New tools for targeting sympathetic neurons that control blood pressure

描述(由申请人提供)：这项探索性提案的目标是发现其表达定义血管运动性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血压控制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鼠。信使RNA将从人工分离的鉴定神经元中提纯，然后用微阵列进行扩增和分析，以检测基因表达。这一提议基于一个新的概念，即整个交感神经流出的峰活动有一半起源于神经节，而不是中枢神经系统，并假设血管运动性交感神经元功能亚群中的突触放大是根据不同血管床的需要进行调节的。实验将针对一个特定的目标，即发现区分血管运动性和非血管运动性神经元的基因，并识别控制心血管功能不同方面的交感神经元亚群。将分析控制大脑循环、心脏、肾脏和四肢肌肉血管的四个不同神经节的基因表达。通过使用逆行示踪剂反向标记控制肌肉、皮肤和肾脏循环的神经元，以及通过分析单细胞中的基因表达，将进一步完善表达分析。验证研究将采用定量聚合酶链式反应、免疫细胞化学和电生理。这个项目有可能产生很大的影响。现在有强有力的证据表明，血管运动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功能衰竭相关的问题。这些情况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。找到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，将促进对集成自主神经生理学的基本科学和医学理解。

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.