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

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

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

Effect of Kir2.1 expression on the functional expression of K_Ca currents in embryonic lumbar motoneurons developing in vivo.

<p><i>A, B)</i> Outward currents in E11 motoneurons from chicken embryos infected with RCASBP(B) or RCASBP(B)-Kir2.1 constructs, respectively. Outward currents were evoked in control and Ca²⁺-free saline (right traces in <i>A</i> and <i>B</i>) following a 25 ms depolarizing step to +30 mV from a holding potential of −40 mV (stimulation protocol is shown as the left bottom trace in <i>A</i>). Net Ca²⁺-dependent outward currents were obtained by digital subtraction of raw traces (right traces in <i>A</i> and B). <i>C)</i> Kir2.1 expression resulted in a significant inhibition of K_Ca current density in E11 lumbar motoneurons. Notice that at K_Ca expression is significantly low in chicken motoneurons isolated at E8 in both RCASBP(B) or RCASBP(B)-Kir2.1 infected embryos. K_Ca expression increased ∼3 fold in RCASBP(B)-infected embryos by E11 but was significantly reduced in RCASBP(B)-Kir2.1 infected embryos. * represents p≤0.05 vs. E8 RCASBP(B), ** p≤0.05 vs. E11 RCASBP(B)-Kir2.1.</p

Effect of Kir2.1 expression on cell capacitance and sodium current amplitude in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos.

<p><i>A)</i> Cell capacitance is not affected by inhibition of spinal cord activity in RCASBP(B)-Kir2.1 infected embryos. <i>B)</i> 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, I_Na) that precedes opening of potassium channels and the generation of an outward potassium current. <i>C)</i> 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.</p

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

<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

Effect of Kir2.1 expression on embryonic movement at E8 and E11 in chicken embryos infected with RCASBP(B), RCASBP(B)-GFP, or RCASBP(B)-Kir2.1.

<p>Controls consisted of non-injected embryos. <i>A & B)</i> Average number of kicks in a 3 min observation period in E8 (A) and E11 (B) chicken embryos injected with different RCASBP(B) constructs. The number of embryos analyzed under different condition is given above each bar. <i>C)</i> Plot showing the distribution of the number of kicks in RCASBP(B) and RCASBP(B)-Kir2.1 infected embryos at E8.</p