Grøndahl T, Langmoen I A
Institute for Surgical Research, Department of Opthalmology, Rikshospitalet, National Hospital, University of Oslo, 0027 Oslo, Norway.
Brain Res. 1998 Feb 23;785(1):58-65. doi: 10.1016/s0006-8993(97)01367-x.
An increase in intracellular calcium during cerebral ischemia has been proposed as a common final pathway underlying the events leading to neuronal death. Intracellular calcium has been measured with ion selective electrodes during energy deprivation (ED) in hippocampal slices and with fluorescent techniques in neuronal cultures. In the present study, we describe a novel method to visualize and quantify changes in intracellular calcium in brain slices using Confocal Laser Scanning Microscopy (CLSM). CA 1 pyramidal neurons in hippocampal slices were filled by intracellular injection with a 1:2 mixture of the fluorescent dyes Fluo 3 and Fura Red. The neurons were then visualized using CLSM, and the ratio of the fluorescence from each probe used to quantify intracellular calcium concentrations before and during ED. The free intracellular calcium concentration was 60 nM prior to ED and increased to 24 microM during ED. These results demonstrates that CLSM and fluorescent probes can be used in functional neuronal networks in addition to cell cultures as previously described.
脑缺血期间细胞内钙的增加被认为是导致神经元死亡的一系列事件的共同最终途径。在海马切片能量剥夺（ED）期间，已使用离子选择性电极测量细胞内钙，在神经元培养物中则使用荧光技术进行测量。在本研究中，我们描述了一种使用共聚焦激光扫描显微镜（CLSM）可视化和量化脑切片中细胞内钙变化的新方法。通过细胞内注射荧光染料Fluo 3和Fura Red的1:2混合物，使海马切片中的CA1锥体神经元充满染料。然后使用CLSM对神经元进行可视化，并使用每个探针的荧光比率来量化ED之前和期间的细胞内钙浓度。ED之前细胞内游离钙浓度为60 nM，ED期间增加到24 μM。这些结果表明，除了如前所述用于细胞培养外，CLSM和荧光探针还可用于功能性神经元网络。
