LOTOS-based two-photon calcium imaging of dendritic spines in vivo
LOTOS-based two-photon calcium imaging of dendritic spines in vivo
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DOI：
10.1038/nprot.2012.106
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发表时间：
2012-10-01
期刊：
影响因子：
14.8
通讯作者：
Konnerth, Arthur
中科院分区：
文献类型：
--
作者：
Chen, Xiaowei;Leischner, Ulrich;Konnerth, Arthur
哺乳动物大脑中的神经元在其树突上接收数千个突触输入。在许多类型的神经元中，例如皮质锥体神经元，兴奋性突触形成于称为棘的细小树突突起上。通常，单个棘与传入轴突形成单个突触接触。在这个协议中，我们描述了一个最近建立的实验程序，通过使用双光子显微镜和全细胞膜片钳记录相结合，测量细胞内钙信号从树突棘在体内皮层神经元。我们已经使用小鼠作为实验模型系统，但该方案可以很容易地适用于其他物种。这种方法涉及在高帧速率和低激发激光功率下的数据采集，并且被称为低功率时间过采样（LOTOS）。由于其高灵敏度的荧光检测和降低的光毒性，LOTOS允许在体内长时间和稳定的钙成像。该方案的关键方面可以在5-6小时内完成，包括使用高速双光子成像的变体，精细的手术程序和优化的组织稳定。
Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.