Optogenetic analysis of the functional role of Mauthner cell on the sound/vibration-evoked fast escapes in larval zebrafish

Tanimoto Masashi, Sugimoto Atsuko, Yokomichi Sena, Asakawa Kazuhide, Kawakami Koichi, Oda Yoichi

Analyses of behaviorally relevant identifiable neural circuits help us understand mechanisms of animal behaviors. Mauthner (M-) cells, a bilateral pair of giant reticulospinal neurons in the teleost fish hindbrain, are believed to play a central role on initiating fast startle responses with C-shaped body bending in response to an abrupt sound/vibration stimulus (Zottoli, 1977; Kohashi et al., 2012). M-cell ablation delays the behavior onset, but it does not completely eliminate the fast escapes in goldfish (Zottoli et al., 1999) and zebrafish (Kimmel et al., 1980). Our previous studies showed that larval zebrafish with bilateral M-cell ablation reduced escape probability and never escaped with the shortest onset latency, whereas they still exhibited escapes with the onset delayed by 5 ms. In the present study, we further examined the functional role of the M-cell by optogenetic silencing in larval zebrafish. Transgenic lines with M-cells expressing light-evoked proton pump Archearhodopsin3 (Arch) were generated by using Gal4/UAS binary expression system and Tol2-mediated transgenesis. In vivo patch-clamp recording showed that green light rapidly and continuously hyperpolarized the Arch-expressing M-cell, while it also evoked transient depolarization sensitive to glutamatergic transmission antagonists at the onset and offset of the light. Threshold current for the action potential generation increased during the green light, suggesting that the firing of Arch-expressing M-cells can be optogenetically silenced. Behavioral analysis showed that head-restrained zebrafish reduced the probability of fast escapes or delayed the behavior onset by 5 ms during the green light, whereas they exhibited normal escapes in the absence of the light. These results confirm the crucial role of the M-cell on the initiation of sound/vibration-evoked fast escapes and show that optogenetic manipulation of neural circuits in zebrafish is an effective approach to address working mechanisms of multiplex, hierarchical or functionally redundant motor control systems of intact animals.