Yu Xinzhu, Zuo Yi
Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz.
Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz;
J Vis Exp. 2014 May 12(87):51520. doi: 10.3791/51520.
In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.
在哺乳动物的皮质中，神经元形成极其复杂的网络，并在突触处交换信息。突触强度的变化以及突触的添加/去除以经验依赖的方式发生，为神经元可塑性提供了结构基础。作为皮质中大多数兴奋性突触的突触后成分，树突棘被认为是突触的良好代表。利用小鼠遗传学和荧光标记技术，可以在完整大脑中标记单个神经元及其突触结构。在这里，我们介绍一种使用双光子激光扫描显微镜的经颅成像方案，以便在体内随时间跟踪荧光标记的突触后树突棘。该方案采用薄颅骨制备方法，保持颅骨完整，避免因脑膜和皮质暴露引起的炎症效应。因此，手术后可立即采集图像。实验过程可以在从数小时到数年的各种时间间隔内重复进行。这种制备方法的应用还可以扩展到研究生理和病理条件下的不同皮质区域和层，以及其他细胞类型。
