Catecholaminergic Modulation of Taste Circuitry Using Optogenetics

描述（由申请人提供）：该提案描述了一个高风险、高影响的项目，该项目使用光遗传学方法来确定与饱腹感相关的内脏神经元如何调节味觉系统。我们已经创建了一种新的病毒载体（AAV-9，PRSx 8-ChR 2（h134 r）-mCherry），其在标记含有去甲肾上腺素的儿茶酚胺能神经元和其他非GABA能内脏感觉神经元的Phox 2选择性启动子的控制下表达通道视紫红质-2（Chr 2）和mCherry。第二种光遗传学方法将使用在多巴胺-β羟化酶启动子（DβHCre/0）和驱动ChR 2表达的cre依赖性病毒控制下表达Cre的转基因小鼠更特异性地靶向儿茶酚胺神经元。尾侧脑干中的神经元包括儿茶酚胺神经元与饱腹感机制和葡萄糖诱导的进食相关的内脏信号密切相关，但它们在何处以及如何调节味觉信号尚不清楚。虽然儿茶酚胺神经元成组聚集，例如A1和A2，但这些簇嵌入更异质的细胞群中，使得难以研究特定的神经通路。光遗传学技术允许特定的神经元类型被特异性激活。通过插入表达能够使神经元膜去极化的光敏通道（蛋白质）的基因来修饰神经元DNA。在用遗传构建体进行神经元转导后，用调谐到光敏通道的最佳频率的激光或高强度LED进行光学刺激，随后将刺激和去激活由启动子遗传上限定的那些神经元。最近的工作表明，重要的相互作用尾孤核神经元敏感的内脏信号和喂养电路发生通过本地脑干通路，包括intrasolitary途径。因此，我们建议的结果将（1）提供一种新的技术方法来确定一组特定的内脏敏感神经元如何调节味觉神经元，（2）确定以前未探索的脑干内通路是否有助于味觉/内脏整合。

DESCRIPTION (provided by applicant): This proposal describes a high risk, high impact project using an optogenetic approach to determine how visceral neurons associated with satiey modulate the taste system. We have created a novel viral vector (AAV-9, PRSx8-ChR2(h134r)-mCherry) that expresses channelrhodopsin-2 (Chr2) and mCherry under the control of a Phox2-selective promoter that labels catecholaminergic neurons containing norepinephrine and other non-GABAergic viscerosenory neurons. A second optogenetic approach will target catecholamine neurons more specifically using a transgenic mouse that expresses Cre under the control of a dopamine-β hydroxylase promoter (DβHCre/0) and a cre-dependent virus driving ChR2 expression. Neurons in the caudal brainstem including catecholamine neurons are intimately associated with visceral signaling underlying satiey mechanisms and glucoprivic-induced feeding, but where and how they modulate taste signals is unknown. Although catecholamine neurons are clustered in groups, e.g. A1 & A2, these clusters are embedded in a more heterogeneous population of cells making it difficult to study specific neural pathways. Optogenetic techniques allow defined neuron types to be specifically activated. Neuronal DNA is modified by inserting a gene expressing a light-sensitive channel (protein) capable of depolarizing the neuron membrane. Following neuronal transduction with the genetic construct, optical stimulation with a laser or high-intensity LED tuned to the optimal frequency of the light-sensitive channel will subsequently stimulate and depolarize those neurons genetically defined by the promoter. Recent work suggests that significant interactions between caudal solitary nucleus neurons sensitive to visceral signals and feeding circuits occur through local brainstem pathways including intrasolitary pathways. The outcomes of our proposal will thus (1) provide a novel technical approach to determine how a specific group of visceral-sensitive neurons modulate gustatory neurons and (2) determine if a previously unexplored brainstem intrasolitary pathway contibutes to taste/visceral integration.