New tools for targeting sympathetic neurons that control blood pressure

描述（由申请人提供）：本探索性建议的目标是发现其表达定义血管交感神经元特化的基因。这些基因代表了在实验研究中操纵交感神经元和血压控制以及开发新的治疗策略的新工具。该方法将利用转基因报告小鼠，其中NPY启动子序列驱动绿色荧光蛋白的表达。信使RNA将从人工分选的识别神经元中纯化，然后扩增并用微阵列分析以检测基因表达。该建议是基于一个新的概念，即整个交感神经流出的尖峰活动的一半起源于神经节，而不是中枢神经系统，并假设突触放大的功能子集的血管交感神经元是调整到不同的血管床的需要。实验将解决一个具体的目标，这是发现基因，区分血管神经元和非血管神经元，并确定控制心血管功能的不同方面的交感神经元的子集。基因表达将在控制脑循环、心脏、肾脏和四肢肌肉血管的四个不同神经节中进行分析。表达分析将通过使用逆行示踪剂来标记控制肌肉，皮肤和肾脏循环的神经元，并通过分析单细胞中的基因表达来进一步完善。 验证研究将采用定量PCR、免疫细胞化学和电生理学。这个项目有可能产生重大影响。强有力的证据表明，血管节后交感神经元的过度活跃预示着50%的人类高血压，并进一步导致与心力衰竭和肾衰竭相关的问题。这些病症造成了巨大的公共卫生负担，然而用于高血压临床管理的现有疗法仍然不足。寻找有选择地操纵调节特定血管床的细胞类型中的节后交感神经活动的工具将推进对综合自主生理学的基础科学和医学理解。

DESCRIPTION (provided by applicant): The goal of this exploratory proposal is to discover genes whose expression defines the specialization of vasomotor sympathetic neurons. Such genes represent new tools for manipulating sympathetic neurons and blood pressure control in experimental studies and for developing new therapeutic strategies. The approach will exploit a transgenic reporter mouse in which NPY-promoter sequences drive expression of green fluorescent protein. Messenger RNA will be purified from manually sorted identified neurons, then amplified and analyzed with microarrays to detect gene expression. The proposal is predicated on the novel concept that half of the entire sympathetic outflow of spike activity originates in the ganglia, not the central nervous system, and upon the hypothesis that synaptic amplification in functional subsets of vasomotor sympathetic neurons is tuned to the needs of different vascular beds. Experiments will address one specific aim, which is to discover genes that distinguish vasomotor from non-vasomotor neurons and that identify subsets of sympathetic neurons controlling different aspects of cardiovascular function. Gene expression will be analyzed in four different ganglia that control brain circulation, the heart, the kidney and limb muscle vasculature. The expression analysis will be further refined by using a retrograde tracer to back label neurons that control circulation in muscles, the skin and the kidney and through analysis of gene expression in single cells. Validation studies will employ quantitative PCR, immunocytochemistry and electrophysiology. This project has potential for high-impact. Strong evidence now indicates that hyperactivity in vasomotor postganglionic sympathetic neurons presages 50% of human hypertension and further contributes to problems associated with heart failure and renal failure. These conditions impose a tremendous public health burden, yet existing therapies for clinical management of hypertension remain inadequate. Finding tools for selectively manipulating postganglionic sympathetic activity in cell types that regulate specific vascular beds will advance fundamental scientific and medical understanding of integrated autonomic physiology.