Electrophysiological reporter for monitoring gene manipulations

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): This proposal is an innovative effort to develop a novel tool that could revolutionize the execution and application of in vivo sharp electrode intracellular electrophysiology, the fundamental approach for examining cellular and synaptic mechanisms of systems-level functions. This tool is a reporter gene that will be detectible by electrophysiological methods in real-time by sharp electrodes in vivo, without the need for secondary factors (i.e., light stimulus or exogenous ligands). This could provide a necessary link to enable a more thorough incorporation of classic-type systems physiology with the reduced preparations and lower species that make up the majority of current experimental approaches used to understand genetic manipulations. An ideal electrophysiological reporter is one that is genetically-encoded, is "visible" to electrophysiological methods, has a signal that is easily recognized and distinguished from native processes in real time, and does not interfere with normal physiological function. We have identified a lab-generated mutant ion channel that fits these criteria, and appears to be an excellent candidate. We will make use of new viral vector technology to deliver candidate gene to adult rats. Aim 1 will use both in vivo and in vitro single-cell electrophysiological approaches to determine if the signal from the reporter gene can be detected against the well characterized background of the model systems (dorsal root ganglion sensory neurons and spinal motoneurons). Aim 2 is hypothesis driven and will determine if constitutive signaling by trkC, the receptor for neurotrophin-3, plays a necessary role in maintenance of the cellular and synaptic properties of adult motoneurons. The issue of maintenance of cellular properties by target-derived factors is in a question of fundamental importance in terms of the principles of neuroscience/neurobiology, and also in terms of biomedical issues in the adult nervous system such as aging, neurodegeneration, traumatic injury, and learning and memory.