Genetic approach for transneuronal NA circuitry mapping

描述(由申请人提供)： 通过特定突触连接进行信息处理是大脑功能的基础。去甲肾上腺素(NA)是一种重要的神经递质，对多种脑功能有深远的影响。虽然NA通路已经得到了广泛的研究，但其精确的连接和突触变化却知之甚少，这主要是由于缺乏选择性地将示踪剂分子输送到NA神经元的方法。基于最近对NA神经元发育和多巴胺B-羟基酶基因转录的分子机制的研究进展，用一种新的遗传学方法来研究NA回路是一个引人注目的研究机会。利用这些分子信息，我们建议开发最佳的遗传工具来研究跨神经元NA回路，如下所示。首先，我们将开发一种最佳的基因传递系统，该系统能够以细胞类型特异的、长期的和可诱导的方式将基因表达靶向NA神经元。为了实现这一目标，我们将通过基因工程来优化Phox2结合基序的合成启动子。最佳合成启动子将在腺病毒和慢病毒主干中进行检查，也将与四环素诱导系统一起进行测试。其次，我们优化的病毒载体系统(S)将用于表达跨神经元示踪分子，即小麦胚凝集素(WGA)和与破伤风毒素无毒片段融合的绿色荧光蛋白(GFP-TTC)。这些示踪剂将与一个参考固定分子(B-半乳糖苷酶)一起表达，该分子将识别示踪剂起源的初级神经元。利用已开发的病毒系统的立体定向注射，我们将研究起源于蓝斑(LC)和孤束核(NTS)以及延髓头端腹外侧(RVLM)的NA回路。最后，我们将开发转基因小鼠模型，使用优化的DBH启动子，可以用于系统和可重复地绘制NA回路的图谱，并用于监测突触的变化。将选择在NA神经元中表现出B-半乳糖苷酶表达的转基因动物，并将用于精确的神经解剖定位研究。在GFP-TTC转基因小鼠的情况下，我们将能够通过荧光检测来制备切片培养，这将使进一步的NA回路系统的功能和电生理研究成为可能。这个项目将提供一个框架，通过这个框架，特定亚型的神经元启动子可以被工程和优化，以产生有效的遗传系统来描绘特定的神经元回路。利用开发的基因工具，将仔细检查LC、NTS和RVLM中的跨突触NA连通性，所有这些都对神经系统功能至关重要。因此，这些方法将成为阐明正常和疾病大脑中NA回路的功能和调节的宝贵工具。

DESCRIPTION (provided by applicant): Information processing via specific synaptic connections is the basis of the brain function. Noradrenaline (NA) is an important neurotransmitter and profoundly influences diverse brain functions. Although NA circuitry has been extensively studied, its precise connectivity and synaptic changes are poorly understood, largely due to the lack of methods to deliver the tracer molecules selectively to NA neurons. Based on recent progress on molecular mechanisms underlying NA neuron development and dopamine B-hydroxylase gene transcription, there is a compelling research opportunity to investigate the NA circuitry with a novel genetic approach. Using this molecular information, we propose to develop optimal genetic tools to study the transneuronal NA circuitry as follows. First, we will develop an optimal gene delivery system that can target gene expression to NA neurons in a cell type-specific, long-term, and inducible manner. Toward this goal, we will optimize synthetic promoters by genetic engineering of Phox2-binding motif. The optimal synthetic promoter will be examined in adenoviral and lentiviral backbone and also be tested in combination with tetracycline-inducible system. Second, our optimized viral vector system(s) will be used to express transneuronal tracer molecules, i.e., wheat germ agglutinin (WGA) and green fluorescent protein fused to a nontoxic fragment of tetanus toxin (GFP-TTC). These tracers will be expressed along with a reference stationary molecule (B-galactosidase) that will identify the primary neurons from which tracer is originated. Using stereotactic injection of the developed viral systems, we will investigate the NA circuitry originating from the locus coeruleus (LC) and the nucleus of solitary tract (NTS), as well as from the rostral ventrolateral medulla (RVLM). Finally, we will develop transgenic mice models that can be used for systematic and reproducible mapping of the NA circuitry and for monitoring synaptic changes, using optimized DBH promoters. Transgenic animals will be selected that exhibit B-galactosidase expression in NA neurons and will be used for precise neuroanatomical mapping studies. In case of GFP-TTC transgenic mice, we will be able to prepare slice cultures by fluorescence detection, which will allow further functional and electrophysiological studies of NA circuitry systems. This project will provide a frame by which subtype-specific neuronal promoters can be engineered and optimized to generate efficient genetic systems to delineate specific neuronal circuitry. Using developed genetic tools, the transsynaptic NA connectivity will be carefully examined in the LC, NTS, and RVLM, all of which are crucial for nervous system functions. Therefore, these approaches will serve as invaluable tools to elucidate the function and regulation of NA circuitry in the normal and diseased brain.