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${it In vivo}$ calcium imaging of motor circuit in freely moving animals using improved G-CaMPs

Ando, Keiko*; Nagamura, Yuko*; Okura, Masamichi*; Fei, X.*; Suzuki, Michiyo; Hashimoto, Koichi*; Nakai, Junichi*

${it Caenorhabditis elegans}$ is a powerful model to investigate how the nervous system generates and regulates locomotion behavior. We previously showed that both body wall muscles and GABAergic DD/VD motoneurons in the ventral nerve cord exhibited increase in Ca$$^{2+}$$ levels during backward locomotion in unrestrained worms expressing G-CaMP4. To further analyze functional dynamics of the neuromuscular circuit, we have developed a new imaging device in which an auto-tracking unit is integrated into a fast-scanning confocal laser microscope to enable prolonged recording of multicolor fluorescence and transmitted images from freely moving worms. We also applied our newly designed G-CaMPs (G-CaMP6 and G-CaMP7) that allow greater signals than previous versions, to reliably obtain high-resolution images on the single cell levels. Using our imaging system, we found that, in contrast with the DD/VD motoneurons, GABAergic RME motoneurons that innervate the head muscles were persistently inhibited during spontaneous backward movement. Further studies for GABAergic regulation in the locomotion behavior are in progress.

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