Birth order specified recruitment of motor circuits during spontaneous neural activity in zebrafish embryo

Modular organization of spinal neural circuits control dynamic regulation of locomotion. However, it is unknown when or how the distinct microcircuits emerge during development. We carried out high-resolution calcium imaging of neural activity driving the first motor behavior in the zebrafish embryo. During this period, at least two waves of neurogenesis occur to generate primary and secondary motoneurons. We found that embryos containing only primary motoneurons exhibit a single highly synchronized rhythmic circuit between the interneurons and motoneurons. Later, embryos with both primary and secondary motoneurons have two distinct interneuron-motoneuron circuits, one containing primary motoneurons displaying low-frequency activity and the other containing secondary motoneurons with high-frequency activity. The results indicate a mode of birth order determined microcircuits where neurons that are born together are recruited together. Nicotine affected neuronal activity frequency, revealing a functional role for cholinergic signaling in the emergence of patterned spinal microcircuits. Indeed, we found aberrant arrhythmic synchronized activity in mutants for cholineacetyltransferase-a where acetylcholine is no longer synthesized. Overall, we reveal the sequential recruitment of birth order specified microcircuits during the emergence of the earliest motor behavior and highlight a conserved role for cholinergic signaling in regulating rhythmic neural activity in the embryonic spinal cord.