Imaging Taste in Ensembles of Afferent Neurons

描述(由申请人提供)：这个多PI R21方案描述了一个高风险、高影响的项目，该项目引入了一种记录味觉传入神经元味觉诱发活动的新方法。这项工作代表了PI以及化学感官社区的一个主要新方向，因此非常适合R21机制。该项目将引入一种新的记录技术，以产生关于大型膝状神经节神经元集合的味觉敏感性和递质系统的新数据。我们将采用一种新的基因工程小鼠品系，它在感觉(包括味觉)神经节神经元中选择性地表达荧光功能报告GCaMP3。我们建议开发一种新的记录技术来成像麻醉小鼠的荧光膝状神经节神经元的味觉诱发活动。膝状体神经元将用扫描激光共聚焦显微镜成像，并对荧光变化进行量化，以单细胞分辨率同时测量大量神经元的活动。神经节细胞会被典型的甜味、苦味、咸味、酸味、鲜味和脂肪味刺激所刺激，并在口腔中传递。我们将测量广度 味觉膝状神经节细胞的调谐、浓度-反应关系和熵(目标1)。将分离具有功能特征的神经元，并进行单细胞RT-PCR，以检测功能不同的味觉神经元(AIM2)所使用的神经递质系统。这两个目标的成功完成将有四个结果：第一，我们将开发一种全新的方法来记录大量味觉神经元的传入感觉活动~第二，我们将积累味觉传入轴突的反应图谱的大型数据库，提供其调谐和熵宽度的全面目录~第三，我们将确定不同类别的感觉神经元(“多面手”~“专家”~甜味、酸味等)。有不同的分子表达谱，如独特的递质系统、转录因子等，最后，是否存在一类独特的脂肪感受性膝状神经节神经元。这些数据将对味觉如何由感觉传入进行编码(例如，标记线VS，组合编码)产生影响，并将极大地增加我们对感觉神经元如何处理味觉信号的理解。 与公共健康相关：这项研究是一个高风险、高回报的项目，旨在开发一种新的方法，利用基因工程小鼠记录来自外周味觉受体的感觉输入。这些实验将有助于解释甜味、酸味、咸味、苦味、鲜味和肥味是如何被检测和区分的。这些发现与人类的饮食选择和生活质量有关。

DESCRIPTION (provided by applicant): This multi-PI R21 proposal describes a high-risk, high-impact project that introduces a novel method for recording taste-evoked activity in gustatory afferent neurons. The work represents a major new direction for the PIs as well as for the chemical senses community, and thus is ideally suited for the R21 mechanism. The project will introduce a novel recording technique to produce new data on the taste sensitivities and transmitter systems of large ensembles of geniculate ganglion neurons. We will employ a new strain of genetically-engineered mice that express a fluorescent functional reporter, GCaMP3, selectively in sensory (including gustatory) ganglion neurons. We propose to develop a new recording technique to image taste-evoked activity in fluorescent geniculate ganglion neurons in anesthetized mice. Geniculate neurons will be imaged with scanning laser confocal microscopy and changes in fluorescence quantified to simultaneously measure activity in large ensembles of neurons with single cell resolution. Ganglion cells will be excited by prototypic sweet, bitter salty, sour, umami and fat taste stimuli, delivered in the oral cavity. We will measure the breadth of tuning, concentration-response relations and entropy for gustatory geniculate ganglion cells (Aim 1). Functionally characterized neurons will be isolated and single cell RT-PCR will be carried out to examine the neurotransmitter systems employed by functionally distinct taste neurons (Aim2). Successful completion of the two aims will have 4 outcomes: first, we will have developed a completely new method for recording afferent sensory activity in large numbers of gustatory neurons~ second, we will accumulate a large database of response profiles for gustatory afferent axons, providing a comprehensive catalog of their breadth of tuning and entropies~ third, we will determine whether different classes of sensory neurons ("generalists"~ "specialists"~ sweet-, sour-sensing, etc.) have different molecular expression profiles, such as distinctive transmitter systems, transcription factors, and so forth~ and finally, whether there i a unique class of fat-sensing geniculate ganglion neurons. The data will have implications for how taste is coded by sensory afferents (e.g., labeled line vs, combinatorial coding) and will tremendously increase our understanding about how sensory neurons process gustatory signals. PUBLIC HEALTH RELEVANCE: This research is a high-risk, high-reward project aimed at developing a new method for recording sensory input from peripheral taste receptors using genetically engineered mice. The experiments will help explain how sweet, sour, salty, bitter, umami and fat tastes are detected and discriminated. The findings are relevant to human diet selection and quality of life.