Limits...
Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila.

Ping Y, Waro G, Licursi A, Smith S, Vo-Ba DA, Tsunoda S - PLoS ONE (2011)

Bottom Line: Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents.Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested.Based on our results, Shal/K(v)4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT

Background: Rhythmic 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 (I(A)), encoded by the highly conserved Shal/K(v)4 gene, in neuronal firing patterns and repetitive behaviors. While I(A) is present in nearly all neurons across species, elimination of I(A) has been complicated in mammals because of multiple genes underlying I(A), and/or electrical remodeling that occurs in response to affecting one gene.

Methodology/principal findings: In Drosophila, the single Shal/K(v)4 gene encodes the predominant I(A) current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v)4 neurons display multiple defects during prolonged stimuli. DNK(v)4 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/K(v)4 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/K(v)4 function. Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested.

Conclusions/significance: Based on our results, Shal/K(v)4 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/K(v)4 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.

Show MeSH

Related in: MedlinePlus

Loss of Kv4 Function Affects Repetitive Firing in Motoneurons, Larval and Adult Locomotion, and Grooming.A, Shown are voltage responses of representative wild-type (wt) and DNKv4 (DN) motoneurons, identified by RRa-Gal4:UAS-CD8-GFP, to repeated 500 ms pulses of 100 pA current injections, with intervals of 500 ms; this protocol mimics stimuli during fictive larval locomotion (see text). Note the loss of latency to firing, and loss of excitability for repetitive firing until the end of each stimulus, in the DN motoneuron. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the dotted lines. Scale bars represent 10 mV and 100 ms. B, Average amplitudes of the interburst AHP are shown; no significant difference was observed between wt and DN motoneurons (N = 5 for wt, N = 6 for DN). C, Crawling speed of individual third instar larvae was measured as the number of 0.5×0.5 cm squares crossed on an agarose plate in a five minute period. Averages from UAS-DNKv4 (UAS), elav-Gal4:UAS-DNKv4 (elav), c164-Gal4:DNKv4, and 109(80)-Gal4:DNKv4 (md) larvae are shown; 14–15 larvae from each genotype were tested. Note that all DN genotypes, whether driven in the entire nervous system (elav), in all motoneurons (mn), or in all multi-dendritic sensory neurons (md), displayed significantly slower crawling speeds from wt and the UAS background stock. D, Adult locomotion was tested in a climbing assay on adult wt, ShKS133 (Sh), eag1 (eag), UAS-DNKv4#14 (US, #14), elav-Gal4:UAS-DNKv4#14 (DN, #14), UAS-DNKv4#20 (US, #20), elav-Gal4:UAS-DNKv4#20 (DN, #20), and c164-Gal4: DNKv4#20 (mn, #20) flies (see Materials and Methods); each fly was given one point for every two tubes they climbed out of. The mean score of flies from each group was noted; this was then repeated for ten groups for each genotype with averages shown. All DN stocks, driven in the whole nervous system or in all motoneurons, displayed significantly lower scores than their respective background stocks. (E) Adult flies were tested for grooming (see Materials and Methods). Average percent clean flies at time points from 0 to 120 minutes are shown; three to four groups were examined for each time point. Time-courses for wt, UAS-DNKv4#14 (UAS), and elav-Gal4;UAS-DNKv4#14 (DN) flies are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3022017&req=5

