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

Vertebrate rhythmic motor behaviour is generated by the central pattern generator (CPG) located in the spinal cord. However, the development of the CPG has not been elucidated at the single-neuron level. We found that a single pair of motor neurons (A10.64/MN2) constitutes the CPG and regulates rhythmic early motor behaviour in the proto-chordate, Ciona. This pair of cells exhibited Ca2+ oscillation with an 80-sec interval at the mid-tailbud stage and 25 sec at the late tailbud stage. The Ca2+ oscillation occurred independently in a dissociated single cell. In the late tailbud stage, the Ca2+ oscillation began to coincide in phase with the ipsilateral tail muscle contraction, which corresponds to rhythmic early motor behaviour. Interestingly, the number and frequency of tail muscle contractions gradually coincided with spikes in the burst of membrane potential as the embryos developed toward late tailbud stage. Photoablation of A10.64/MN2 abolished the rhythmic early motor behaviour. These findings indicate that the early spontaneous rhythmic motor behaviour of Ciona is directly regulated by A10.64/MN2 as an essential component of the CPG. Our findings shed light on the development and evolution of chordate rhythmic behaviours.