Probing ASIC1 function in vivo using novel genetic tools

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 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.