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受的分子机制的理解有所进展，但味觉信息如何在神经系统中传递和编码仍不清楚。我们的长期目标是了解味觉信息的编码系统，从味觉感受细胞到中枢神经系统。在这项提案中，我们的目标是开发遗传工具，这将大大有助于我们的理解，同时阐明精确的味觉神经通路在同一动物，然后使用这些工具来测试我们的假设，味觉信息不同的味觉方式是通过特定的上行神经通路传输。一种众所周知的遗传示踪剂是小麦胚芽凝集素（WGA），它是十年前开发的。在本研究中，我们将设计整合Myc肽、FLAG肽和增强型绿色荧光蛋白等特定标签的各种WGA，并通过分子动力学模拟评估其三级结构。我们将评估所得的工程WGA在其细胞内的稳定性和凝集素活性在体外。然后，我们将在小鼠中诱导它们在味觉受体相关基因的启动子/增强子控制下的味觉受体细胞亚群中的表达，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，其可用于同时且可重复地标记多个味觉通路。这些示踪剂不仅在我们未来的味觉研究中，而且在嗅觉和躯体感觉系统的研究以及许多其他研究相互关联的通路的遗传学的神经科学研究领域中，都应该有很大的用处。 公共卫生相关性：这项拟议的研究旨在开发新的遗传工具，以标记多个神经回路同时和可重复。这些工具将有助于阐明不同味觉形态的多个神经通路之间的关系。他们还将为其他神经科学研究做出贡献，包括嗅觉和体感系统的研究。

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.