Electrophysiological reporter for monitoring gene manipulations

描述（由申请人提供）：该提案是一项创新的努力，旨在开发一种新的工具，可以彻底改变体内尖锐电极细胞内电生理学的执行和应用，这是检查细胞和突触机制的基本方法。该工具是一种报告基因，可以通过电生理方法在体内通过尖锐电极实时检测到，而不需要次要因素（即光刺激或外源配体）。这可以提供一个必要的联系，使经典型系统生理学与减少的准备和较低的物种构成了目前用于理解遗传操作的大多数实验方法的更彻底的结合。理想的电生理报告器是经过基因编码的，对电生理方法是“可见的”，具有易于识别和实时区分的信号，并且不干扰正常的生理功能。我们已经确定了一个实验室生成的突变离子通道，符合这些标准，似乎是一个很好的候选者。我们将利用新的病毒载体技术将候选基因传递给成年大鼠。目的1将使用体内和体外单细胞电生理方法来确定是否可以在模型系统（背根神经节感觉神经元和脊髓运动神经元）的良好特征背景下检测到来自报告基因的信号。Aim 2是假设驱动的，将确定trkC（神经营养因子-3的受体）的构成信号是否在维持成年运动神经元的细胞和突触特性中起必要作用。通过靶源性因子维持细胞特性的问题在神经科学/神经生物学原理方面具有根本性的重要性，并且在成人神经系统的生物医学问题（如衰老，神经变性，创伤性损伤，学习和记忆）方面也具有重要意义。

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.