Imaging Taste in Ensembles of Afferent Neurons

描述（由申请人提供）：该多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和化学感觉界的一个主要新方向，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递质系统的新数据。我们将采用一种新的基因工程小鼠，表达荧光功能的报告，GCaMP 3，选择性地在感觉（包括味觉）神经节神经元。我们建议开发一种新的记录技术，在麻醉小鼠的荧光膝状体神经节神经元的图像味觉诱发活动。膝状体神经元将用扫描激光共聚焦显微镜成像，并定量荧光的变化，以同时测量具有单细胞分辨率的大型神经元集合中的活性。 神经节细胞将被口腔中递送的原型甜味、苦味、咸味、酸味、鲜味和脂肪味刺激物兴奋。我们将测量 味觉膝状体神经节细胞的调谐、浓度-反应关系和熵（目的1）。将分离功能表征的神经元，并进行单细胞RT-PCR以检查功能不同的味觉神经元（Aim 2）所采用的神经递质系统。成功完成这两个目标将产生4个结果：首先，我们将开发出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，我们将积累一个味觉传入轴突反应谱的大型数据库，提供其调谐宽度和熵的全面目录~第三，我们将确定不同类别的感觉神经元（“通才”~“专家”~甜味、酸味感觉等）是否有不同的分子表达谱，如独特的递质系统，转录因子，等等，最后，是否有一类独特的脂肪感觉膝状神经节神经元。这些数据将对味觉如何由感觉传入编码产生影响（例如，标记线vs，组合编码），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。 公共卫生相关性：这项研究是一项高风险、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外周味觉受体的感觉输入。这些实验将有助于解释甜、酸、咸、苦、鲜味和脂肪的味道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.