pone-0016043-g007: Loss of Kv4 Function Affects Repetitive Firing in Motoneurons, Larval and Adult Locomotion, and Grooming.A, Shown are voltage responses of representative wild-type (wt) and DNKv4 (DN) motoneurons, identified by RRa-Gal4:UAS-CD8-GFP, to repeated 500 ms pulses of 100 pA current injections, with intervals of 500 ms; this protocol mimics stimuli during fictive larval locomotion (see text). Note the loss of latency to firing, and loss of excitability for repetitive firing until the end of each stimulus, in the DN motoneuron. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the dotted lines. Scale bars represent 10 mV and 100 ms. B, Average amplitudes of the interburst AHP are shown; no significant difference was observed between wt and DN motoneurons (N = 5 for wt, N = 6 for DN). C, Crawling speed of individual third instar larvae was measured as the number of 0.5×0.5 cm squares crossed on an agarose plate in a five minute period. Averages from UAS-DNKv4 (UAS), elav-Gal4:UAS-DNKv4 (elav), c164-Gal4:DNKv4, and 109(80)-Gal4:DNKv4 (md) larvae are shown; 14–15 larvae from each genotype were tested. Note that all DN genotypes, whether driven in the entire nervous system (elav), in all motoneurons (mn), or in all multi-dendritic sensory neurons (md), displayed significantly slower crawling speeds from wt and the UAS background stock. D, Adult locomotion was tested in a climbing assay on adult wt, ShKS133 (Sh), eag1 (eag), UAS-DNKv4#14 (US, #14), elav-Gal4:UAS-DNKv4#14 (DN, #14), UAS-DNKv4#20 (US, #20), elav-Gal4:UAS-DNKv4#20 (DN, #20), and c164-Gal4: DNKv4#20 (mn, #20) flies (see Materials and Methods); each fly was given one point for every two tubes they climbed out of. The mean score of flies from each group was noted; this was then repeated for ten groups for each genotype with averages shown. All DN stocks, driven in the whole nervous system or in all motoneurons, displayed significantly lower scores than their respective background stocks. (E) Adult flies were tested for grooming (see Materials and Methods). Average percent clean flies at time points from 0 to 120 minutes are shown; three to four groups were examined for each time point. Time-courses for wt, UAS-DNKv4#14 (UAS), and elav-Gal4;UAS-DNKv4#14 (DN) flies are shown.

Mentions: To examine how the loss of Kv4 function in motoneurons would likely affect their motor output, we applied repeated 500 ms current injections, at frequency of 1 Hz, to simulate the synaptic input motoneurons would likely receive during crawling, as previously described [34]. During these repeated depolarizations, wild-type neurons displayed bursts of firing which began after a constant delay and then continued until the end of the burst (Figure 7A). In contrast, DNKv4 motoneurons fired bursts with no delay to the first AP, but adapted easily and did not fire continuously until the end of the burst like wild-type (Figure 7A). Note that the AHP between bursts was still present in DNKv4 motoneurons (Figure 7A), and indistinguishable from wild-type (Figure 7B), consistent with the report that this AHP is due to the Na+-K+ pump [34]. Kv4, in contrast, is responsible for repolarization of the membrane after AP firing during repetitive firing, and it is this membrane repolarization that is required for maintaining excitability during bursts of AP firing.


Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila.

Ping Y, Waro G, Licursi A, Smith S, Vo-Ba DA, Tsunoda S - PLoS ONE (2011)

Loss of Kv4 Function Affects Repetitive Firing in Motoneurons, Larval and Adult Locomotion, and Grooming.A, Shown are voltage responses of representative wild-type (wt) and DNKv4 (DN) motoneurons, identified by RRa-Gal4:UAS-CD8-GFP, to repeated 500 ms pulses of 100 pA current injections, with intervals of 500 ms; this protocol mimics stimuli during fictive larval locomotion (see text). Note the loss of latency to firing, and loss of excitability for repetitive firing until the end of each stimulus, in the DN motoneuron. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the dotted lines. Scale bars represent 10 mV and 100 ms. B, Average amplitudes of the interburst AHP are shown; no significant difference was observed between wt and DN motoneurons (N = 5 for wt, N = 6 for DN). C, Crawling speed of individual third instar larvae was measured as the number of 0.5×0.5 cm squares crossed on an agarose plate in a five minute period. Averages from UAS-DNKv4 (UAS), elav-Gal4:UAS-DNKv4 (elav), c164-Gal4:DNKv4, and 109(80)-Gal4:DNKv4 (md) larvae are shown; 14–15 larvae from each genotype were tested. Note that all DN genotypes, whether driven in the entire nervous system (elav), in all motoneurons (mn), or in all multi-dendritic sensory neurons (md), displayed significantly slower crawling speeds from wt and the UAS background stock. D, Adult locomotion was tested in a climbing assay on adult wt, ShKS133 (Sh), eag1 (eag), UAS-DNKv4#14 (US, #14), elav-Gal4:UAS-DNKv4#14 (DN, #14), UAS-DNKv4#20 (US, #20), elav-Gal4:UAS-DNKv4#20 (DN, #20), and c164-Gal4: DNKv4#20 (mn, #20) flies (see Materials and Methods); each fly was given one point for every two tubes they climbed out of. The mean score of flies from each group was noted; this was then repeated for ten groups for each genotype with averages shown. All DN stocks, driven in the whole nervous system or in all motoneurons, displayed significantly lower scores than their respective background stocks. (E) Adult flies were tested for grooming (see Materials and Methods). Average percent clean flies at time points from 0 to 120 minutes are shown; three to four groups were examined for each time point. Time-courses for wt, UAS-DNKv4#14 (UAS), and elav-Gal4;UAS-DNKv4#14 (DN) flies are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3022017&req=5

