? 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. ? ?
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