Weimer Robby M, Hill Travis C, Hamilton Andrew M, Zito Karen
Cold Spring Harb Protoc. 2012 Jul 1;2012(7):771-7. doi: 10.1101/pdb.prot070029.
Considerable evidence has accumulated that structural changes in dendritic spines and their synapses are associated with adaptive functional changes in cortical circuits, such as during circuit refinement in young animals and in learning and memory in adults. Understanding the mechanisms of circuit plasticity requires detailed investigation of the structural dynamics of dendritic spines and how they are regulated by neural activity and sensory experience. Studying the dynamic localization of synaptic proteins in dendritic spines and how their stabilization and exchange rates influence spine structural plasticity is also important. This protocol describes imaging approaches to study synaptic protein dynamics in dendritic spines of the rodent cerebral cortex. It gives a strategy for generating photoactivatable green fluorescent protein (PA-GFP)-tagged synaptic proteins and in vitro and in vivo transfection methods for coexpression of these proteins with a spectrally separable cell-filling marker (DsRed-Express). Methods for tracking synaptic protein localization using photoactivation and time-lapse imaging of PA-GFP in spiny pyramidal neuron dendrites are given. A discussion of imaging hardware and software preferences is also included. The methods described here can be used to study the dynamic processes underlying spine synapse development during the formation and plasticity of neural circuits in the mammalian brain.
大量证据表明，树突棘及其突触的结构变化与皮质回路的适应性功能变化相关，比如在幼小动物的回路精细化过程以及成年动物的学习和记忆过程中。理解回路可塑性的机制需要详细研究树突棘的结构动态以及它们如何受神经活动和感觉经验的调节。研究突触蛋白在树突棘中的动态定位以及它们的稳定和交换速率如何影响棘突结构可塑性也很重要。本方案描述了用于研究啮齿动物大脑皮质树突棘中突触蛋白动态的成像方法。它给出了生成光激活绿色荧光蛋白（PA-GFP）标记的突触蛋白的策略以及这些蛋白与光谱可分离的细胞填充标记物（DsRed-Express）共表达的体外和体内转染方法。还给出了使用PA-GFP在棘状锥体神经元树突中的光激活和延时成像来追踪突触蛋白定位的方法。此外还包括对成像硬件和软件偏好的讨论。这里描述的方法可用于研究哺乳动物大脑神经回路形成和可塑性过程中棘突突触发育的动态过程。
