The therapeutic mode of action of 4-aminopyridine in cerebellar ataxia

Abstract

Episodic ataxia type 2 (EA2) is a hereditary cerebellar ataxia associated with mutations in the P/Q-type voltage-gated calcium (Ca(2+)) channels. Therapeutic approaches for treatment of EA2 are very limited. Presently, the potassium (K(+)) channel blocker 4-aminopyridine (4-AP) constitutes the most promising treatment, although its mechanism of action is not understood. Here we show that, in contrast to what is commonly believed, therapeutic concentrations of 4-AP do not increase the inhibitory drive of cerebellar Purkinje cells. Instead, 4-AP restores the severely diminished precision of pacemaking in Purkinje cells of EA2 mutant mice by prolonging the action potential and increasing the action potential afterhyperpolarization. Consistent with this mode of action, the therapeutic efficacy of 4-AP was comparable, and not additive, to chlorzoxazone, an activator of Ca(2+)-dependent K(+) channels that also restores the precision of Purkinje cell pacemaking. The likely target of 4-AP at the concentrations used are the K(v)1 family of K(+) channels, possibly the K(v)1.5 subtype. Because at higher concentrations 4-AP blocks a large array of K(+) channels and is a proconvulsant, use of selective K(v)1 channel blockers is likely to be a safer substitute for treatment of cerebellar ataxia.

Figure 1.

Therapeutic concentrations of 4-AP do not affect the activity in Purkinje cells. A, The firing rate of a cerebellar Purkinje cells was monitored by extracellular recording and sequentially higher concentrations of 4-AP were bath applied. B, Histograms of the interspike interval distributions obtained from the cell shown in A in each condition. C, Average Purkinje cell firing rates in control condition and in the presence of 4-AP. *p < 0.05. D, Distribution of interspike intervals from a Purkinje cell recorded in the presence of blockers of excitatory and inhibitory synaptic transmission under control conditions and when the cell was exposed to 4-AP. E, Average Purkinje cell firing rates in control condition and in the presence of 4-AP with synaptic transmission blocked. n.s., Not significant.

Figure 2.

Therapeutic concentrations of 4-AP do not alter neurotransmitter release probability at parallel fiber synapses. A, Example traces of the response of an extracellularly recorded Purkinje cell to the electrical stimulation of PFs, in control conditions (Ctrl) and in increasing concentrations of 4-AP. B, Average Purkinje cell maximum firing rate in response to PF stimulation under various conditions (n = 8 cells). *p < 0.05, ***p < 0.001 (one-way ANOVA with Bonferroni's post test for multiple comparisons). C, Parallel fiber-evoked EPSCs recorded from a voltage-clamped Purkinje cell, under control conditions and sequential application of increasing concentrations of 4-AP. D, Average EPSC amplitudes in each condition reported above (n = 12 cells). *p < 0.05, ***p < 0.001. E, A comparison of the efficacy of 4-AP in increasing the amplitude of PF-evoked EPSCs versus PF-evoked increases in the firing rate of Purkinje cells. F, Paired-pulse ratio of the amplitudes of the two EPSCs evoked by paired activation of PFs (50 ms apart) in voltage-clamped Purkinje cells. 4-AP did not alter this ratio at any of the concentrations examined. G, Examples of mossy fiber (MF)-evoked EPSCs in voltage-clamped Purkinje cells under control conditions and in the presence of 5 μm 4-AP. H, Average of the MF-evoked EPSCs before and after application of 5 μm 4-AP. n.s., Not significant. p > 0.85 (paired t test).

Figure 3.

Therapeutic concentrations of 4-AP restores the precision of Purkinje cell pacemaking in the ataxic tg/tg mouse. A, Left, Raw traces from extracellular recordings obtained from tg/tg and wt Purkinje cells. Right, The corresponding autocorrelogram of the spontaneous activity of each cell. Note the increased variability in the interspike interval in the tg/tg cell. B, Coefficient of variation of interspike intervals and the firing rate of an extracellularly recorded tg/tg Purkinje cell under control conditions, and with sequential application of therapeutic concentrations of 4-AP. C, Average and individual values of coefficient of variation of interspike intervals in tg/tg Purkinje cells in control conditions and in the presence of 4-AP. The coefficient of variation of wt Purkinje cells is plotted for comparison. D, Average and individual values of the corresponding firing rates for the same cells shown in C. **p < 0.01; ***p < 0.001 (one-way ANOVA followed by Bonferroni's post test for multiple comparisons). n.s., Not significant.

