dcf1基因剔除介导焦虑行为机制的研究

Mental illness is closely related to brain specific neural circuit function,while anxiety disorders are one of the most common mental illness. However, understanding the pathogenesis of this disease is not yet clear. Our previous study found that when dcf1 gene knockout, cortical neurons migration in mouse was obstacled, diaphragm shrinking and the lateral ventricles increasing, resulting in an anxiety behavior.It suggests that the neural circuit dysfunction may be an important reason. Using dcf1 knockout mouse model in this project, we will combine the optogenetics and nerve electrophysiological testing to study neural circuits on the cortex , the diaphragm, the hippocampus induced by lack of dcf1. By embryo electroporation, cortical cell types in migrate disorder will be classified. By AAV-ChR2 vector, neurons projecting will be indicated among the cortex, diaphragm, hippocampal atrophy area. AAV-Cre-inducible system is used to demonstrate neural circuits projection and signal changes. Through specific activation and inhibition to specific genetic neural circuits by optogenetics, anxiety behavior will be tested in Elevated Plus to determine anxiety behavior change. Return dcf1 by viral vector gene is used to rescue electrophysiological signals and anxious behavior, and ultimately to clarify anxious behavior mediated mechanism mediated by dcf1 knockout.

精神类疾病与大脑特定神经回路功能密切相关，焦虑症是最为常见精神疾病之一，然而，对此病发生机制的认识尚未清楚。我们的前期研究发现当dcf1基因敲除，小鼠大脑皮层神经细胞迁移障碍，隔膜萎缩，侧脑室显著增大，小鼠伴随焦虑行为，提示神经回路功能异常可能是重要原因。本项目利用dcf1基因剔除小鼠模型，将光遗传技术与神经电生理检测相结合，研究基于dcf1缺失导致的皮层、隔膜、海马发育障碍的神经回路。通过胚胎电转，归属皮层迁移障碍的细胞类型；采用AAV-ChR2载体，研究皮层间以及隔膜、海马萎缩区域的神经元投射；采用AAV-Cre-inducible系统证实神经回路投射与信号改变；通过光遗传特异性激活与抑制特定神经回路，进行高架十字、开场行为测试，判断焦虑行为变化。通过病毒载体送还dcf1基因营救（rescue），实现电生理信号、焦虑行为的恢复，最终阐明Dcf1缺失介导焦虑行为机制。

本项目通过胚胎电转、免疫组化、光遗传操作等方法证实dcf1基因剔除引发大脑皮层发育障碍、神经投射的影响以及焦虑行为机制，完成了立项预期目标，为深入理解大脑皮层发育、神经回路建成与行为机制提供了新的科学依据。.采用光遗传操作技术，本项目新发现前额叶（PLC）向海马（CA1）的神经投射，dcf1缺失能阻断这PLC-CA1投射,小鼠出现空间记忆障碍和焦虑行为。 在PLC-CA1终末端光遗传激活可以逆转空间记忆缺陷和焦虑。这是具有原创意义的发现，为认识学习记忆、情感控制的大脑工作原理提出了新线索，为深入研究疾病的发生与预防及治疗提供了新的借鉴。通过胚胎电转、免疫组化的方法，证实DCF1基因敲除小鼠皮层神经元数目减少，迁移减慢。为了追溯这些差异的起源，进一步发现发育早期（E14.5,P1,P7）DCF1基因敲除导致小鼠皮层区域ERK蛋白的磷酸化水平增加的，从分子机制明确了DCF1基因调控皮层发育是通过MAPK信号通路中的ERK途径发挥其功能。从细胞数量、迁移速率以及皮层结构建成与神经回路关系的源头上提出了具有重要科学价值的依据，证实了DCF1在皮层发育过程中起重要作用。与此同时，还观察到DCF1基因敲除小鼠海马神经元活性的降低，增进了dcf1在维持大脑功能的认识。Dcf1缺失从电生理层面也得以互证。电生理研究进一步证实dcf1基因敲除小鼠mEPSC频率显著降低，LTP下调，焦虑症标志蛋白上调。从Dcf1缺失小鼠行为异常在电生理层面互证了分子机制，为认识空间记忆障碍与焦虑的病理机制提出了新的理论依据，为推进dcf1在大脑功能的认识，深入研究大脑发育与神经回路建成奠定了基础。