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蛋白在体内神经元连接中的特异性功能，本研究尝试应用基于生物分子荧光互补（BiFC）的新技术--GRAPHIC系统标记所有神经元之间的连接，首先，为了确定GRAPHIC系统是否能够检测所有神经元之间的连接，包括突触和非突触连接，我将GRAPHIC系统应用于多巴胺中皮质边缘通路，即从腹侧被盖区（VTA）到丘脑核（NAc）的输入，因为以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通信。通过表达C-GRAPHIC标记的VTA的多巴胺轴突与AAV-EF 1a-DIO-mCherry结合并与AAV-TH-Cre共表达，并且通过用hSynapsinI启动子表达N-GRAPHIC-mTagBFP 2标记NAc中的神经元。在病毒感染后一个月，我证实了在mCherry下表达的GRAPHIC信号（多巴胺神经元的来源）和蓝色mTagBFP 2（目标神经元）颜色以及有或没有突触后密度的表达，这表明GRAPHIC系统可以成功标记突触和非突触连接位点。在这一年中，我尝试重新设计我的实验建议的GRAPHIC系统，并在体内测试其效率。通过在多巴胺能通路中建立GRAPHIC系统，我首次展示了多巴胺神经元与其他神经元进行突触和非突触相互作用的证据。

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.