DrosophilaVoltage-Gated Sodium Channels Are Only Expressed in Active Neurons and Are Localized to Distal Axonal Initial Segment-like Domains

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 intronicMinos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaVchannel in both male and femaleDrosophila,para. Despite being the only NaVchannel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS expresspara, while in the adult CNSparais broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identifyparaexpressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaVlocalization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENTThe site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly,para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally,parais only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows thatparaexpression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire NaV-dependent APs.