近年来，NeuroD1等转录因子介导的中枢神经系统胶质细胞原位转分化技术发展迅猛，然而我们对原位转分化产生的神经元如何整合融入神经环路所知甚少，重要原因之一是目前缺乏显示胶质细胞原位转分化并建立功能性突触连接的实时影像证据。通过小鼠躯体感觉皮层长时程颅窗双光子成像，我们成功记录到过表达NeuroD1的胶质细胞的形态逐步向神经元形态转变的过程。据此我们提出假设：NeuroD1诱导原位转分化产生的神经元将迁移至特定脑区和细胞层，生成合理的轴/树突投射，建立成熟的突触结构，整合融入局部神经环路，最终参与环路功能的执行。本项目拟采用颅窗(海马窗)双光子成像、跨突触神经环路示踪、活体双光子钙成像等技术，从结构和功能影像学的角度，尝试验证上述假设，确认在体转分化新生的神经元可以替代损伤和死亡的神经元行使功能，为解决原位再生的神经元如何重塑疾病和损伤的神经网络这一再生医学领域的关键科学问题提供理论依据。

The last few years have witnessed many breakthroughs in the research on transcription factor-induced in vivo reprogramming of endogenous glial cells into functional neurons in the central nervous system. However, we still have very few clues about how these newly converted neurons manage to integrate structurally and functionally into existing neural circuits. A significant portion of this knowledge gap can be attributed to a lack of long-term in vivo optical brain imaging evidence regarding how the neurons reprogrammed from glial cells establish appropriate synaptic connections in live animals. By imaging the somatosensory cortex of mice transduced with recombinant retrovirus or AAVs carrying CAG or GFAP-driven transcription factor NeuroD1 through open-skull cranial windows using a two-photon microscope every day for 45 days, we have successfully captured the step-by-step transitional process of a morphologically typical astrocyte converting into a cell with unipolar (or bipolar in some cases) neuronal morphology in situ in the mouse brains. Therefore, our scientific hypothesis is that glia-converted neurons induced by transcription factor NeuroD1 can survive long term in mouse brains, migrate to specific brain regions and layers, extend long axonal and elaborate dendritic processes projecting to particular brain areas, assemble functional synapses with the right upstream and downstream targets, and eventually carry out certain neural circuit functions. In this research project, we plan to test the hypothesis proposed above by applying chronic two-photon imaging through a cranial window or a chronic hippocampal window, trans-synaptic circuit tracing, chronic in vivo two-photon calcium imaging and other biomedical techniques, so this research will offer the first continuous in vivo live imaging evidence on the formation of functional synaptic connections of the newborn reprogrammed neurons derived from glias in the brain. The result of this study will serve as a blueprint for solving one of the key scientific and technical challenges in the field of regenerative medicine: how to rebuild neural circuits after diseases and injuries of the central nervous system with neurons generated through in vivo reprogramming.