Abstract
In this protocol, we describe steps that utilize the optical clarity of the zebrafish larvae and the stereotyped motor neuron axon structure in the trunk to measure spontaneous or evoked motor neuron axon activity. This activity is detected with transgenic fluorescent indicators introduced into the larvae by zygotic injection. Fluorescent indicator intensity changes in the small neuromuscular junctions are quantified to measure the presynaptic calcium activity and consequent synaptic vesicle release. For complete details on the use and execution of this protocol, please refer to Mandal et al. (2020).