采用光电联用技术对谷氨酸能与GABA能突触囊泡回收机制的异质性研究

To maintain the normal synaptic transmission, continuous synaptic vesicular recycling by efficient exocytosis or release and endocytosis plays a critical and essential role. Four modes of retrieval machinery have been proposed to operate at synaptic terminals of brain’s neurons, including kiss-and-run, clathrine-mediated endocytosis, activity dependent bulk endocytosis and ultrafast endocytosis. However, the relative importance of these has been controversial, despite intensive studies, especially among different kinds of synaptic terminals. Besides, it is also not clear whether readily release pool of synaptic vesicles (SVs) are replenished by maturation of endocytic vesicles, or through endosomes. Using electrophysiology to compare presynaptic vesicle recycling property of Glutamatergic and GABAergic synapses, we found that synaptic transmissions acting on two types of CNS synapses showed different depletion and recovery rate in response to high frequency stimulation. Ultrastructural analyses using STEM Tomography and 3D remodeling also showed significant difference in vesicle size and distribution profiles between glutamatergic and GABAergic synapses. Here, we planned to clearly display the different organelle composition and properties of retrieval machineries between native glutamatergic and GABAergic synapses with multiple light and electron microscopy techniques, which are comprised of high potassium induced FM dye uptake, correlative light and cryo-electron tomography (CLCEM) at cultured neurons on grids, and optogenetics to stimulate cultured hippocampal neurons with different kinds of stimulus, followed by rapidly freeze them and examine the ultrastructure using electron tomopraphy (FIB/SEM). This study will not only provide an important basis for us to uncover the different electric functions of glutamatergic and GABAergic transmission, but to lay foundations to the molecular regulation and physiological significance of synaptic vesicle recycling between two types of synapses in the future.

突触囊泡的释放与回收循环机制包括kiss-and-run、CME、ADBE和超快内吞，但究竟以哪种模式为主仍存在争议，各种模式与刺激强度间的关联以及不同类型突触内参与的模式有无区别都不清楚。谷氨酸能和GABA能突触分别是中枢神经系统中最重要的兴奋性和抑制性突触，我们前期的电生理研究发现两种突触囊泡可释放池的清空能力和恢复能力都存在显著差异，扫描透射电镜和活动依赖性HRP内吞实验也都显示两种突触囊泡特性和回收机制上有很大区别。为了直观且透彻地解析两种突触内囊泡回收循环机制的异质性，拟在培养的海马神经元上，采用活动依赖性FM染料标记实验、光电联用技术、光遗传学调控与快速冷冻电镜联合技术等手段，对两种突触结构的异质性进行详细的比较，并对两种类型突触囊泡的内吞回收过程进行细致地分析，为深入了解两种突触传递的不同特性提供重要的依据，也为研究两种突触囊泡循环在分子调控机制上的差异和生理意义打下基础。