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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.

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Kv4 is Required for Maintaining Excitability in Drosophila Neurons.A, Representative firing patterns recorded with current injections of 40, 60, and 100 pA from wild-type (wt) and DNKv4 (DN) neurons. With increasing current injections, wt neurons display a reduction in the delay to the first AP and an augmentation in AP firing frequency, maintaining excitability for the duration of the 500 ms stimulus. DN neurons display little delay to the first AP, and subsequent peaks decrease in amplitude, compared with wt. B, Plotted are amplitudes of the first 10 peaks, representing APs or graded potentials, normalized to the first AP (N = 30 for wt, N = 20 for DN); a pronounced adaptation of peaks in DN neurons is observed. C, The average amplitude of the first AP in DN neurons (N = 20) is significantly greater than wt (N = 30). D, The interspike interval (ISI), measured as the time between the peaks of the first and second APs, is significantly decreased in DN neurons (N = 20) compared to wt (N = 30). E, Current-clamp recording was performed (100 pA, 500 ms), followed by a voltage-clamp recording to isolate the Kv4 current, as described in Figure 1A. Shown is the normalized charge carried by the Kv4 current plotted against the average interspike interval between each AP fired during the stimulus. A positive correlation (r = 0.78, N = 15) is seen between Kv4 current charge and average ISI. F–G, Shown are voltage responses of representative wt and DN neurons to a pair of 500 ms current injections of 100 pA, with an interval of 500 ms. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the doted lines. No significant difference in this “interburst” AHP was seen between wild-type and DNKv4 neurons (N = 9 for wt, N = 11 for DN) (G). Scale bars represent 10 mV and 100 ms.
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pone-0016043-g004: Kv4 is Required for Maintaining Excitability in Drosophila Neurons.A, Representative firing patterns recorded with current injections of 40, 60, and 100 pA from wild-type (wt) and DNKv4 (DN) neurons. With increasing current injections, wt neurons display a reduction in the delay to the first AP and an augmentation in AP firing frequency, maintaining excitability for the duration of the 500 ms stimulus. DN neurons display little delay to the first AP, and subsequent peaks decrease in amplitude, compared with wt. B, Plotted are amplitudes of the first 10 peaks, representing APs or graded potentials, normalized to the first AP (N = 30 for wt, N = 20 for DN); a pronounced adaptation of peaks in DN neurons is observed. C, The average amplitude of the first AP in DN neurons (N = 20) is significantly greater than wt (N = 30). D, The interspike interval (ISI), measured as the time between the peaks of the first and second APs, is significantly decreased in DN neurons (N = 20) compared to wt (N = 30). E, Current-clamp recording was performed (100 pA, 500 ms), followed by a voltage-clamp recording to isolate the Kv4 current, as described in Figure 1A. Shown is the normalized charge carried by the Kv4 current plotted against the average interspike interval between each AP fired during the stimulus. A positive correlation (r = 0.78, N = 15) is seen between Kv4 current charge and average ISI. F–G, Shown are voltage responses of representative wt and DN neurons to a pair of 500 ms current injections of 100 pA, with an interval of 500 ms. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the doted lines. No significant difference in this “interburst” AHP was seen between wild-type and DNKv4 neurons (N = 9 for wt, N = 11 for DN) (G). Scale bars represent 10 mV and 100 ms.

Mentions: We then used longer pulses (500 ms) of current injection and compared firing patterns in wild-type and DNKv4 neurons. While wild-type neurons displayed clear latencies to firing, one of the most pronounced differences in DNKv4 neurons was the near absence of a delay to the first AP in all DNKv4 neurons (Figure 3A–B, 4A). Consistent with this decreased latency to AP firing, we also found that DNKv4 neurons had a lower threshold for inducing repetitive firing. This was evident in the amount of current injection required to induce repetitive firing during both prolonged stimuli; wild-type neurons consistently required larger current injections to induce firing (Figure 3C–D, 4A). When the current injection was ramped from 0 to 150 pA, firing also induced earlier in DNKv4 neurons, compared with wild-type. The requirement for higher stimuli to induce repetitive firing was not due to a difference in resting potential since membrane potentials at rest were not significant different between wild-type and DNKv4.


