In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos -Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila , para . Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para , while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of lar...