A new platform for long-term tracking and recording of neural activity and simultaneous optogenetic control in freely behaving Caenorhabditis elegans

Background Real-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 method Here, 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. Results Using 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 GABAergic motor neurons in conjunction with behavior monitoring. 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. Conclusion Our 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.