Effect of prolonged riluzole exposure on cultured motoneurons in a mouse model of ALS

Abstract

Riluzole is the only FDA-approved drug to treat amyotrophic lateral sclerosis, but its long-term effects on motoneurons are unknown. Therefore, we treated primary mouse spinal cord cultures with 2 μM riluzole for 4-9 days and then used whole cell patch clamp to record the passive and active properties of both wild-type and SOD1(G93A) motoneurons. At this concentration, riluzole blocks >50% of the sodium component of a persistent inward current that plays a major role in determining motoneuron excitability. Prolonged riluzole treatment significantly decreased the amplitude of the persistent inward current. This effect was specific for SOD1(G93A) motoneurons, where the amplitude decreased by 55.4%. In addition, prolonged treatment hyperpolarized the resting membrane potential as well as the voltage onset and voltage maximum of the persistent inward current (∼2-3 mV in each case). These effects appeared to offset one another and resulted in no change in the firing properties. In a subset of cells, acute reapplication of 2 μM riluzole during the recording decreased repetitive firing and the persistent inward current, which is consistent with the normal effects of riluzole. The downregulation of the persistent inward current in response to prolonged riluzole administration is in contrast to the strong upregulation of this same current after descending neuromodulatory drive to the cord is lost following spinal injury. This dichotomy suggests that decreased activation of G protein-coupled pathways can induce upregulation in the persistent inward current but that direct channel block is ineffective.

Fig. 1.

Wild-type (wt) and superoxide dismutase 1 mutant (SOD1) current-voltage (IV) relationships. A: TTX-insensitive (TTX-ins) currents for both a wt (black) and a SOD1 (blue) neuron. Note that the SOD1 TTX-ins current has a more depolarized voltage onset and smaller amplitude. B: comparison of the persistent inward current (PIC) traces for the same wt (black, trace 1) and SOD1 (blue, trace 2) neurons as in A. Compare to the TTX-ins traces from the wt (light gray, trace 3) and SOD1 (light blue, trace 4). C: sodium PIC (NaPIC) for the same neurons: wt NaPIC (from B: trace 1 − trace 3). and SOD1 NaPIC (from B: trace 2 − trace 4). Note that the NaPIC amplitudes are nearly identical.

Fig. 2.

PIC and NaPIC onset and max voltages by drug condition. A: onset voltages for the PIC (left) and the NaPIC (right) by drug condition. B: max voltage for the PIC (left) and NaPIC (right) by drug condition. Each is presented as a box plot with median (thick line), 25% (top line), 75% (bottom line), 10% (top whiskers), and 90% (bottom whiskers). Box plots instead of means and SDs were used because the NaPIC onset voltage was nonparametric and to better show the actual distributions. Data shown are pooled across wt and SOD1 neurons as there was no significant main effect of genotype. See Table 2 for data separated by genotype and drug condition (4 groups) and ANOVA results. Significance: *0.05 ≥ P > 0.01, **0.01 ≥ P > 0.001, ****P ≤ 0.0001.

Fig. 3.

PIC and NaPIC amplitudes by drug condition and genotype. A: PIC amplitude with genotype [wt (left) and SOD1 (right)] by drug condition with pairwise differences. Note the decrease in PIC amplitude in SOD1 neurons treated with riluzole. B: NaPIC amplitude with genotype [wt (left) and SOD1 (right)] by drug condition. Note the lack of significant pairwise differences. Each is presented as a box plot with median (thick line), 25% (top line), 75% (bottom line), 10% (top whiskers), and 90% (bottom whiskers). Box plots instead of means and SDs were used because the PIC amplitude was nonparametric and to better show the actual distributions. See Table 2 for ANOVA results. Significance: *0.05 ≥ P > 0.01, **0.01 ≥ P > 0.001.

Fig. 4.

Reapplication of riluzole. Riluzole was reapplied to both wt and SOD1 neurons previously exposed to the chronic riluzole treatment. For A and B, all responses before reapplication of riluzole are on left; responses during reapplication are on right. Insets on left for A and B show the current input. All traces are from the same neuron. A: voltage response to a current ramp. Note the decrease in firing, higher current threshold, and smaller action potential (AP) height. B: voltage response to a current step. Note the decrease in firing rate and AP failure. Inset on right shows that the height of the initial AP is not altered. C and D: current response to a voltage ramp. C: current trace before riluzole (black, trace 1), after riluzole application (gray, trace 2), and after TTX application (light gray, trace 3). D: NaPIC amplitudes before and after riluzole application: NaPIC (black; from C: trace 1 − trace 3) and +2 Riluzole NaPIC (gray; from C: trace 2 − trace 3).