Imaging neuronal activity with voltage-sensitive Green Fluorescent Protein

描述（由申请人提供）：候选人研究计划的长期目标是尽可能完整地了解简单无脊椎动物——线虫——秀丽隐杆线虫的整个行为的神经元基础。为此，他开发了几项重要的新技术，推进了他和其他实验室的研究目标。然而，要实现对行为的几乎完整的描述，将需要秀丽隐杆线虫的另一种新技术：对完整、行为动物的多个神经元的膜电位进行光学记录。该候选人寻求摆脱教学和减少管理，以开发基于绿色荧光蛋白（GFP）的膜电位分子探针，并使用这些探针进一步实现他对秀丽隐杆线虫空间定向行为的神经元基础的持续研究目标。 职业发展计划有四个组成部分。第一部分是协调和参与由三名 PI 和六名顾问组成的新联盟，以重新设计 FlaSh 蛋白，这是一种有前途的电压依赖性 GFP 形式。该联盟汇集了结构生物学、生物物理学、荧光探针和光学记录领域的世界领先者，旨在提高原始 FlaSh 分子的速度和灵敏度。如果成功，重新设计的蛋白质将用于阐明线虫中趋化性的神经元基础，趋化性是一种广泛的空间定向形式。拟议的研究意义重大，因为它可能会产生新的神经元活动基因靶向探针，可用于许多其他重要的实验系统。此外，这项工作将加深我们对共同目标导向行为背后的神经计算的理解。第二部分和第三部分分别是技术课程和对光学记录技术前沿实验室的辅导参观。这两项活动将为 PI 提供完成第一部分工作所需的新技术背景。第四部分是 PI 将利用他在分析哲学方面的广泛背景讲授研究伦理学课程。

DESCRIPTION (provided by applicant): The long-term goal of the candidate's research program is to understand as completely as possible the neuronal basis of the entire behavioral repertoire of a simple invertebrate--the nematode worm C. elegans. To this end he has developed several important new techniques that have advanced the research goals of his and other labs. However, achieving a virtually complete account of behavior will require another new technique in C. elegans: optical recording of membrane potential from multiple neurons in intact, behaving animals. The candidate seeks release from teaching and reduced administration in order to develop molecular probes for membrane potential based on green fluorescent protein (GFP), and to use these probes to further the goals of his continuing research on the neuronal basis of spatial orientation behavior in C. elegans. The career development plan has four components. Component I is to coordinate and participate in a new consortium of three PI's and six consultants to re-engineer the FlaSh protein, a promising form of voltage-dependent GFP. The consortium brings together world leaders in structural biology, biophysics, fluorescent probes, and optical recording with the aim of enhancing the speed and sensitivity of the original FlaSh molecule. If successful, the re-engineered protein will be used to elucidate the neuronal basis of chemotaxis, a widespread form of spatial orientation, in the nematode C. elegans. The proposed research is significant because it will likely lead to new genetically targetable probes of neuronal activity that can be used in many other important experimental systems. In addition, this work will deepen our understanding of neural computations underlying a common goal-directed behavior. Components II and III are, respectively, technical courses and tutorial visits to laboratories at the leading edge of optical recording technology. These two activities will give the PI new technical background needed to complete the work of Component I. Component IV is a course the PI will teach in research ethics, drawing on his extensive background in analytical philosophy.