神経ネットワーク形成における微小管結合タンパク質タウのアイソフォーム特異的な役割

为了阐明tau同工异构体在体内神经元连接中的特异性功能，我尝试使用基于生物分子荧光互补（bioc）的新技术GRAPHIC系统来标记所有神经元之间的连接，该技术已经发展到可以标记所有神经元连接。首先，为了确定GRAPHIC系统是否能够检测到包括突触和非突触的所有神经元连接，我将GRAPHIC系统应用于多巴胺中脑皮质边缘通路，即从腹侧被盖区（VTA）到伏隔核（NAc）的输入，因为之前的研究假设多巴胺神经元可以与其他神经元形成突触和非突触的通信。表达C- GRAPHIC标记的VTA多巴胺轴突与AAV- EF1a-DIO-mCherry结合并与AAV- th - cre共表达，而表达N- GRAPHIC- mtagbfp2并表达hSynapsinI启动子标记的NAc神经元。病毒感染一个月后，我证实了在mCherry（多巴胺神经元来源）和蓝色mTagBFP2（目标神经元）颜色下表达的GRAPHIC信号，以及有无突触后密度的表达，这表明GRAPHIC系统可以成功标记突触和非突触连接位点。在这一年里，我尝试重新设计我的实验方案GRAPHIC系统，并在体内测试了它的效率。通过在多巴胺能通路中建立GRAPHIC系统，我首次证明了多巴胺神经元与其他神经元之间存在突触性和非突触性相互作用。

To clarify the tau isoform specific function in neuronal connection in vivo, I tried to apply the GRAPHIC system to label all neuron to neuron connections which is new technique based on biomolecular fluorescence complementation (BiFC) and has been developed to label all neurons connections.Firstly, to determine whether the GRAPHIC system could able to detect all neuronal connections that included both synaptic and non-synaptic, I applied the GRAPHIC system to the dopamine mesocorticolimbic pathway, the input from the ventral tegmental area (VTA) into nucleus accumbens (NAc), because of previous studies assumed that dopamine neurons could be formed both synaptic and non-synaptic communication with other neurons. Dopamine axons of VTA labeled by expressing C- GRAPHIC binds with AAV- EF1a-DIO-mCherry and co-expressing with AAV-TH-Cre, and neurons in NAc labeled by expressing N- GRAPHIC-mTagBFP2 with hSynapsinI promoter. After one month virus post-infection, I confirmed that the GRAPHIC signal expressed under mCherry (the source of dopamine neuron) and blue mTagBFP2 (the target neuron) colors as well as expressed with or without postsynaptic density, which indicated the GRAPHIC system could successfully label synaptic and non-synaptic connections sites.During this year, I tried to re-design the GRAPHIC system for my experimental propose and tested its efficiency in vivo. By establishing the GRAPHIC system in the dopaminergic pathway, I showed the evidence for the first time the dopamine neurons make both synaptic and non-synaptic interactions with other neurons.