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

开发了一种光学记录系统，可用于电生理实验，利用无脊椎动物的整个动物制备来可视化细胞内钙浓度与突触活动相关的变化。该系统包括一个用于激发Fura-2的荧光装置，该装置通过离子电泳注射到细胞中，一个工作距离为187mm的解剖显微镜，一个peltier冷却CCD摄像机，以及一台装有新开发的图像分析程序的个人计算机。为了测试该记录系统的性能，我们将小龙虾腹末节的神经细胞装载染料，在CCD曝光时间为16秒的情况下从背侧观察。在解剖显微镜下，在380nm或340nm的激发下，可以原位观察到包括体细胞、树突和轴突在内的整个细胞结构以及电极尖端。当在暴露时间内以1Hz重复刺激感觉神经束时，在细胞的特定树突状区域检测到染料在380nm激发下的荧光减少。然而，在突触激活过程中，由于340nm激发的荧光强度相对较弱，无法检测到380nm与340nm激发的荧光比例有明显变化。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，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浓度变化以及整个细胞结构来定位细胞内的功能突触区域。

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.