制。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，但本研究将通过揭示中枢神经系统中的作用部位来提供证据，以帮助改变这种范式。这项工作对那些对SFO或NPY调节基本体内平衡功能感兴趣的人，以及对人类疾病心血管调节生物学感兴趣的人来说，将具有重要意义。