Genetic approach for transneuronal NA circuitry mapping

描述（由申请人提供）： 通过特定的突触连接进行的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，深刻地影响着多种脑功能。虽然NA电路已被广泛研究，其精确的连接和突触的变化知之甚少，主要是由于缺乏方法来提供示踪分子选择性NA神经元。基于NA神经元发育和多巴胺B-羟化酶基因转录的分子机制的最新进展，有一个令人信服的研究机会，调查NA电路与一种新的遗传方法。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电路如下。首先，我们将开发一种最佳的基因递送系统，可以以细胞类型特异性，长期和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优化Phox 2结合基序的合成启动子。将在腺病毒和慢病毒骨架中检查最佳合成启动子，并结合四环素诱导系统进行测试。第二，我们优化的病毒载体系统将用于表达跨神经元示踪分子，即，小麦胚芽凝集素（WGA）和绿色荧光蛋白融合到破伤风毒素的无毒片段（GFP-TTC）。这些示踪剂将与参考固定分子（B-半乳糖苷酶）一起沿着表达，所述参考固定分子将鉴定示踪剂来源的原代神经元。使用立体定向注射发达的病毒系统，我们将调查NA电路起源于蓝斑（LC）和孤束核（NTS），以及从延髓头端腹外侧（RVLM）。最后，我们将开发转基因小鼠模型，可用于NA电路的系统和可重复的映射和监测突触的变化，使用优化的DBH启动子。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，并将其用于精确的神经解剖学映射研究。在GFP-TTC转基因小鼠的情况下，我们将能够通过荧光检测制备切片培养物，这将允许NA电路系统的进一步功能和电生理学研究。该项目将提供一个框架，通过该框架，亚型特异性神经元启动子可以被工程化和优化，以产生有效的遗传系统来描绘特定的神经元回路。使用发达的遗传工具，跨突触NA连接将在LC，NTS和RVLM中仔细检查，所有这些都对神经系统功能至关重要。因此，这些方法将作为宝贵的工具来阐明NA电路在正常和患病的大脑中的功能和调节。

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.