In vivo two-photon calcium imaging of neuronal networks
In vivo two-photon calcium imaging of neuronal networks
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DOI：
10.1073/pnas.1232232100
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发表时间：
2003-06-10
影响因子：
11.1
通讯作者：
Konnerth, A
中科院分区：
文献类型：
--
作者：
Stosiek, C;Garaschuk, O;Konnerth, A
双光子钙成像是监测活体脑组织中不同神经元活性的有力手段。在哺乳动物的大脑中，这样的成像研究主要局限于神经元的钙记录，这些神经元通过微电极单独携带染料。以前尝试使用膜穿透形式的荧光钙指示剂来负载神经元群体，只在细胞培养或未成熟脑组织切片中产生了令人满意的结果。在这里，我们介绍了一种在大量细胞中装载膜透意荧光指示剂染料的通用方法。我们建立了一种基于压力喷射的局部染料传递方案，该方案可用于大光谱的膜穿透指示染料，包括钙绿-1乙酰氧甲基(AM)酯、Fura-2 AM、Fluo-4 AM和Indo-1 AM我们在体内和体外成功地将这种染料负载方案应用于小鼠从新生儿到成年的任何发育阶段的脑组织中。在活体中，通过完整的头骨成像获得的双光子钙离子记录表明，胡须偏转引起的钙瞬变发生在桶形皮质2/3层神经元的一个子集中。因此，我们的结果证明了这项技术适用于对单个细胞分辨率的完整神经元电路的实时分析。
Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes. Previous attempts to use membrane-permeant forms of fluorometric calcium indicators to load populations of neurons have yielded satisfactory results only in cell cultures or in slices of immature brain tissue. Here we introduce a versatile approach for loading membrane-permeant fluorescent indicator dyes in large populations of cells. We established a pressure ejection-based local dye delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including calcium green-1 acetoxymethyl (AM) ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. We applied this dye-loading protocol successfully in mouse brain tissue at any developmental stage from newborn to adult in vivo and in vitro. In vivo two-photon Ca2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex. Thus, our results demonstrate the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.