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Inhibition of electrical activity by retroviral infection with Kir2.1 transgenes disrupts electrical differentiation of motoneurons.

Yoon YJ, Kominami H, Trimarchi T, Martin-Caraballo M - PLoS ONE (2008)

Bottom Line: Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo.Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels.Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Vermont, Burlington, Vermont, United States of America.

ABSTRACT
Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K(+) currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.

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Effect of Kir2.1 expression on cell capacitance and sodium current amplitude in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos.A) Cell capacitance is not affected by inhibition of spinal cord activity in RCASBP(B)-Kir2.1 infected embryos. B) Current generated by a 25 ms-depolarizing step to 0 mV from a holding potential of −80 mV (stimulation protocol is shown as bottom trace). Note the presence of a fast inward current (or sodium current, INa) that precedes opening of potassium channels and the generation of an outward potassium current. C) No changes in sodium current amplitude were detected in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Recordings were performed in acutely isolated motoneurons at E8.
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pone-0002971-g010: Effect of Kir2.1 expression on cell capacitance and sodium current amplitude in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos.A) Cell capacitance is not affected by inhibition of spinal cord activity in RCASBP(B)-Kir2.1 infected embryos. B) Current generated by a 25 ms-depolarizing step to 0 mV from a holding potential of −80 mV (stimulation protocol is shown as bottom trace). Note the presence of a fast inward current (or sodium current, INa) that precedes opening of potassium channels and the generation of an outward potassium current. C) No changes in sodium current amplitude were detected in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Recordings were performed in acutely isolated motoneurons at E8.

Mentions: We have previously demonstrated that pharmacological inhibition of ongoing electrical activity in the chicken spinal cord plays a significant role in regulating electrical differentiation of developing LMNs [2], [37]. To determine whether decreased electrical activity in chicken embryos results in a significant change in the electrical properties of LMNs we conducted whole cell recordings of acutely isolated cells. Motoneurons were isolated at E8 or E11 from embryos infected with the RCASBP(B) open vector or RCASBP(B)-Kir2.1 constructs. Infection of chicken embryos with RCASBP(B)-Kir2.1 did not result in a significant change in cell size as revealed by measurements of cell capacitance in E8 motoneurons (cap RCASBP(B) = 21.4±2.5 pF, cap RCASBP(B)-Kir2.1 = 16.4±1.3 pF, Fig. 10A). Cell capacitance in RCASBP(B) or RCASBP(B)-Kir2.1-injected embryos was similar to that previously reported for non-injected spinal cord neurons [2]. In the chicken spinal cord, Na+ currents mediate action potential spikes at E6 [38]. To determine whether paralysis of spinal cord activity with Kir2.1 has any effect on Na+ conductances we measured the current generated by injection of depolarizing voltage step to 0 mV from a holding potential of −80 mV. Expression of Kir2.1 transgene did not alter the amplitude of voltage-gated sodium currents in acutely isolated motoneurons (INa RCASBP(B) = −663±176 pA, INa RCASBP(B)-Kir2.1 = −547±147 pA, Fig. 10 B & C). Expression of Kir2.1 transgene, however, did have a noticeable effect on two types of potassium conductances. Previous findings indicate that expression of Ca2+-dependent K+ (KCa) channels is developmentally regulated in chicken spinal motoneurons [2]. Thus, the functional expression of KCa channels increases ∼3 fold between E8 and E11 (Fig. 11C). Similar to our previous findings in non-infected chicken embryos, in spinal cords transfected with RCASBP(B) open vector we also observed a near 3 fold increase in KCa channel density (IKCa E8 RCASBP(B) = 7.5±2.9 pA/pF, IKCa E11 RCASBP(B) = 19.4±3.1 pA/pF, Fig. 11A, B, C). The age-dependent increase in KCa expression was reversed in chicken embryos infected with RCASBP(B)-Kir2.1 suggesting that inhibition of electrical activity prevents normal development of KCa channels (IKCa E8 RCASBP(B)-Kir2.1 = 5.5±0.7 pA/pF, IKCa E11 RCASBP(B)-Kir2.1 = 7.3±2.4 pA/pF, Fig. 11A, B, C). KCa channel activation required Ca2+ influx via voltage-activated Ca2+ currents. Therefore, inhibition of KCa expression could be attributed to activity-evoked changes in voltage-activated Ca2+ currents. To investigate this possibility, we recorded voltage-activated Ca2+ currents in E11 motoneurons isolated from embryos infected with RCASBP(B) or RCASBP(B)-Kir2.1. Under our recording conditions (see Methods), Ca2+ current densities did not change significantly between RCASBP(B) and RCASBP(B)-Kir2.1-infected neurons. In RCASBP(B)-infected embryos, Ca2+ current density was −10.6±2.6 pA/pF (n = 10), whereas in RCASBP(B)-Kir2.1 infected embryos Ca2+ current density was −8.7±2.8 pA/pF (n = 9, p>0.05). These results suggest that changes in KCa expression are not due to changes in Ca2+ influx via voltage-activated Ca2+ channels.


