Shal/Kv4 Channels Are Required for Maintaining Excitability during Repetitive Firing and Normal Locomotion in Drosophila

Abstract

BackgroundRhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K+ current (IA), encoded by the highly conserved Shal/Kv4 gene, in neuronal firing patterns and repetitive behaviors. While IA is present in nearly all neurons across species, elimination of IA has been complicated in mammals because of multiple genes underlying IA, and/or electrical remodeling that occurs in response to affecting one gene.Methodology/Principal FindingsIn Drosophila, the single Shal/Kv4 gene encodes the predominant IA current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNKv4), we show that IA is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNKv4 neurons display multiple defects during prolonged stimuli. DNKv4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/Kv4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/Kv4 function. Further, knock-out of Shal/Kv4 function specifically in motoneurons significantly affects the locomotion behaviors tested.Conclusions/SignificanceBased on our results, Shal/Kv4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/Kv4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors.

Effects of manipulating slowpoke calcium-dependent potassium channel expression on rhythmic locomotor activity inDrosophilalarvae

“…The role of MN currents in shaping rhythmic motor activity is a subject of active research (Harris-Warrick, 2002; Heckman et al, 2009; Kiehn et al, 2000; Marder & Goaillard, 2006). Much of the recent work has focused on the role of persistent inward currents in MNs (Enríquez Denton et al, 2012; Manuel et al, 2012; Revill & Fuglevand, 2011), but outward currents have also been shown to shape MN firing during rhythmic activity (Hooper et al, 2009; Manuel et al, 2012; Ping et al, 2011; Schaefer, Worrell & Levine, 2010). In particular, a recent study in Drosophila showed that elimination of Shal -mediated currents in MNs, via expression of a dominant negative construct under the control of RRA-GAL4, altered the frequency of larval crawling (Ping et al, 2011).…”

“…Much of the recent work has focused on the role of persistent inward currents in MNs (Enríquez Denton et al, 2012; Manuel et al, 2012; Revill & Fuglevand, 2011), but outward currents have also been shown to shape MN firing during rhythmic activity (Hooper et al, 2009; Manuel et al, 2012; Ping et al, 2011; Schaefer, Worrell & Levine, 2010). In particular, a recent study in Drosophila showed that elimination of Shal -mediated currents in MNs, via expression of a dominant negative construct under the control of RRA-GAL4, altered the frequency of larval crawling (Ping et al, 2011). The results from the RNAi experiments reported herein add to an increasing body of work suggesting that MN outward currents, and BK currents in particular, may enable MNs to shape the timing of rhythmic motor output.…”

Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

“…Dominant negative versions of the tetrameric potassium channels Shaker, Eag, Shaw, and Shal have been made by truncation of the wild-type channels, usually after the N-terminal multimerization domain (Broughton et al, 2004;Hodge et al, 2005;Mosca et al, 2005;Ping et al, 2011). RNAi constructs against Shaw also increase neural activity (Hodge and Stanewsky, 2008).…”

Inactivity-induced increase in nAChRs upregulates Shal K+ channels to stabilize synaptic potentials

“…Electrodes resistances for all perforated–patch and whole–cell recordings were 3–8 MΩ; gigaohm seals were obtained in all cases. For cell–attached recordings, electrode resistances were 7–9 MΩ, with > 5 GΩ seals; tips were fire–polished and inspected for uniform diameter. We isolated Shal currents by prepulse protocol, as previously described 15,46 . Briefly, we obtained total whole–cell K + currents using a voltage jump to +50 mV, following a 500 ms prepulse of –125 mV.…”

“…We isolated Shal currents by prepulse protocol, as previously described 15,46 . Briefly, we obtained total whole–cell K + currents using a voltage jump to +50 mV, following a 500 ms prepulse of –125 mV.…”