New Genetic Tracers for Concurrent Labeling of Multiple Gustatory Neural Pathways

描述（由申请人提供）：尽管近几十年来我们对味觉接收的分子机制的理解有了进步，但味觉信息是如何在神经系统中传递和编码的仍然不清楚。我们的长期目标是了解味觉信息的编码系统，从味觉受体细胞到中枢神经系统。在这个提议中，我们的目标是开发遗传工具，通过同时阐明同一动物的精确味觉神经通路，从而极大地有助于我们的理解，然后使用这些工具来验证我们的假设，即不同味觉模式的味觉信息是通过特定的上升神经通路传递的。小麦胚芽凝集素（WGA）是一种众所周知的遗传示踪剂，是十年前开发出来的。在我们提出的研究中，我们将设计各种整合特定标签（如Myc肽，FLAG肽和增强型绿色荧光蛋白）的WGAs，并将通过分子动力学模拟评估它们的三级结构。我们将根据其细胞内稳定性和体外凝集素活性来评估由此产生的工程WGAs。然后，我们将在味觉接收相关基因的启动子/增强子控制下，在小鼠味觉受体细胞亚群中诱导它们的表达，并评估它们在体内的效用。从这些研究中，我们期望开发至少两种标记的基于WGA的遗传示踪剂，可用于同时标记多种味觉途径并可区分。这些示踪剂不仅在我们未来的味觉研究中，而且在嗅觉和体感系统的研究以及许多其他研究相互关联通路遗传学的神经科学研究领域中都有很大的用处。

DESCRIPTION (provided by applicant): Although in recent decades our understanding of the molecular mechanisms of taste reception has advanced, how the taste information is transmitted and coded in the nervous system remains unclear. Our long-term objective is to understand the coding system of gustatory information, from taste receptor cells to the central nervous system. In this proposal, we aim to develop genetic tools that will greatly contribute to our understanding by concurrently elucidating precise gustatory neural pathways within the same animal, and then use these tools to test our hypothesis that gustatory information for different taste modalities is transmitted via specific ascending neural pathways. One well-known genetic tracer is wheat germ agglutinin (WGA), which was developed ten years ago. In the research we propose here, we will engineer various WGAs that integrate specific tags such as Myc peptide, FLAG peptide, and enhanced green fluorescent protein, and will evaluate their tertiary structure by molecular dynamic simulation. We will evaluate the resultant engineered WGAs in terms of their intracellular stability and lectin activity in vitro. We will then induce their expression in mice in a subset of taste receptor cells under the control of promoters/enhancers of taste reception-related genes and evaluate their utility in vivo. From these studies, we expect to develop a minimum of two tagged WGA- based genetic tracers that are available to label multiple gustatory pathways concurrently and distinguishably. These tracers should be of great use not only in our future gustatory research but in studies of olfactory and somatosensory systems and many other areas of neuroscience research that study the genetics of interconnected pathways. PUBLIC HEALTH RELEVANCE: This proposed research aims to develop new genetic tools to label multiple neural circuitries concurrently and distinguishably. These tools will contribute to elucidating relationships between multiple neural pathways for different taste modalities. They will also contribute to other neuroscience research, including studies of olfactory and somatosensory systems.