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异构体在活体神经元连接中的特定功能，我尝试应用图形系统来标记所有神经元到神经元的连接，这是一种基于生物分子荧光互补(BIFC)的新技术，已经发展起来用于标记所有神经元的连接。首先，为了确定图形系统是否能够检测包括突触和非突触的所有神经元连接，我将图形系统应用于多巴胺中皮质边缘通路，即从腹侧被盖区(VTA)到伏核(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。C-Graphic标记的VTA的多巴胺轴突与AAV-EF1a-DIO-mCherry结合并与AAV-TH-Cre共表达，N-Graphic-mTagBFP2结合hSynapsinI启动子标记NAC内的神经元。在病毒感染一个月后，我证实了图形信号在mCherry(多巴胺神经元的来源)和蓝色mTagBFP2(靶神经元)颜色下表达，以及在有或没有突触后密度的情况下表达，这表明图形系统可以成功地标记突触和非突触连接部位。在这一年里，我试图为我的实验方案重新设计图形系统，并在体内测试其效率。通过建立多巴胺能通路的图形系统，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.