Probing ASIC1 function in vivo using novel genetic tools

 描述(申请人提供)：ASIC或酸感应离子通道是一种质子门控钠通道，属于脊椎动物和无脊椎动物表达的Deg/ENaC离子通道大家族。尽管ASIC的生理作用仍不明确，但它们与各种神经疾病有关。阐明ASIC功能的进展缓慢，部分原因是改变大脑选定区域的间质pH值的技术困难。到目前为止，大多数激活ASIC的尝试都是使用酸性溶液灌流或诱导缺血；然而，这种操作对质子浓度的时空控制性较低，而且往往会抑制ASIC的活动，因为它们导致脱敏。为了克服这个问题，我们实施了一种基于光遗传学的方法，以高时空分辨率酸化CNS中选定的细胞外空间区域，从而能够在大脑的任何结构中激活ASIC。该方法包括在星形胶质细胞中表达光驱动的质子泵ARCHT，在光照时释放质子，降低附近神经元周围的pH。酸化激活ASIC并启动动作电位的尖峰。该领域的另一个障碍是无处不在的表达 中枢神经系统中的ASIC--几乎所有独立于位置或功能特化的神经元都表达ASIC；因此，微电路中的所有神经元都受到pH下降的影响，从而使困难 梳理出单个神经元对行为反应的贡献。为了解决这个问题，我们开发了一种基因编码的ASIC特异性抑制物；它由一个与氨基末端结合并抑制通道活动的‘纳米体’组成。这项提议旨在通过检查ASIC在定义明确的神经元微电路中的贡献来验证新开发的工具；具体地说，我们将研究ASIC如何调制杏仁基底外侧核中编码恐惧学习和记忆的信号的处理。虽然ASIC存在于大脑的大部分区域，但我们将重点放在杏仁核，因为之前的报告表明，它调节恐惧反应，尽管涉及的细胞类型和机制在很大程度上仍不清楚。这项工作将证明这些新的遗传学方法在研究ASIC行为方面的价值，并为下一阶段探索这些通道在哺乳动物大脑中的生理和病理作用奠定基础。

 DESCRIPTION (provided by applicant): ASIC or Acid Sensing Ion Channel is a proton-gated sodium channel that belongs to the large family of Deg/ENaC ion channels expressed in vertebrates and invertebrates. ASIC has been implicated in various neurological disorders though their physiological roles remain poorly defined. Slow progress elucidating ASIC functions stems in part from technical difficulties of changing the interstitial pH in selected areas of the brain. To date most attempts to activate ASIC have used perfusion of acid solutions or induction of ischemia; however such maneuvers have low spatial and temporal control of the concentration of protons and also tend to suppress ASIC activity because they induce desensitization. To overcome this problem we have implemented an optogenetic-based method that acidifies selected areas of the extracellular space in the CNS with high temporospatial resolution enabling activation of ASIC in any structure of the brain. The method consists on expressing the light-driven proton pump ArchT in astrocytes that upon illumination extrude protons lowering the pH surrounding nearby neurons. The acidification activates ASIC and initiates spiking of action potentials. Another hurdle in the field is the ubiquitous expression of ASIC in the CNS - almost all neurons independent of location or functional specialization express ASIC; thus, all the neurons in a microcircuit are affected by drops in pH, making difficult to tease out the contribution of individual neurons to a behavioral response. To address this problem we have developed a genetically encoded specific inhibitor of ASIC; it consists of a `nanobody' that binds to the aminoterminus and inhibits channel activity. This proposal aims to validate the newly developed tools by examining the contribution of ASIC in a well-defined neuronal microcircuit; specifically, we will investigate how ASIC modulates processing of signals encoding fear learning and memory in the basolateral amygdala. Although ASIC is present in most areas of the brain, we will focus in the amygdala because previous reports suggest that it modulates fear responses, albeit the cell types involved and the mechanism remain largely unknown. The work will prove the value of these novel genetic approaches to studying ASIC in behaviors and sets the stage for a next phase of exploration of the physiological and pathological roles of these channels in the mammalian brain.