Jaffe D B, Brown T H
Department of Psychology, Yale University, New Haven, Connecticut 06520.
Microsc Res Tech. 1994 Nov 1;29(4):279-89. doi: 10.1002/jemt.1070290404.
Changes in the intracellular Ca2+ concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca(2+)-sensitive fluorescent indicators. The imaging was performed in thick hippocampal brain slices while simultaneously measuring or controlling electrical activity with sharp microelectrodes or whole-cell patch-clamp electrodes. The combination of imaging and electrophysiology was essential for interpreting the changes in [Ca2+]i. We compared the increases in [Ca2+]i produced by either of two methods--direct depolarization of the cell via the somatic electrode or high-frequency stimulations of synaptic inputs. The increases in [Ca2+]i in the soma and proximal dendrites caused by both methods were of comparable magnitude and they always decayed within seconds in healthy cells. However, the spatial patterns of distal Ca2+ increases were different. Separate sets of synaptic inputs to the same cell resulted in different spatial patterns of [Ca2+]i transients. We isolated and observed what appeared to be a voltage-independent component of the synaptically mediated [Ca2+]i transients. This work demonstrates that the combination of neurophysiology and simultaneous confocal microscopy is well suited for visualizing and analyzing [Ca2+]i changes within highly localized regions of neurons in thick brain slices. The approach should allow further analysis of the relative contribution of voltage- and agonist-dependent influences on [Ca2+]i within neurons throughout the CNS and it raises the possibility of routinely relating subcellular [Ca2+]i changes to structural and functional modifications.
利用共聚焦激光扫描显微镜结合钙敏感荧光指示剂，对海马CA1区锥体神经元内的细胞内钙离子浓度（[Ca2+]i）变化进行成像。成像在厚海马脑片中进行，同时用尖锐微电极或全细胞膜片钳电极测量或控制电活动。成像与电生理的结合对于解释[Ca2+]i的变化至关重要。我们比较了两种方法所引起的[Ca2+]i增加——通过体细胞电极直接使细胞去极化或高频刺激突触输入。两种方法引起的胞体和近端树突中[Ca2+]i的增加幅度相当，并且在健康细胞中它们总是在数秒内衰减。然而，远端钙离子增加的空间模式不同。对同一细胞的不同组突触输入导致[Ca2+]i瞬变的不同空间模式。我们分离并观察到了突触介导的[Ca2+]i瞬变中似乎与电压无关的成分。这项工作表明，神经生理学与同步共聚焦显微镜的结合非常适合于可视化和分析厚脑片中神经元高度局部区域内的[Ca2+]i变化。该方法应能进一步分析电压依赖性和激动剂依赖性影响对整个中枢神经系统神经元内[Ca2+]i的相对贡献，并增加了将亚细胞[Ca2+]i变化与结构和功能修饰常规关联的可能性。