Inhibition of electrical activity by retroviral infection with Kir2.1 transgenes disrupts electrical differentiation of motoneurons.

Yoon YJ, Kominami H, Trimarchi T, Martin-Caraballo M - PLoS ONE (2008)

Effect of Kir2.1 expression on cell capacitance and sodium current amplitude in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos.A) Cell capacitance is not affected by inhibition of spinal cord activity in RCASBP(B)-Kir2.1 infected embryos. B) Current generated by a 25 ms-depolarizing step to 0 mV from a holding potential of −80 mV (stimulation protocol is shown as bottom trace). Note the presence of a fast inward current (or sodium current, INa) that precedes opening of potassium channels and the generation of an outward potassium current. C) No changes in sodium current amplitude were detected in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Recordings were performed in acutely isolated motoneurons at E8.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2500219&req=5

pone-0002971-g010: Effect of Kir2.1 expression on cell capacitance and sodium current amplitude in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos.A) Cell capacitance is not affected by inhibition of spinal cord activity in RCASBP(B)-Kir2.1 infected embryos. B) Current generated by a 25 ms-depolarizing step to 0 mV from a holding potential of −80 mV (stimulation protocol is shown as bottom trace). Note the presence of a fast inward current (or sodium current, INa) that precedes opening of potassium channels and the generation of an outward potassium current. C) No changes in sodium current amplitude were detected in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Recordings were performed in acutely isolated motoneurons at E8.
Mentions: We have previously demonstrated that pharmacological inhibition of ongoing electrical activity in the chicken spinal cord plays a significant role in regulating electrical differentiation of developing LMNs [2], [37]. To determine whether decreased electrical activity in chicken embryos results in a significant change in the electrical properties of LMNs we conducted whole cell recordings of acutely isolated cells. Motoneurons were isolated at E8 or E11 from embryos infected with the RCASBP(B) open vector or RCASBP(B)-Kir2.1 constructs. Infection of chicken embryos with RCASBP(B)-Kir2.1 did not result in a significant change in cell size as revealed by measurements of cell capacitance in E8 motoneurons (cap RCASBP(B) = 21.4±2.5 pF, cap RCASBP(B)-Kir2.1 = 16.4±1.3 pF, Fig. 10A). Cell capacitance in RCASBP(B) or RCASBP(B)-Kir2.1-injected embryos was similar to that previously reported for non-injected spinal cord neurons [2]. In the chicken spinal cord, Na+ currents mediate action potential spikes at E6 [38]. To determine whether paralysis of spinal cord activity with Kir2.1 has any effect on Na+ conductances we measured the current generated by injection of depolarizing voltage step to 0 mV from a holding potential of −80 mV. Expression of Kir2.1 transgene did not alter the amplitude of voltage-gated sodium currents in acutely isolated motoneurons (INa RCASBP(B) = −663±176 pA, INa RCASBP(B)-Kir2.1 = −547±147 pA, Fig. 10 B & C). Expression of Kir2.1 transgene, however, did have a noticeable effect on two types of potassium conductances. Previous findings indicate that expression of Ca2+-dependent K+ (KCa) channels is developmentally regulated in chicken spinal motoneurons [2]. Thus, the functional expression of KCa channels increases ∼3 fold between E8 and E11 (Fig. 11C). Similar to our previous findings in non-infected chicken embryos, in spinal cords transfected with RCASBP(B) open vector we also observed a near 3 fold increase in KCa channel density (IKCa E8 RCASBP(B) = 7.5±2.9 pA/pF, IKCa E11 RCASBP(B) = 19.4±3.1 pA/pF, Fig. 11A, B, C). The age-dependent increase in KCa expression was reversed in chicken embryos infected with RCASBP(B)-Kir2.1 suggesting that inhibition of electrical activity prevents normal development of KCa channels (IKCa E8 RCASBP(B)-Kir2.1 = 5.5±0.7 pA/pF, IKCa E11 RCASBP(B)-Kir2.1 = 7.3±2.4 pA/pF, Fig. 11A, B, C). KCa channel activation required Ca2+ influx via voltage-activated Ca2+ currents. Therefore, inhibition of KCa expression could be attributed to activity-evoked changes in voltage-activated Ca2+ currents. To investigate this possibility, we recorded voltage-activated Ca2+ currents in E11 motoneurons isolated from embryos infected with RCASBP(B) or RCASBP(B)-Kir2.1. Under our recording conditions (see Methods), Ca2+ current densities did not change significantly between RCASBP(B) and RCASBP(B)-Kir2.1-infected neurons. In RCASBP(B)-infected embryos, Ca2+ current density was −10.6±2.6 pA/pF (n = 10), whereas in RCASBP(B)-Kir2.1 infected embryos Ca2+ current density was −8.7±2.8 pA/pF (n = 9, p>0.05). These results suggest that changes in KCa expression are not due to changes in Ca2+ influx via voltage-activated Ca2+ channels.

Bottom Line: Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo.Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels.Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Vermont, Burlington, Vermont, United States of America.

ABSTRACT
Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K(+) currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.

Show MeSH
Related in: MedlinePlus