视皮层发育突触稳态机制的研究

Visual experience plays a pivotal role in refining the connectivity of primary visual cortex, but the synaptic plasticity mechanisms that contribute to this refinement are still under debate. Turrigino has recently demonstrated that homeostatic synaptic plasticity scales excitatory and inhibitory synaptic strengths up and down in the cortical networks in vitro. A similar phenomenon has also found in vivo. Furthermore, homeostatic synaptic scaling in experience-dependent plasticity has been demonstrated in vivo using a classic sensory deprivation paradigm, monocular deprivation (MD) and binocular deprivation (BD) using lid suture. However those studies were not able to dissect presynaptic mechanism due to lack of proper tools. Using cultured hippocampal networks we demonstrated that the presynaptic mechanism is involved in homeostatic synaptic plasticity, that is presynaptic calcium signal is a key control point for regulating synaptic strength in response to chronic changes in network activity. Therefore we will approach this question by manipulating activity in rodent visual cortex through MD, then using our presynaptic and postsynaptic calcium（SypGCaMPs,PSD-95-GCaMPs） and synaptic vesicle fusion reports（SypHy） to measure paired synaptic transmission in vitro and in vivo， which were measured in zebrafish retina，optic tectum and rat viusal cortex in vivo. We will address whether the presyanptic mechanism and paired synaptic transmission for excitatory and inhibitory synapses are scaled in the opposite direction in response to altered visual input, and whether the rules for synaptic scaling are specific for particular classes of excitatory and inhibitory inputs. These experiments will provide fundamental understanding the detailed changes in visual cortical circuitry that arise as a result of altered sensory experience, and will have important implications for the mechanisms of visual abnormalities such as amblyopia.

突触稳态可塑性（HSP）作为视皮层发育突触可塑性主要调控机制，通过调节神经环路整体兴奋性，从而保持神经网络适度兴奋，防止长时间处于过度兴奋或抑制的状态。由于研究手段的匮乏，在活体水平研究HSP中突触前膜的调控，及兴奋性和抑制性神经元二者之间相互作用的研究仍为空白。借助我们对研究技术瓶颈的突破,使用SypGCaMP2（突触前钙离子）和SypHy（递质释放）我们在体外模型中首次证实突触前参与了HSP，并揭示突触前钙离子为HSP突触前调节机制的关键。该课题将使用更灵敏、新一代SypGCaMP5/PSD-85-GCaMP5和SypHy结合在体转基因、双光子图像技术，以视皮层发育关键期谷氨酸能和GABA能突触及神经元为研究对象，研究突触前、后钙离子在视皮层发育HSP调节机理。并在离体和活体模型系统地研究突触稳态可塑性，尤其兴奋性或抑制性突触前、后膜及胞体的功能变化及二者之间相互作用的时空关系。

突触稳态可塑性（HSP）作为视皮层突触可塑性主要调控机制，通过调节神经环路整体兴奋性，从而保持神经网络适度兴奋，防止长时间处于过度兴奋或抑制的状态。由于研究手段的匮乏，在研究HSP 中突触前膜的调控，及兴奋性和抑制性神经元二者之间相互作用的研究仍为空白。1）我们首先构建和鉴定了Syp-GCaMP3、5、6 ，从而获得了高信噪比的突触前钙信号工具。2）使用Syp-GCaMP2、3、5分别研究了海马神经元和视皮层突触稳态可塑性，证实突触前机制参与了突触稳态可塑性，而且证实突触前钙信号是HSP的关键因素。3）结合全细胞膜片钳近一步揭示兴奋性和抑制性网络都可因突触前钙内流变化导致的突触递质释放改变而参与突触稳态可塑性。4）使用新的突触前钙信号指示蛋白和数据分析，我们意外的发现突触兴奋的异质性存在突触前机制，提示突触对动作电位数字编码转换为模拟编码存在有突触前机制。.我们的研究证实突触前机制参与了稳态可塑性，为弱视的视觉训练提供了实验证据。我们发现存在突触前异质性，该发现提示神经编码在突触前即存在数字编码向模拟编码转换，而且可能存在两种形式的混合编码。