Persistent sodium currents participate in fictive locomotion generation in neonatal mouse spinal cord

Abstract

The persistent sodium current (I(Na(P))) has been implicated in the regulation of synaptic integration, intrinsic membrane properties, and rhythm generation in many types of neurons. We characterized I(Na(P)) in commissural interneurons (CINs) in the neonatal (postnatal days 0-3) mouse spinal cord; it is activated at subthreshold potentials, inactivates slowly, and can be blocked by low concentrations of riluzole. The role of I(Na(P)) in locomotor pattern generation was examined by applying riluzole during fictive locomotion induced by NMDA, serotonin, and dopamine or by stimulation of the cauda equina. Blockade of I(Na(P)) has marginal effects on the locomotion frequency but progressively weakens the rhythmic firing and locomotor-related membrane oscillation of CINs and motoneurons (MNs) and the locomotor-like bursts in ventral roots, until the motor pattern ceases. Riluzole directly affects the intrinsic firing properties of CINs and MNs, reducing their ability to fire repetitively during tonic depolarizations and raising their spike threshold. At the same time, riluzole has little effects on the strength of spike-evoked synaptic transmission onto CINs and MNs. Our results suggest that I(Na(P)) is essential for the generation of the locomotor pattern and acts in part by regulating the frequency of interneuron firing in the central pattern generator for locomotion.

Figure 1.

Experimental setup. A, Stimulation suction electrodes were placed on the rostral and caudal contralateral hemicords at the level of T13–L1 and L4–L5, respectively, to stimulate the contralateral projecting axons of CINs. Additionally, three suction recording electrodes were placed to monitor nerve activities from lL2, rL2, and lL5. A patch-clamp electrode was inserted in a small slit made in the L2 segment for neuronal recording. Bi, Extracellular recordings from lL2, rL2, and lL5 ventral roots after application of 6 μm NMDA, 12 μm 5-HT, and 18 μm DA, showing locomotor-like activity characterized by left–right (Bii) and flexor–extensor (Biii) alternation in a P2 spinal cord. The dotted circle indicates the level of statistical significance (p < 0.05). Ci, Di, A typical firing pattern of a dCIN and an MN during NMDA, 5-HT, and DA-induced locomotor-like activity. Cii, Dii, Circular lots of the phase and rhythmicity of the dCIN and MN from Ci and Di derived from circular statistics. Each black circle represents one action potential. The open square is the average phase vector point for this neuron. The direction of the vector represents the preferred phase of firing of the neuron, and the distance from the center indicates the statistical significance of the rhythmicity of the neuron. Circular plots are read in a clockwise direction with 0 at the 12 o'clock position. Ciii, Diii, Pooled data from the circular analysis showing the distribution of the preferred firing phase of all rhythmically active CINs and MNs. The dotted circle indicates the level of statistical significance (p < 0.05). Each small square represents the vector point for one experiment. Gray-shaded areas represent the ipsilateral L2 flexor phase. l-r, Left–right; F-E, flexor–extensor; c, contralateral.

Figure 2.

Riluzole blocks I_Na(P) in CINs. A, I_Na(P) recordings by a ramp protocol from −90 to 10 mV at the rate of 20 mV/s from a dCIN in control and different concentrations of riluzole (Ril). B, Riluzole-sensitive inward currents. C, Dose–response curve showing the effects of riluzole on I_Na(P). The curve was fit with the equation 1/(1 + (([D]/IC₅₀)ⁿ), where [D] is the riluzole concentration, IC₅₀ is the dose for half-inhibition, and n is the Hill coefficient. IC₅₀ of 2.8 ± 0.2 μm.

Figure 3.

Riluzole reversibly inhibits fictive locomotion induced by application of chemicals or stimulation of the cauda equina. A, Locomotor-like activity was recorded from lL2, rL2, and lL5 after application of 6 μm NMDA, 12 μm 5-HT, and 18 μm DA in a P2 isolated spinal cord; fictive locomotion was reversibly weakened and eventually inhibited by 5 μm riluzole (Ril). B, Riluzole did not affect the left–right (l-r) and flexor–extensor (F-E) alternation until the ventral root bursts became undetectable. C, The time course of effects of riluzole on the burst frequency (filled circles) and amplitude (filled triangles). D, Locomotor-like activities were recorded from lL2, rL2, and lL5 by stimulating cauda equina in a P1 isolated mouse spinal cord; the evoked fictive locomotion was reversibly inhibited by 5 μm riluzole.