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)

Kv4 is Required for Maintaining Excitability in Drosophila Neurons.A, Representative firing patterns recorded with current injections of 40, 60, and 100 pA from wild-type (wt) and DNKv4 (DN) neurons. With increasing current injections, wt neurons display a reduction in the delay to the first AP and an augmentation in AP firing frequency, maintaining excitability for the duration of the 500 ms stimulus. DN neurons display little delay to the first AP, and subsequent peaks decrease in amplitude, compared with wt. B, Plotted are amplitudes of the first 10 peaks, representing APs or graded potentials, normalized to the first AP (N = 30 for wt, N = 20 for DN); a pronounced adaptation of peaks in DN neurons is observed. C, The average amplitude of the first AP in DN neurons (N = 20) is significantly greater than wt (N = 30). D, The interspike interval (ISI), measured as the time between the peaks of the first and second APs, is significantly decreased in DN neurons (N = 20) compared to wt (N = 30). E, Current-clamp recording was performed (100 pA, 500 ms), followed by a voltage-clamp recording to isolate the Kv4 current, as described in Figure 1A. Shown is the normalized charge carried by the Kv4 current plotted against the average interspike interval between each AP fired during the stimulus. A positive correlation (r = 0.78, N = 15) is seen between Kv4 current charge and average ISI. F–G, Shown are voltage responses of representative wt and DN neurons to a pair of 500 ms current injections of 100 pA, with an interval of 500 ms. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the doted lines. No significant difference in this “interburst” AHP was seen between wild-type and DNKv4 neurons (N = 9 for wt, N = 11 for DN) (G). Scale bars represent 10 mV and 100 ms.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016043-g004: Kv4 is Required for Maintaining Excitability in Drosophila Neurons.A, Representative firing patterns recorded with current injections of 40, 60, and 100 pA from wild-type (wt) and DNKv4 (DN) neurons. With increasing current injections, wt neurons display a reduction in the delay to the first AP and an augmentation in AP firing frequency, maintaining excitability for the duration of the 500 ms stimulus. DN neurons display little delay to the first AP, and subsequent peaks decrease in amplitude, compared with wt. B, Plotted are amplitudes of the first 10 peaks, representing APs or graded potentials, normalized to the first AP (N = 30 for wt, N = 20 for DN); a pronounced adaptation of peaks in DN neurons is observed. C, The average amplitude of the first AP in DN neurons (N = 20) is significantly greater than wt (N = 30). D, The interspike interval (ISI), measured as the time between the peaks of the first and second APs, is significantly decreased in DN neurons (N = 20) compared to wt (N = 30). E, Current-clamp recording was performed (100 pA, 500 ms), followed by a voltage-clamp recording to isolate the Kv4 current, as described in Figure 1A. Shown is the normalized charge carried by the Kv4 current plotted against the average interspike interval between each AP fired during the stimulus. A positive correlation (r = 0.78, N = 15) is seen between Kv4 current charge and average ISI. F–G, Shown are voltage responses of representative wt and DN neurons to a pair of 500 ms current injections of 100 pA, with an interval of 500 ms. The AHP that follows each stimulus “burst” is measured as the hyperpolarization beyond the membrane potential before the stimulus, as indicated by the doted lines. No significant difference in this “interburst” AHP was seen between wild-type and DNKv4 neurons (N = 9 for wt, N = 11 for DN) (G). Scale bars represent 10 mV and 100 ms.
Mentions: We then used longer pulses (500 ms) of current injection and compared firing patterns in wild-type and DNKv4 neurons. While wild-type neurons displayed clear latencies to firing, one of the most pronounced differences in DNKv4 neurons was the near absence of a delay to the first AP in all DNKv4 neurons (Figure 3A–B, 4A). Consistent with this decreased latency to AP firing, we also found that DNKv4 neurons had a lower threshold for inducing repetitive firing. This was evident in the amount of current injection required to induce repetitive firing during both prolonged stimuli; wild-type neurons consistently required larger current injections to induce firing (Figure 3C–D, 4A). When the current injection was ramped from 0 to 150 pA, firing also induced earlier in DNKv4 neurons, compared with wild-type. The requirement for higher stimuli to induce repetitive firing was not due to a difference in resting potential since membrane potentials at rest were not significant different between wild-type and DNKv4.

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