Figure 4.

4-AP broadens action potentials in Purkinje cells. A, Averaged extracellularly recorded action potentials in a tg/tg Purkinje cell under control conditions and in the presence of different concentrations of 4-AP. Inset, The average of the normalized waveforms in each condition. B, Average and individual values of time to peak positive deflection, measured as indicated at the top of the inset in A. C, Comparison of the efficacy of 4-AP in improving the precision of Purkinje cell pacemaking (represented by the coefficient of variation of interspike intervals) and its efficacy in prolonging the duration of the spike. Red trace represents the linear fit (r = −0.99; p = 0.007). D, Averaged action potentials recorded in whole-cell configuration in a wt juvenile rat Purkinje cell, in control conditions, and after the application of therapeutic concentrations of 4-AP. E, Average and individual values of maximum AHP potentials in six Purkinje cells. F, Average and individual values of the AHP amplitudes of the same cells in E. G, Average and individual values of the action potential width of the same cells in E. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Bonferroni's post test for multiple comparisons).

Figure 5.

Blockade of K_v1.5 channels mimics the effects of 4-AP. A, Coefficient of variation of interspike intervals an extracellularly recorded Purkinje cell under control conditions, and with application of increasing concentrations of cadmium to partially block Ca²⁺ channels and make the firing rate of the cell irregular. Bath perfusion of the selective K_v1.5 K⁺ channel blocker DPO restored the precision of pacemaking. Subsequent addition of 10 μm 4-AP did not improve the precision of pacemaking beyond that done by DPO. B, C, Average and individual values of coefficient of variation of interspike intervals (B) and firing rate (C) in Purkinje cells in control conditions, in the presence of DPO, and with subsequent addition of 4-AP. ***p < 0.001 (one-way ANOVA followed by Bonferroni's correction). n.s., Not significant.

Figure 6.

The therapeutic efficacy of orally administered 4-AP in improving basal motor performance in tg/tg mice is not additive to that of CHZ. A, The performance of tg/tg (n = 13) and wt mice (n = 14) was measured using the accelerating rotarod paradigm. After 8 d of receiving normal drinking water, the drinking water was supplemented with 4-AP, 4-AP and CHZ combined, and CHZ alone as denoted with colored vertical bars. Each treatment lasted for 2 weeks. B, Maximum speed achieved on the rotarod during each treatment. The average value was obtained by averaging the second week of each treatment period. **p < 0.01; ***p < 0.001 (one-way ANOVA with Bonferroni's correction). n.s., Not significant.

Figure 7.

The therapeutic efficacy of orally administered 4-AP in reducing the frequency and severity of stress-induced episodes of dyskinesia in tg/tg mice is not additive to that of CHZ. A, The stress-triggered attacks of dyskinesia were quantified at 10 min intervals for the same mice examined using the accelerating rotarod paradigm reported in Figure 4. The beginning and end of each treatment is indicated with colored bars on top. B, The average severity of dyskinesia during each period for the same tg/tg mice shown in A. C, The average frequency of occurrence of an episode of dyskinesia after the rotarod session, regardless of the severity of the attack. D, The average severity of all attacks of dyskinesia before, during, and after the three different treatments. E, The duration of all attacks of dyskinesia shown in D. F, The average frequency of occurrence of a severe episode of dyskinesia (dyskinesia score ≥3.5) triggered by the rotarod session. G, The average severity of the attacks shown in F. H, Average duration of severe episodes of dyskinesia. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA followed by Bonferroni's post test). ns, Not significant. I, Summary comparison of the relative efficacies of 4-AP and CHZ in reducing stress-evoked attacks of dyskinesia in tg/tg mice. Frequency of sessions in which tg/tg mice were free of symptoms (no attack) or had an episode of dyskinesia with a score <3.5 (mild) or one with a score ≥3.5 (severe) with various treatments.