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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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Kir2.1 expression in the chicken spinal cord.A) Typical melting curve from samples obtained from an RCASBP(B)-infected embryo (continuous line) or an RCASBP(B)-Kir2.1-infected embryo (discontinuous line). Fluorescence melting peaks were obtained by plotting the negative derivative of the fluorescence signal over temperature (-dF/dT) as a function of temperature (T). Notice the sharp peak in the sample obtained from an RCASBP(B)-Kir2.1- but not RCASBP(B)-infected embryo, indicating the presence of one PCR product. B) Quantification of Kir2.1 mRNA expression by real time PCR in chicken ventral spinal cords isolated at E8. C & D) Whole cell recordings from RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Currents were evoked by an 850 ms-voltage ramp from −130 mV to +40 mV. Notice that incubation with 100 µM barium ions causes a significant reduction in the inwardly rectifying current (in D). Chicken embryos were infected with RCASBP(B)-Kir2.1 or RCASBP(B) open vector at E2. Controls consisted of non-injected embryos. Motoneurons were acutely isolated at E8.
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pone-0002971-g004: Kir2.1 expression in the chicken spinal cord.A) Typical melting curve from samples obtained from an RCASBP(B)-infected embryo (continuous line) or an RCASBP(B)-Kir2.1-infected embryo (discontinuous line). Fluorescence melting peaks were obtained by plotting the negative derivative of the fluorescence signal over temperature (-dF/dT) as a function of temperature (T). Notice the sharp peak in the sample obtained from an RCASBP(B)-Kir2.1- but not RCASBP(B)-infected embryo, indicating the presence of one PCR product. B) Quantification of Kir2.1 mRNA expression by real time PCR in chicken ventral spinal cords isolated at E8. C & D) Whole cell recordings from RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Currents were evoked by an 850 ms-voltage ramp from −130 mV to +40 mV. Notice that incubation with 100 µM barium ions causes a significant reduction in the inwardly rectifying current (in D). Chicken embryos were infected with RCASBP(B)-Kir2.1 or RCASBP(B) open vector at E2. Controls consisted of non-injected embryos. Motoneurons were acutely isolated at E8.

Mentions: Does infection of chicken spinal cord with the RCASBP(B)-Kir2.1 construct result in increased Kir2.1 expression? Expression of Kir2.1 in chicken embryos was quantified by real time PCR using E8 ventral spinal cords. As shown in Fig. 4A (discontinuous line) from a representative sample of a chick embryo infected with RCASBP(B)-Kir2.1, the melting curve show a single peak, indicating the presence of a single PCR product. No amplification product was detected in samples obtained from RCASBP(B)-infected embryos (Fig. 4A, continuous line) or non-infected controls (not shown). Quantification of Kir2.1 mRNA expression by real time PCR shows no detectable levels of Kir2.1 mRNA in non-injected controls and embryos injected with the RCASBP(B) open vector (Fig. 4B). Kir2.1 mRNA expression was only detected in embryos infected with the RCASBP(B)-Kir2.1 construct (Fig. 4B). Little expression of Kir2.1 mRNA seems to confirm data from our electrophysiological recordings that show no inward rectification in acutely isolated lumbar motoneurons from non-injected embryos or embryos injected with the RCASBP(B) open vector (Fig, 4C). Functional expression of Kir2.1 channels was assessed in isolated lumbar motoneurons by recording whole cell currents generated by injection of an 850 ms-voltage ramp from −130 to +20 mV (Fig. 4C & D). In chicken embryos injected with RCASBP(B) open vector, injection of hyperpolarizing voltage steps or a voltage ramp did not show any inward current at hyperpolarizing voltage potentials between −130 and −60 mV (total of 28 neurons recorded from 4 different embryos, Fig. 4C). Consistent with an increased expression of Kir2.1 mRNA, whole cell recordings revealed the presence of an inward rectifying current in motoneurons from RCASBP(B)-Kir2.1 injected embryos (n = 12 out of 28 neurons recorded, Fig. 4D). Kir2.1 positive motoneurons were defined as those having an inward current above 50 pA at −110 mV and a non flat IV between −130 and −40 mV. The inwardly rectifying current reverses in polarity at −70.3±6.2 mV (n = 7), close to the potassium reversal potential calculated according to the Nernst equation (−80.8 mV) under our specific recording conditions. Consistent with the high sensitivity of Kir2.1 channels to low concentrations of barium ions [31], application of 100 µM barium blocked the inwardly rectifying current by ∼80% (76.8±15.2% at −110 mV, n = 4; Fig. 4D).


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)

Kir2.1 expression in the chicken spinal cord.A) Typical melting curve from samples obtained from an RCASBP(B)-infected embryo (continuous line) or an RCASBP(B)-Kir2.1-infected embryo (discontinuous line). Fluorescence melting peaks were obtained by plotting the negative derivative of the fluorescence signal over temperature (-dF/dT) as a function of temperature (T). Notice the sharp peak in the sample obtained from an RCASBP(B)-Kir2.1- but not RCASBP(B)-infected embryo, indicating the presence of one PCR product. B) Quantification of Kir2.1 mRNA expression by real time PCR in chicken ventral spinal cords isolated at E8. C & D) Whole cell recordings from RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Currents were evoked by an 850 ms-voltage ramp from −130 mV to +40 mV. Notice that incubation with 100 µM barium ions causes a significant reduction in the inwardly rectifying current (in D). Chicken embryos were infected with RCASBP(B)-Kir2.1 or RCASBP(B) open vector at E2. Controls consisted of non-injected embryos. Motoneurons were acutely isolated at E8.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2500219&req=5

pone-0002971-g004: Kir2.1 expression in the chicken spinal cord.A) Typical melting curve from samples obtained from an RCASBP(B)-infected embryo (continuous line) or an RCASBP(B)-Kir2.1-infected embryo (discontinuous line). Fluorescence melting peaks were obtained by plotting the negative derivative of the fluorescence signal over temperature (-dF/dT) as a function of temperature (T). Notice the sharp peak in the sample obtained from an RCASBP(B)-Kir2.1- but not RCASBP(B)-infected embryo, indicating the presence of one PCR product. B) Quantification of Kir2.1 mRNA expression by real time PCR in chicken ventral spinal cords isolated at E8. C & D) Whole cell recordings from RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos. Currents were evoked by an 850 ms-voltage ramp from −130 mV to +40 mV. Notice that incubation with 100 µM barium ions causes a significant reduction in the inwardly rectifying current (in D). Chicken embryos were infected with RCASBP(B)-Kir2.1 or RCASBP(B) open vector at E2. Controls consisted of non-injected embryos. Motoneurons were acutely isolated at E8.
Mentions: Does infection of chicken spinal cord with the RCASBP(B)-Kir2.1 construct result in increased Kir2.1 expression? Expression of Kir2.1 in chicken embryos was quantified by real time PCR using E8 ventral spinal cords. As shown in Fig. 4A (discontinuous line) from a representative sample of a chick embryo infected with RCASBP(B)-Kir2.1, the melting curve show a single peak, indicating the presence of a single PCR product. No amplification product was detected in samples obtained from RCASBP(B)-infected embryos (Fig. 4A, continuous line) or non-infected controls (not shown). Quantification of Kir2.1 mRNA expression by real time PCR shows no detectable levels of Kir2.1 mRNA in non-injected controls and embryos injected with the RCASBP(B) open vector (Fig. 4B). Kir2.1 mRNA expression was only detected in embryos infected with the RCASBP(B)-Kir2.1 construct (Fig. 4B). Little expression of Kir2.1 mRNA seems to confirm data from our electrophysiological recordings that show no inward rectification in acutely isolated lumbar motoneurons from non-injected embryos or embryos injected with the RCASBP(B) open vector (Fig, 4C). Functional expression of Kir2.1 channels was assessed in isolated lumbar motoneurons by recording whole cell currents generated by injection of an 850 ms-voltage ramp from −130 to +20 mV (Fig. 4C & D). In chicken embryos injected with RCASBP(B) open vector, injection of hyperpolarizing voltage steps or a voltage ramp did not show any inward current at hyperpolarizing voltage potentials between −130 and −60 mV (total of 28 neurons recorded from 4 different embryos, Fig. 4C). Consistent with an increased expression of Kir2.1 mRNA, whole cell recordings revealed the presence of an inward rectifying current in motoneurons from RCASBP(B)-Kir2.1 injected embryos (n = 12 out of 28 neurons recorded, Fig. 4D). Kir2.1 positive motoneurons were defined as those having an inward current above 50 pA at −110 mV and a non flat IV between −130 and −40 mV. The inwardly rectifying current reverses in polarity at −70.3±6.2 mV (n = 7), close to the potassium reversal potential calculated according to the Nernst equation (−80.8 mV) under our specific recording conditions. Consistent with the high sensitivity of Kir2.1 channels to low concentrations of barium ions [31], application of 100 µM barium blocked the inwardly rectifying current by ∼80% (76.8±15.2% at −110 mV, n = 4; Fig. 4D).

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