In Vivo Imaging of Neuronal Plasticity in Visual Cortex

本研究的目的是通过结合神经元和神经元之间的相互作用来阐明皮质结构可塑性的机制。 创新的体内成像技术与经典的视觉操作。这一综合性方法适用于 有可能彻底改变我们对自适应电路修改的理解，这是 大脑功能我们以前的研究表明，成年人视皮层2/3层锥体神经元的视皮层神经元中， 随着时间的推移，分支尖端长度几乎没有变化，GABA能非锥体中间神经元显示出显著的 树突状分支尖端重塑由视觉经验以输入和回路特异性方式驱动。的事实 中间神经元的结构可塑性在成年期是连续的，这提出了一个有趣的可能性， 抑制性连接的局部重塑可能是成人皮质可塑性的基础。然而，经验如何改变 抑制回路尚不清楚，抑制回路和兴奋回路的改变如何局部协调 仍然没有解决。 在前一个资助期，我们开发了一种标记体内抑制性突触的方法， 同时监测抑制性突触和树突棘重塑在整个树突乔木， 在正常和改变的视觉体验中的体内皮质层2/3锥体神经元。我们发现 抑制性突触和树突棘的重排是局部聚集的，主要在每个10 μ m内 其他，这种集群是受经验的影响。在这一建议中，我们寻求以高 时间分辨率兴奋性突触和相邻突触的协调插入和移除的性质 新皮层回路中的抑制性突触。为此，我们将实施新开发的三色 标记系统，以独立和同时监测树突的形成和消失， 棘沿着出现或去除这些棘中的突触后致密物，并且这些棘的出现和 沿着同一树突移除沿着抑制性突触。使用光谱分辨双光子显微镜， 将1）在整个过程中监测体内抑制性和兴奋性突触重塑的时间顺序， L2/3锥体神经元的树突状乔木在短时间间隔; 2）监测经验的影响- 抑制性和兴奋性突触重塑协调的依赖性可塑性; 3）检查特异性 兴奋性/抑制性突触对的传入输入。4、我们将制定和实施 光谱分辨多焦点多光子显微术，以进一步提高成像速度并允许询问 突触动力学在更短的时间间隔。

The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Our previous findings show that while pyramidal neurons in layer 2/3 of adult visual cortex show little, if any, change in branch tip length over time, GABAergic non-pyramidal interneurons display significant dendritic branch tip remodelling driven by visual experience in an input and circuit-specific manner. The fact that structural plasticity of interneurons is continuous through adulthood raises the intriguing possibility that local remodelling of inhibitory connections may underlie adult cortical plasticity. Yet, how experience alters inhibitory circuitry is unclear, and how modifications to inhibitory and excitatory circuits are locally coordinated remains unaddressed. In the previous funding period we developed a method for labeling inhibitory synapses in vivo and simultaneously monitored inhibitory synapse and dendritic spine remodeling across the entire dendritic arbor of cortical layer 2/3 pyramidal neurons in vivo during normal and altered visual experience. We found that the rearrangements of inhibitory synapses and dendritic spines are locally clustered, mainly within 10 ¿m of each other, and that this clustering is influenced by experience. In this proposal we seek to characterize with high temporal resolution the nature of the coordinated insertion and removal of excitatory synapses and neighboring inhibitory synapses in the neocortical circuit. To this purpose we will implement a newly developed three-color labeling system to independently and simultaneously monitor the formation and disappearance of dendritic spines along with appearance or removal of the post-synaptic density in these spines, and the appearance and removal of inhibitory synapses along the same dendrites. Using spectrally resolved two-photon microscopy we will 1) monitor the temporal sequence of inhibitory and excitatory synapse remodeling in vivo across the full dendritic arbor of L2/3 pyramidal neurons at short time intervals; 2) monitor the effects of experience- dependent plasticity on coordination of inhibitory and excitatory synapse remodeling; 3) examine the specificity of afferent inputs to coordinated excitatory/inhibitory synaptic pairs. Further, 4) we will develop and implement spectrally resolved multifocal multiphoton microscopy to further enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals.