A patch clamp set-up and thermocycler for polymerase chain reaction: tools for understanding neuronal excitability and gene expression

动物必须消耗能量来平衡消耗，但是告诉动物何时进食的信号是什么呢？这些信号是如何传递到大脑的呢？神经元如何对这些信号作出反应？这些问题对于理解饥饿的感觉和感知以及能量稳态的调节至关重要。大脑中的一组神经元，被称为皮层下器官（SFO）神经元，专门用于检测血液中循环的激素水平，包括调节饥饿和进食的激素（如瘦素和胰岛素）。我的初步实验表明，大鼠SFO神经元在禁食48小时后表现出基因表达的变化。两个下调的基因编码电压门控Na+通道，已知蛋白质直接控制神经元的电兴奋性。本提案的目的是了解观察到的SFO神经元基因表达变化的生理作用。这项建议概述了三个具体目标。第一个目标是确定大鼠SFO中哪些神经元在48小时的禁食后发生了变化，并详细描述这些神经元的电特性变化。第二个目标是调查调节饥饿和进食的激素的变化是否可能导致观察到的基因表达和电特性的变化。第三个目标是确定一种下调的Na+通道蛋白对SFO神经元电活动的调节的具体贡献。这些目标将通过免疫组织化学、膜片钳电生理记录、逆转录偶联聚合酶链反应（RT-PCR）、实时PCR和siRNA介导的基因表达抑制等方法的组合来实现。本提案请求资金用于购买电生理设备和热循环器（用于RTPCR），这是开展本研究计划所必需的。总之，这项研究将增加我们对中枢神经系统如何感知饥饿信号和调节能量平衡的分子生理学的理解。

Animals must consume energy to balance expenditures, but what are the signals that tell animals when to eat?  How are these signals communicated to the brain?  How do neurons respond to these signals?  These questions are central to understanding sensation and perception of hunger, and regulation of energy homeostasis.      A group of neurons in the brain, called subfornical organ (SFO) neurons are specialized to detect levels of hormones circulating in the blood, including hormones that regulate hunger and feeding (such as leptin and insulin).  My preliminary experiments indicate that rat SFO neurons exhibit changes in gene expression after a 48 hour fast.  Two of the down-regulated genes encode voltage gated Na+ channels, proteins known to directly control neuronal electrical excitability.  The goal of this proposal is to understand physiological roles of observed changes in SFO neuron gene expression.  This proposal outlines three specific objectives.  The first objective is to identify which population of neurons in the rat SFO is altered by a 48h fast, and to describe in detail changes in the electrical properties of these neurons.  The second objective is to investigate whether changes in hormones that regulate hunger and feeding may be responsible for driving the observed changes in gene expression and electrical properties.  The third objective is to determine the specific contributions of one of the down-regulated Na+ channel proteins to the regulation of electrical activity of SFO neurons.  These objectives will be accomplished using a combination of methods including immunohistochemistry, patch clamp electrophysiological recording, reverse transcription coupled polymerase chain reaction (RT-PCR), real time PCR and siRNA mediated inhibition of gene expression.        This proposal is requesting funds for the purchase of electrophysiology equipment and a thermocycler (for RTPCR) that are essential to carry out this research program.  Together, this research will increase our understanding of the molecular physiology of how the central nervous system percieves hunger signals and regulates energy balance.