Marandi Nima, Konnerth Arthur, Garaschuk Olga
Institut für Physiologie, Ludwig-Maximilians Universität München, Pettenkoferstrasse 12, 80336 Munich, Germany.
Pflugers Arch. 2002 Dec;445(3):357-65. doi: 10.1007/s00424-002-0933-7. Epub 2002 Sep 20.
Two-photon laser scanning microscopy has been used successfully for imaging activity-dependent changes of intracellular calcium and sodium levels. Here we introduce a simple technique for two-photon chloride imaging in intact neurons. It involves the use of the membrane-permeable Cl(-) indicator dye MQAE [N-(6-methoxyquinolyl) acetoethyl ester]. Brief incubation with MQAE produced a robust loading of cells in slices from various brain regions including hippocampus, cortex and cerebellum. In contrast to conventional fluorescence measurements using MQAE, two-photon imaging was not affected in a major way by dye bleaching and phototoxic damage. Instead, it allowed prolonged recordings of time-resolved changes in intracellular chloride concentration in somata and dendrites. As an example of an application we imaged GABA-mediated Cl(-) transients in pyramidal cells of cortical and hippocampal slices as well as in cerebellar Purkinje neurons. By combining Cl(-) imaging with the gramicidin-based perforated-patch-clamp technique we showed that changes in MQAE fluorescence are proportional to the magnitudes of GABA-evoked transmembrane Cl(-) fluxes. Thus, MQAE-based two-photon microscopy promises to be a valuable technique for many applications requiring chloride imaging in single cells.
双光子激光扫描显微镜已成功用于对细胞内钙和钠水平的活动依赖性变化进行成像。在此，我们介绍一种在完整神经元中进行双光子氯化物成像的简单技术。该技术涉及使用膜通透性Cl(-)指示剂染料MQAE [N-(6-甲氧基喹啉基)乙酰乙酯]。用MQAE短暂孵育能使来自包括海马体、皮层和小脑等不同脑区的切片中的细胞大量负载染料。与使用MQAE的传统荧光测量不同，双光子成像在很大程度上不受染料漂白和光毒性损伤的影响。相反，它能够长时间记录胞体和树突中细胞内氯化物浓度的时间分辨变化。作为一个应用实例，我们对皮层和海马体切片的锥体细胞以及小脑浦肯野神经元中GABA介导的Cl(-)瞬变进行了成像。通过将Cl(-)成像与基于短杆菌肽的穿孔膜片钳技术相结合，我们发现MQAE荧光的变化与GABA诱发的跨膜Cl(-)通量大小成正比。因此，基于MQAE的双光子显微镜有望成为许多需要对单细胞进行氯化物成像的应用中的一项有价值的技术。
