New tools for targeting sympathetic neurons that control blood pressure

描述（由申请人提供）：这个探索性提案的目标是发现其表达定义血管舒缩交感神经元专门化的基因。这些基因代表了在实验研究中操纵交感神经元和控制血压以及开发新的治疗策略的新工具。该方法将利用npy启动子序列驱动绿色荧光蛋白表达的转基因报告小鼠。信使RNA将从人工分类识别的神经元中纯化，然后用微阵列扩增和分析以检测基因表达。该建议基于这样一个新概念，即整个交感神经脉冲活动流出的一半起源于神经节，而不是中枢神经系统，并且基于血管舒缩交感神经元功能亚群的突触扩增被调整到不同血管床的需要的假设。实验将解决一个特定的目标，即发现区分血管舒缩神经元和非血管舒缩神经元的基因，并识别控制心血管功能不同方面的交感神经元亚群。基因表达将在控制大脑循环、心脏、肾脏和肢体肌肉血管系统的四个不同神经节中进行分析。通过使用逆行示踪剂来反向标记控制肌肉、皮肤和肾脏循环的神经元，以及通过分析单个细胞中的基因表达，表达分析将进一步完善。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，节后血管舒张性交感神经元的过度活跃预示着50%的人类高血压，并进一步导致心力衰竭和肾衰竭相关的问题。这些疾病造成了巨大的公共卫生负担，但现有的高血压临床治疗方法仍然不足。在调节特定血管床的细胞类型中寻找选择性操纵节后交感神经活动的工具，将促进对综合自主神经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.