Imaging Taste in Ensembles of Afferent Neurons

描述（由申请人提供）：这个多pi R21提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中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，以产生关于膝状神经节神经元大集合的味觉敏感性和传递系统的新数据。我们将使用一种新的基因工程小鼠，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基因GCaMP3。我们建议开发一种新的记录技术来成像麻醉小鼠膝状神经节荧光神经元的味觉诱发活动。膝状神经元将用扫描激光共聚焦显微镜成像，并定量荧光变化，同时以单细胞分辨率测量神经元大集合的活动。在口腔内传递的甜味、苦味、咸味、酸味、鲜味和脂肪味刺激会使神经节细胞兴奋。我们将测量宽度

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.