Source:Journal of Neuroscience Methods
Author(s): Keiko Gengyo-Ando, Yuko Kagawa-Nagamura, Masamichi Ohkura, Xianfeng Fei, Min Chen, Koichi Hashimoto, Junichi Nakai
BackgroundReal-time recording and manipulation of neural activity in freely behaving animals can greatly advance our understanding of how neural circuits regulate behavior. Ca2+ imaging and optogenetic manipulation with optical probes are key technologies for this purpose. However, integrating the two optical approaches with behavioral analysis has been technically challenging.New methodHere, we developed a new imaging system, ICaST (Integrated platform for Ca2+ imaging, Stimulation, and Tracking), which combines an automatic worm tracking system and a fast-scanning laser confocal microscope, to image neurons of interest in freely behaving C. elegans. We optimized different excitation wavelengths for the concurrent use of channelrhodopsin-2 and G-CaMP, a green fluorescent protein (GFP)-based, genetically encoded Ca2+ indicator.ResultsUsing ICaST in conjunction with an improved G-CaMP7, we successfully achieved long-term tracking and Ca2+ imaging of the AVA backward command interneurons while tracking the head of a moving animal. We also performed all-optical manipulation and simultaneous recording of Ca2+ dynamics from these same GABAergic motor neurons in conjunction with behavior monitoring and manipulation.Comparison with existing method(s)Our system differs from conventional systems in that it does not require fluorescent markers for tracking and can track any part of the worm's body via bright-field imaging at high magnification. Consequently, this approach enables the long-term imaging of activity from neurons or nerve processes of interest with high spatiotemporal resolution.ConclusionOur imaging system is a powerful tool for studying the neural circuit mechanisms of C. elegans behavior and has potential for use in other small animals.
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