pone-0016043-g007: Loss of Kv4 Function Affects Repetitive Firing in Motoneurons, Larval and Adult Locomotion, and Grooming.A, Shown are voltage responses of representative wild-type (wt) and DNKv4 (DN) motoneurons, identified by RRa-Gal4:UAS-CD8-GFP, to repeated 500 ms pulses of 100 pA current injections, with intervals of 500 ms; this protocol mimics stimuli during fictive larval locomotion (see text). Note the loss of latency to firing, and loss of excitability for repetitive firing until the end of each stimulus, in the DN motoneuron. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the dotted lines. Scale bars represent 10 mV and 100 ms. B, Average amplitudes of the interburst AHP are shown; no significant difference was observed between wt and DN motoneurons (N = 5 for wt, N = 6 for DN). C, Crawling speed of individual third instar larvae was measured as the number of 0.5×0.5 cm squares crossed on an agarose plate in a five minute period. Averages from UAS-DNKv4 (UAS), elav-Gal4:UAS-DNKv4 (elav), c164-Gal4:DNKv4, and 109(80)-Gal4:DNKv4 (md) larvae are shown; 14–15 larvae from each genotype were tested. Note that all DN genotypes, whether driven in the entire nervous system (elav), in all motoneurons (mn), or in all multi-dendritic sensory neurons (md), displayed significantly slower crawling speeds from wt and the UAS background stock. D, Adult locomotion was tested in a climbing assay on adult wt, ShKS133 (Sh), eag1 (eag), UAS-DNKv4#14 (US, #14), elav-Gal4:UAS-DNKv4#14 (DN, #14), UAS-DNKv4#20 (US, #20), elav-Gal4:UAS-DNKv4#20 (DN, #20), and c164-Gal4: DNKv4#20 (mn, #20) flies (see Materials and Methods); each fly was given one point for every two tubes they climbed out of. The mean score of flies from each group was noted; this was then repeated for ten groups for each genotype with averages shown. All DN stocks, driven in the whole nervous system or in all motoneurons, displayed significantly lower scores than their respective background stocks. (E) Adult flies were tested for grooming (see Materials and Methods). Average percent clean flies at time points from 0 to 120 minutes are shown; three to four groups were examined for each time point. Time-courses for wt, UAS-DNKv4#14 (UAS), and elav-Gal4;UAS-DNKv4#14 (DN) flies are shown.
Mentions: To examine how the loss of Kv4 function in motoneurons would likely affect their motor output, we applied repeated 500 ms current injections, at frequency of 1 Hz, to simulate the synaptic input motoneurons would likely receive during crawling, as previously described [34]. During these repeated depolarizations, wild-type neurons displayed bursts of firing which began after a constant delay and then continued until the end of the burst (Figure 7A). In contrast, DNKv4 motoneurons fired bursts with no delay to the first AP, but adapted easily and did not fire continuously until the end of the burst like wild-type (Figure 7A). Note that the AHP between bursts was still present in DNKv4 motoneurons (Figure 7A), and indistinguishable from wild-type (Figure 7B), consistent with the report that this AHP is due to the Na+-K+ pump [34]. Kv4, in contrast, is responsible for repolarization of the membrane after AP firing during repetitive firing, and it is this membrane repolarization that is required for maintaining excitability during bursts of AP firing.

Bottom Line: Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents.Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested.Based on our results, Shal/K(v)4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT

Background: Rhythmic 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 (I(A)), encoded by the highly conserved Shal/K(v)4 gene, in neuronal firing patterns and repetitive behaviors. While I(A) is present in nearly all neurons across species, elimination of I(A) has been complicated in mammals because of multiple genes underlying I(A), and/or electrical remodeling that occurs in response to affecting one gene.

Methodology/principal findings: In Drosophila, the single Shal/K(v)4 gene encodes the predominant I(A) current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v)4 neurons display multiple defects during prolonged stimuli. DNK(v)4 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/K(v)4 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/K(v)4 function. Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested.

Conclusions/significance: Based on our results, Shal/K(v)4 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/K(v)4 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.

Show MeSH
Related in: MedlinePlus