One pair of motor neurons provokes early spontaneous motor behavior through periodic bursting in the chordate ascidian embryo

Swimming aquatic animals exhibit spontaneous motor behaviors before the maturation of swimming locomotion. In this study, we demonstrated that a single pair of motor neurons, A10.64/MN2, which exhibits periodic bursting, is essential for early spontaneous motor behaviors in the invertebrate chordate Ciona intestinalis type A (Ciona robusta). The Ca2+ oscillation was first observed only in a pair of cells at mid tailbud II (St.22) with an 80-sec interval, which shortened to 25 sec at late tailbud II (St.24). A dissociation experiment revealed that the Ca2+ oscillation occurred in a single cell independently, without contact with other cells. The start of the Ca2+ oscillation coincided with the Ca2+ elevation in ipsilateral tail muscle cells at late tailbud I (St.23). Cell lineage tracking revealed that a pair of cells exhibiting Ca2+ oscillation corresponded to A10.64/MN2 motor neurons. Simultaneous imaging of Ca2+ and membrane potential demonstrated that the Ca2+ oscillation coupled with a burst firing of membrane potential. Interestingly, the number and frequency of tail muscle contractions initially coincided with those of the burst, but gradually came to coincide with those of spikes in the burst toward late tailbud II (St.24). Finally, single-cell photoablation of A10.64/MN2 abolished early spontaneous motor behaviors until late tailbud II (St.24), suggesting that the early spontaneous motor behavior of Ciona is directly regulated by only a single pair of A10.64/MN2 motor neurons. These findings revealed that the chordate early spontaneous motor behavior was generated by a minimum motor circuit that consist of a pair of motor neurons, exhibiting spontaneous periodic bursts.