This provides evidence that this restricted cell population is likely capable of firing APs, that expression is a potential marker for active AP firing neurons, and that only 23 1% of cells in the third instar larvae fit this criterion. 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 Geldanamycin 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 NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates. SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, is 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 that expression 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. (Huang et al., 2017), the genome of encodes only two genes predicted to encode NaV proteins and (is the putative NaV channel as null animals are viable with no loss of inward sodium currents detected in neurons using patch clamp (Germeraad et al., 1992; Anholt et al., 1996; Kulkarni et al., BGLAP 2002). In contrast, null animals die as first instar larvae with no detectable inward sodium current in neurons using patch clamp (Loughney et al., 1989; O’Dowd et al., 1989; Hong and Ganetzky, 1994). Despite having one NaV gene, compared with nine in mammals, it is possible that a similar degree of channel protein diversity is achieved via alternate splicing. has 60 predicted isoforms, some of which have different developmental expression (Lin et al., 2009; Baines Geldanamycin et al., 2012). Very little is known about the expression pattern or subcellular localization of Para. ISH studies determined that is expressed in the nervous system from embryos to adults (Amichot et al., 1993; Hong and Ganetzky, 1994). Whether is expressed in all or just some cells in the nervous system, and where it is subcellularly localized, remains to be established. To determine the expression pattern and protein localization of NaV channels in neurons, we used previously established tools (Bateman et al., 2006; Venken et al., 2011) to develop two novel fly models: a model where the endogenous Para is tagged with GFP to determine Para subcellular localization and another with replaced with GAL4 to determine gene expression. Surprisingly, we find present in a small fraction of CNS neurons in embryos and third instar larvae, while it is broadly expressed in neurons in the adult CNS. We also generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify that correlates with RNAs of active zone proteins and mature neuron markers; hence, expression is restricted to active, differentiated neurons in larvae. Neurons that coexpress and active zone protein RNAs are abundant in the adult CNS but only represent 23 1% of neurons in third instar larvae. In neurons where is expressed, Para protein is enriched at an AIS-like region in axons distal to where the dendritic tree connects to the axons in a distal axonal segment (DAS). Para localized far from the soma is functionally verified electrophysiologically. In longer neurons, Para Geldanamycin is expressed throughout the axon, likely to maintain AP propagation to the synapses. Materials and Methods Reagent and resource sharing Further information and requests for resources and reagents should be directed to and will be fulfilled by H.J.B. Flies generated in this study will be deposited to the Bloomington Drosophila Stock Center. Model and subject details For experiments using gene or protein-trapped alleles, all stocks were kept at room temperature (22C), all crosses were performed at 25C, and both male and female flies were used for imaging experiments. For the single-cell RNA sequencing experiments on the larval brain, flies were Geldanamycin raised on a yeast-based medium at 25C on a 12 h/12 h day/night light cycle. All lines used in the single-cell RNA-seq experiments are derived from the DGRP collection. One hybrid was created by crossing different DGRP lines,.
Posted in Non-selective Muscarinics.