Development of an optical recording system for neuronal network activities of invertebrates

一种可以用于电生理实验的光学记录系统已经被开发出来，该系统可以使用整个无脊椎动物的动物标本来可视化与突触活动相关的细胞内钙浓度的变化。该系统由一个用于激发Fura-2的荧光装置、一个工作距离为187 mm的解剖显微镜、一个Peltier冷却的CCD摄像机和一台装有新开发的图像分析程序的个人计算机组成。为了测试该记录系统的性能，将染料负载到小龙虾腹神经节末梢的神经细胞中，从背侧观察，CCD曝光时间为16秒。在380 nm和340 nm激发下，均可在解剖显微镜下原位观察到整个细胞结构，包括胞体、树突和轴突以及电极尖端。当在暴露时间内以1 Hz的频率重复刺激感觉神经束时，该染料对380 nm激发的荧光在细胞的特定树突区域被检测到减弱。然而，在突触激活过程中，380 nm和340 nm激发的荧光比值没有检测到任何明显的变化，可能是由于后者的强度相对较弱。结论：虽然灵敏度和时间分辨率不足以量化细胞内钙离子的动态变化，但目前开发的系统可以通过可视化突触活动引起的细胞内钙离子浓度的变化和整个细胞结构来定位细胞内的功能性突触区域。

An optical recording system that can be used in electrophysiological experiments using the whole animal preparation of invertebrates has been developed to visualize the change in intracellular calcium concentration associated with the synaptic activity. The system consists of an epifluorescence device for exciting Fura-2 that is iontophoretically injected into a cell, a dissecting microscope with the working distance of 187mm, a Peltier-cooled CCD video camera, and a personal computer with newly developed programs for image analysis. To test the performance of this recording system, nerve cells in the terminal abdominal ganglion of crayfish were loaded with the dye and observed from the dorsal side with the CCD exposure time of 16sec. The whole cell structure having the soma, dendrites and axon could be visualized in situ together with the electrode tip under the dissecting microscope by either 380nm or 340nm excitation. When the sensory nerve bundle was stimulated repeatedly at 1Hz during the exposure time, a decrease in the fluorescence of the dye to excitation at 380nm was detected in a specific dendritic region of the cell. However, any significant change in the ratio of fluorescence by 380nm to that by 340nm excitation during synaptic activation could not be detected probably due to the relatively weak strength of the latter. It is concluded that although the sensitivity and time resolution are not enough to quantify the dynamics of intracellular calcium ions, the presently developed system can be used to localize the functional synaptic region within a cell by visualizing the change in intracellular calcium concentration due to synaptic activity together with the whole cell structure.