Figure 4.

Effects of riluzole on rhythmic activity of MNs during fictive locomotion induced by a combined application of NMDA, 5-HT, and DA. A, Intracellular recordings from an MN in a P1 isolated spinal cord in current clamp, with iL2 and contralateral L2 (cL2) ventral root bursts. B, Riluzole (Ril) blocks the rhythmic bursting of the MN in current clamp in parallel with its block of ventral root bursts. C, Voltage-clamp recordings from the same MN and the ipsilateral and contralateral L2 ventral root bursts, showing rhythmic EPSCs driving the MN bursts. D, Riluzole blocks the rhythmic synaptic input to the MN in parallel with its blockade of fictive locomotion.

Figure 5.

Role of I_Na(P) in controlling the intrinsic membrane properties of partially synaptically isolated MNs. A, Tonic responses of an MN to a series of current injections in the presence of 50 μm AP-5, 15 μm CNQX, 10 μm strychnine, and 10 μm picrotoxin in a P3 spinal cord. B, Phasic responses of the MN to a series of current injections during application of 5 μm riluzole (Ril). C, Comparison of the action potentials elicited by 5 Hz transient inputs during control and riluzole application. D, Action potentials triggered by 5 Hz transient inputs during riluzole application. E, F, Action potentials triggered by 50 Hz transient inputs during control and riluzole application, respectively.

Figure 6.

Effect of I_Na(P) on synaptic inputs to MNs. A, A reflex EPSP in a P3 spinal cord evoked by stimulating the dorsal iL2 root. B, Riluzole (5 μm) (Ril) has no effect on the reflex EPSP. C, AP-5 at 50 μm and 15 μm CNQX block the reflex EPSP. D, A synaptically driven action potential in an MN shown in the gray square and amplified below, evoked by stimulating the dorsal iL2 root in a P2 spinal cord. Subsequent square pulse current injections evoke tonic spiking in the MN. E, Riluzole (5 μm) has no effect on the synaptically driven action potential from the same MN as D (gray square, amplified below), although it eliminates tonic spiking in response to tonic current injection. F, After washout of riluzole, tonic spiking responses to current injection are restored, and 50 μm AP-5 and 15 μm CNQX eliminate the synaptically driven action potential in the same MN.

Figure 7.

Effects of riluzole on the rhythmic activity of CINs during fictive locomotion induced by application of NMDA, 5-HT, and DA. A, Intracellular recordings from a dCIN in a P3 isolated spinal cord in current clamp and the iL2 and contralateral L2 (cL2) ventral root bursts. B, Riluzole (Ril) blocks the rhythmic activity of the dCIN in parallel with the blockade of fictive locomotion measured from the ventral roots. C, Voltage-clamp recordings from the same dCIN, showing rhythmic IPSCs in-phase with the contralateral L2 ventral root bursts. D, Riluzole abolishes the IPSCs in the dCIN in parallel with its reduction in fictive locomotion.

Figure 8.

Role of I_Na(P) in the intrinsic membrane properties of CINs in spinal cord slice preparations. A, Tonic responses to a series of current injections of a dCIN that is partially isolated from synaptic inputs by application of 50 μm AP-5, 15 μm CNQX, 10 μm strychnine, and 10 μm picrotoxin in a P2 slice. B, Riluzole abolishes the tonic responses of the dCIN to a series of current injections. C, Tonic potentials elicited by a square pulse current injection (left) are eliminated by riluzole (middle), but brief transient current pulses can still evoke spikes during the current step in the presence of riluzole.

Figure 9.

Riluzole does not block synaptic transmission to dCINs. A, An EPSP recorded from a dCIN from a P1 spinal cord, evoked by stimulating contralateral rostral hemicord. B, Riluzole (Ril) has no effect on the evoked EPSP. C, CNQX (15 μm) abolishes the EPSP and uncovers a simultaneous IPSP. D, E, Antidromic action potentials triggered by caudal hemicord stimulation are not affected by riluzole application.