Voltage‐gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium

Abstract

This study reports the composition of a chemically defined medium (DDM1) that supports the survival and differentiation of neurons in dissociated cell cultures prepared from midgastrula stage Drosophila embryos. Cells with neuronal morphology that stain with a neural-specific marker are clearly differentiated by 1 day in vitro and can be maintained in culture for up to 2 weeks. Although the whole cell capacitance measurements from neurons grown in DDM1 were 5- to 10-fold larger than those of neurons grown in a conventional serum-supplemented medium, the potassium current densities were similar in the two growth conditions. A small but significant increase in the sodium current density was observed in the neurons grown in DDM1 compared with those in serum-supplemented medium. The majority of neurons grown in DDM1 fired either single or trains of action potentials in response to injection of depolarizing current. Contributing to the observed heterogeneity in the firing properties between individual neurons grown in DDM1 was heterogeneity in the levels of expression and gating properties of voltage-dependent sodium, calcium, and potassium currents. The ability of embryonic Drosophila neurons to differentiate in a chemically defined medium and the fact that they are amenable to both voltage-clamp and current-clamp analysis makes this system well suited to studies aimed at understanding the mechanisms regulating expression of ion channels involved in electrical excitability.

Molecular and functional characterization of voltage-gated sodium channel variants from Drosophila melanogaster

“…1). This observation is consistent with earlier analysis of partial cDNAs from D. melanogaster and D. virilis (O'Dowd et al, 1995;Ganetzky, 1994,1995). No positive or negative associations of these alternative exons with each other were observed.…”

“…Prior to this study, molecular characterization of partial DmNa V cDNA sequences resulted in the identification of eleven alternative exons in the DmNa V transcript (Thackeray and Ganetzky, 1994;O'Dowd et al, 1995;Warmke et al, 1997, Lee et al, 2002. In order to determine the alternative splicing pattern of each full-length DmNa V transcript and to conduct functional expression of each splice type, it was necessary to isolate and characterize a large number of full-length cDNA clones.…”

“…Functionally distinct sodium currents have been documented in various insect neurons, indicating existence of heterogeneous sodium channels with distinct gating properties in vivo (Byerly and Leung, 1988;Lapied et al, 1990Lapied et al, , 1999Wu, 1991, 1993 ;Schafer et al, 1994;O'Dowd, 1995;Le Corronc et al, 1999;Grolleau and Lapied, 2000;Wicher et al, 2001;Defaix and Lapied, 2005). A fundamental question is how insects produce heterogeneous sodium channels from a single gene.…”

“…One possibility is that posttranscriptional modifications of the primary sodium channel transcript result in variations of sodium channel gating properties. Indeed, alternative splicing and RNA editing of the DmNa V transcript have been reported (O'Dowd and Aldrich, 1988;Loughney et al, 1989;Ganetzky, 1994, 1995;O'Dowd, 1995;Warmke et al, 1997;Palladino et al, 2000). The functional consequences of alternative splicing and/or RNA editing of the DmNa V transcript, however, remain largely unknown.…”

The Steroid Hormone 20-Hydroxyecdysone Enhances Neurite Growth ofDrosophilaMushroom Body Neurons Isolated during Metamorphosis

“…This structural fidelity suggests that pupal M B neurons have an endogenous program directing their morphogenetic development, analogous to that proposed for hippocampal pyramidal neurons (Banker and Cowan, 1979;Dotti et al, 1988). Previous studies of cultured Drosophila neurons of various stages from wild type and mutants allowed morphological and electrophysiological assessment, but without specific identification of the neurons beyond their regional origin (Salvaterra et al, 1987;Byerly and Leung, 1988;Solc and Aldrich, 1988;Wu, 1988;Li and Meinertzhagen, 1995;O'Dowd, 1995;Z hao and Wu, 1997). Primary cultures enriched for M B neurons have been established from brains of other insects (Kreissl and Bicker, 1992;Cayre et al, 1998), but the lack of tools for genetic manipulation limits the versatility of these systems.…”