in vivo imaging of inhibitory circuit remodeling in mouse visual cortex

 描述（由申请人提供）：本提案的目标是通过将创新的体内成像技术与经典的视觉操作相结合来阐明皮质结构可塑性的机制。这种综合方法具有革命性的潜力 我们对自适应电路修改的理解，这是大脑功能的一个基本方面。我们以前的研究结果表明，虽然成年小鼠视觉皮层第2/3层（L2/3）的锥体神经元显示分支尖端长度随时间的变化很小（如果有的话），但GABA能非锥体中间神经元显示由视觉体验以输入和回路特异性方式驱动的显著树突分支尖端重塑。中间神经元的结构可塑性在成年期是连续的，这一事实提出了一种有趣的可能性，即抑制性神经元的局部重塑可能是一种潜在的机制。 连接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 目前尚不清楚，抑制性和兴奋性回路的改变如何局部协调仍然没有解决。 最近，我们开发了一种方法，用于标记抑制性突触在体内，同时监测抑制性突触和树突棘重塑在整个树突状乔木的皮质L2/3锥体神经元在体内正常和改变视觉体验。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10 μm的范围内，这是局部细胞内信号机制的空间范围，这种聚集受到经验的影响。然而，以前的成像间隔通常为4天。因此，协调的抑制性和兴奋性突触动力学的性质仍然暂时未解决的两个事件是否同时发生或两个驱动器的变化，而另一个调整它。目前还不清楚是否突触的行为在一个协调的方式是由特定的传入输入驱动，以及视觉体验如何增加兴奋性和抑制性突触变化之间的协调。在这个建议中，我们试图以高时间分辨率的性质的协调插入和删除的兴奋性突触和相邻的抑制性突触在新皮层电路的特点。为此，我们将实施一个新开发的三色标记系统，独立地，同时监测突触后标记，代表完整的突触补体到单个L2/3锥体神经元在小鼠视觉皮层。使用光谱分辨双光子显微镜，我们将1）在短时间间隔内监测L2/3锥体神经元的整个树突状结构中抑制性和兴奋性突触重塑的时间序列; 2）监测经验依赖性可塑性对抑制性和兴奋性突触重塑协调的影响; 3）检查传入输入对协调的兴奋性/抑制性突触对的特异性。此外，4）我们将开发和实施光谱分辨多焦点多光子显微镜，以提高成像速度，并允许在更短的时间间隔内询问突触动力学。

 DESCRIPTION (provided by applicant): 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 (L2/3) of the adult mouse 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. Recently, 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 L2/3 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 µm of each other, the spatial range of local intracellular signaling mechanisms, and that this clustering is influenced by experience. However, previous imaging intervals were typically 4 days. Thus, the nature of the coordinated inhibitory and excitatory synaptic dynamics remained temporally unresolved in terms of whether the two events occur simultaneously or one of the two drives the change, while the other adjusts to it. It is also unclear whether synapses that behave in a coordinated manner are ones driven by specific afferent inputs, and how visual experience increases coordination between excitatory and inhibitory synaptic changes. 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 postsynaptic markers representing the full synaptic complement onto individual L2/3 pyramidal neurons in mouse visual cortex. 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 f 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 enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals.