Inhibition of Electrical Activity by Retroviral Infection with Kir2.1 Transgenes Disrupts Electrical Differentiation of Motoneurons

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 Ca2+-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

Changes in resting membrane potential, input resistance and action potential amplitude in E8 and E11 motoneurons isolated from chicken embryos infected with RCASBP(B) or RCASBP(B)-Kir2.1.

*<p>, p<0.05 vs. E8 RCASBP(B);</p>**<p>, p<0.05 vs. E11 RCASBP(B);</p>#<p>, p<0.05 vs. E8 RCASBP(B)-Kir2.1</p

Effect of Kir2.1 expression on neuronal survival in non-injected, and RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos.

<p><i>A)</i><i> Islet</i> staining in the lumbar spinal cord of a Kir2.1-infected embryo. <i>B)</i> Higher magnification image of the ventral spinal cord cross section represented in <i>A.</i><i> </i><i>C)</i> Inhibition of electrical activity in RCASBP(B)- Kir2.1 infected embryos does not alter motoneuron survival, whereas application of the neuromuscular blocker tubocurare caused a significant increase in the number of surviving neurons. The number of <i>Islet</i>-positive neurons on both sides of the lumbar spinal cord was counted using design-based stereology. Chicken embryos were infected with RCASBP(B)-Kir2.1 or RCASBP(B) open vector at E2 and motoneuron survival was assessed at E10 from six lumbar segments (L1–L6).</p

Expression of GFP transgene in the chicken spinal cord following retroviral infection with an RCASBP(B)-GFP construct.

<p>RCASBP(B)-GFP viral particles were injected into the developing neural tube at E2 (approximately 36 hr after incubation). <i>A)</i> Infected embryos with RCASBP(B)-GFP show strong fluorescent labeling throughout the whole spinal cord cross section. <i>B)</i> High magnification picture of the ventral spinal cord section shown in A. <i>C)</i> Embryos injected with RCASBP(B) open vector show no fluorescent signal. In these experiments, chicken embryos were infected with RCASBP(B)-GFP or RCASBP(B) open vector at E2. Embryos were allowed to develop until E8 (corresponding to stage 34) before tissue isolation and sectioning.</p

Kir2.1 expression in the chicken spinal cord.

<p><i>A)</i> 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. <i>B)</i> Quantification of Kir2.1 mRNA expression by real time PCR in chicken ventral spinal cords isolated at E8. <i>C & D)</i> 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.</p

Typical action potentials generated in E8 and E11 chicken spinal motoneurons infected with RCASBP(B).

<p>Motoneurons were obtained from chicken embryos isolated at E8 or E11. Individual action potentials were generated by injection of 1 ms-depolarizing currents (<i>A</i> & <i>C</i>). Injection of long depolarizing current pulses (50 ms, <i>B</i> & <i>D</i>) only generated one action potential. Bottom trace in <i>A</i>–<i>D</i> represents the stimulation protocol used and the amount of current injected.</p

Immuno-labeling of chicken spinal cord for the viral gag p27 protein and the neuronal marker Hu.

<p><i>A)</i> RCASBP(B)-Kir2.1 infected embryos show significant expression of viral p27 gag throughout the spinal cord. <i>C)</i> Hu staining is found mainly in the gray matter area of the spinal cord and in the DRG. <i>E)</i> Superimposed images from <i>A</i> and <i>C</i> showing double staining of spinal cord neurons with gag p27 viral protein and Hu. <i>B, D, F)</i> Higher magnification image of the ventral spinal cord for the sections represented in <i>A, C, E,</i> respectively. In these experiments, chicken embryos were infected with RCASBP(B)-Kir2.1 at E2. Embryos were allowed to develop until E8 before tissue isolation and sectioning.</p

Effect of Kir2.1 expression on the kinetics of A-type K⁺ channels.

<p><i>A–D)</i> Outward K⁺ currents generated in E8 lumbar motoneurons from chicken embryos injected with the RCASBP(B) and RCASBP(B)-Kir2.1 constructs. Outward K⁺ currents were evoked following a series of 10 mV-depolarizing voltage steps from a holding potential of −100 mV (<i>A</i> and <i>C</i>) or from a holding potential of −40 mV (<i>B</i> and <i>D</i>). Stimulation protocol used in each case is shown as bottom traces in <i>C</i> and <i>D</i>. Net A-type K⁺ currents were obtained by digital subtraction of traces obtained from −100 and −40 mV holding potentials (<i>A–B</i> and <i>C–D</i>). <i>E)</i> Blockade of spinal cord activity has no significant effect on A-type K⁺ current density (<i>p</i> = 0.08). <i>F)</i> Disruption of spinal cord activity results in a significant reduction in the inactivation time constant of A-type K⁺ currents (τ, <i>p</i> = 0.04 vs. RCASBP(B)). <i>G–H)</i> Histograms of inactivation time constants in acutely isolated motoneurons from chicken embryos infected with the RCASBP(B) or RCASBP(B)-Kir2.1 constructs. Changes in A-type K⁺ current density and inactivation time constant were calculated from whole-cell currents evoked by a step pulse to +10 mV.</p

<i>A)</i> Distribution of AChR clusters and presynaptic terminals in a cross section of the iliofibularis (IFIB) muscle as revealed by double labeling with Alexa 488- conjugated α-bungarotoxin and SV2.

<p>Large arrows indicate co-localization of AChR clusters and presynaptic terminals, whereas the short arrow indicates an AChR clusters alone. <i>B)</i> The total number of synapses (as determined by co-localization of AChR clusters and SV2-labeling) was determined in control (non-injected), or chicken embryos infected with RCASBP(B) open vector or RCASBP(B)-Kir2.1. Chicken embryos were isolated at E7. Notice there are no significant differences in the number of synapses between non injected and RCASBP(B)-infected embryos. Infection of chicken embryos with RCASBP(B)-Kir2.1 results in a significant increase in the number of synapses along the IFIB muscle at E7. * denotes <i>p</i>≤0.05 vs. control (non-injected); ** denotes <i>p</i>≤0.05 vs. RCASBP(B)-infected embryos (n = 3).</p