Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts

Abstract

A culture system of "giant" Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiated into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers (Wu et al., 1990). Here, we report that these giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibited distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca(2+)-activated K+ currents. In addition, we found a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke (slo), which is known to lack a component of the Ca(2+)-activated K+ currents in muscles, exhibited prolonged action potentials associated with defects in the Ca(2+)-activated K+ current. This abnormality appeared to be more severe in the neurites than in the soma.

Calcium Current Diversity in Physiologically Different Local Interneuron Types of the Antennal Lobe

“…In all investigated cell types, the current waveforms and physiological properties of I Ca were typical for voltage-activated Ca 2ϩ currents and in the range of other insect preparations [A. mellifera, antennal motorneurons (Kloppenburg et al, 1999a) and Kenyon cells (Schäfer et al, 1994); D. melanogaster, embryonic neurons (Byerly and Leung, 1988;Saito and Wu, 1991); Gryllus bimaculatus, giant interneurons (Kloppenburg and Hörner, 1998); P. americana, embryonic cockroach neurons (Benquet et al, 1999) and DUM neurons (Wicher and Penzlin, 1994); Manduca sexta, motor neurons (Hayashi and Levine, 1992); S. americana, thoracic neurons (Laurent et al, 1993); Schistocerca gregaria, DUM neurons (Heidel and Pflüger, 2006) and thoracic neurons (Pearson et al, 1993)]. However, a quantitative comparison of the functional properties from I Ca between the different cell types revealed significant differences in some physiologically important parameters (for summary, see supplemental Table 1, available at www.jneurosci.org as supplemental material).…”

“…5B). This is a relatively low activation threshold compared with I Ca in uPNs and in many other insect neurons (Byerly and Leung, 1988;Saito and Wu, 1991;Hayashi and Levine, 1992;Laurent et al, 1993;Pearson et al, 1993;Schä-fer et al, 1994;Wicher andPenzlin, 1994, 1997;Kloppenburg and Hörner, 1998;Benquet et al, 1999;Kloppenburg et al, 1999a;Heidel and Pflüger, 2006). In type II LNs, despite the similar activation threshold in both LN types, the half-maximal voltage for activation is significantly more hyperpolarized than in type I LNs (Fig.…”

Impaired clock output by altered connectivity in the circadian network

“…A null mutation in the slo locus strikingly alters the electrical properties of both neurons and muscles (22)(23)(24), affecting neurotransmitter release (25) and thereby causing a variety of behavioral defects among those in courtship behavior (26)(27)(28). We have previously demonstrated that a slo null mutant is behaviorally arrhythmic under free-running [constant darkness (DD)] conditions (13).…”

Reduced Delayed-Rectifier K+ Current in the Learning Mutant rutabaga

“…The steady-state inactivation that was determined here (range 6%-20%) is typical of the delayed-rectifier K + current (Saito and Wu 1991;Alshuaib and Mathew 1998). Although the majority of cells did not display the transient A-type current, pharmacological experiments were carried out to test whether 4-AP affects the difference in K + current between the two genotypes.…”

“…For this reason, we focused mainly on the delayed-rectifier K + current. We applied a voltage protocol that maximizes the delayed-rectifier K + current and diminishes the A-type K + current (holding potential, −80 mV; test pulses, −40 to +60 mV) (Byerly and Leung 1988;Saito and Wu 1991;Alshuaib and Mathew 1998). IK DR was clearly activated at 0 mV, but only weakly activated at −20 mV in wild-type and rutabaga neurons.…”