Keiko Gengyo-Ando1, Yuko Kagawa-Nagamura2, Masamichi Ohkura2, Xianfeng Fei3, Min Chen3, Koichi Hashimoto4, Junichi Nakai5. 1. Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan; Brain and Body System Science Institute, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. Electronic address: andok@mail.saitama-u.ac.jp. 2. Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan; Brain and Body System Science Institute, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. 3. Graduate School of Information Sciences, Tohoku University, Sendai, Miyagi 980-8579, Japan. 4. Graduate School of Information Sciences, Tohoku University, Sendai, Miyagi 980-8579, Japan. Electronic address: koichi@m.tohoku.ac.jp. 5. Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan; Brain and Body System Science Institute, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. Electronic address: jnakai@mail.saitama-u.ac.jp.
Abstract
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.
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.
Authors: Frank K Lee; Jane C Lee; Bo Shui; Shaun Reining; Megan Jibilian; David M Small; Jason S Jones; Nathaniel H Allan-Rahill; Michael Re Lamont; Megan A Rizzo; Sendoa Tajada; Manuel F Navedo; Luis Fernando Santana; Nozomi Nishimura; Michael I Kotlikoff Journal: Elife Date: 2021-10-29 Impact factor: 8.140