Calcium indicator loading of neurons using single-cell electroporation
Calcium indicator loading of neurons using single-cell electroporation
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DOI：
10.1007/s00424-007-0234-2
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发表时间：
2007-07-01
影响因子：
4.5
通讯作者：
Helmchen, Fritjof
中科院分区：
文献类型：
--
作者：
Nevian, Thomas;Helmchen, Fritjof
亚细胞Ca2+信号转导的研究依赖于用荧光Ca2+指示染料标记细胞的方法。在这项研究中，我们证明了使用单细胞电穿孔的Ca2+指示负载的个别神经元和小神经元网络在大鼠新皮层在体外和体内。短暂的电压脉冲通过靠近靶细胞的玻璃移液管传递。这种方法导致可靠和快速（在几秒钟内）加载的胞体和随后的树突和轴突分支的完整标记。通过在脑切片中使用同步全细胞记录，我们直接解决了电穿孔对神经元的影响。细胞活力高（约85%），在一分钟内从膜透化中恢复。电穿孔前后恢复的细胞的电特性是不可区分的。此外，正常外观的Ca2+瞬变可以诱发树突，棘，和轴突终扣的电穿孔细胞。利用胞体的负染色，靶向单细胞电穿孔同样适用于体内。我们的结论是，电穿孔是一种简单的方法，允许Ca2+指示剂负载的多个细胞在短时间内低背景染色，这使得它特别适合于功能成像的亚细胞Ca2+动态小神经元网络。
Studies of subcellular Ca2+ signaling rely on methods for labeling cells with fluorescent Ca2+ indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca2+ indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca2+ transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca2+ indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca2+ dynamics in small neuronal networks.