Reduced sodium current in Purkinje neurons from Nav1.1 mutant mice: implications for ataxia in severe myoclonic epilepsy in infancy

Abstract

Loss-of-function mutations of Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI), which is accompanied by severe ataxia that contributes substantially to functional impairment and premature deaths. Mutant mice lacking Na(V)1.1 channels provide a genetic model for SMEI, exhibiting severe seizures and premature death on postnatal day 15. Behavioral assessment indicated severe motor deficits in mutant mice, including irregularity of stride length during locomotion, impaired motor reflexes in grasping, and mild tremor in limbs when immobile, consistent with cerebellar dysfunction. Immunohistochemical studies showed that Na(V)1.1 and Na(V)1.6 channels are the primary sodium channel isoforms expressed in cerebellar Purkinje neurons. The amplitudes of whole-cell peak, persistent, and resurgent sodium currents in Purkinje neurons were reduced by 58-69%, without detectable changes in the kinetics or voltage dependence of channel activation or inactivation. Nonlinear loss of sodium current in Purkinje neurons from heterozygous and homozygous mutant animals suggested partial compensatory upregulation of Na(V)1.6 channel activity. Current-clamp recordings revealed that the firing rates of Purkinje neurons from mutant mice were substantially reduced, with no effect on threshold for action potential generation. Our results show that Na(V)1.1 channels play a crucial role in the excitability of cerebellar Purkinje neurons, with major contributions to peak, persistent, and resurgent forms of sodium current and to sustained action potential firing. Loss of these channels in Purkinje neurons of mutant mice and SMEI patients may be sufficient to cause their ataxia and related functional deficits.

Figure 1.

Distribution of Na_V1.1, Na_V1.2, Na_V1.3, and Na_V1.6 in cerebellar slices of P14 Na_V1.1 WT, HET, and KO mice. A1–A4, Antibody staining indicated that Na_V1.1 is found in the cell bodies and Na_V1.6 in cells bodies and dendrites of Purkinje neurons. Na_V1.2 and Na_V1.3 were not detected in Purkinje neuron cell bodies or dendrites. However, the marked staining of the molecular layer by the anti-Na_V1.2 indicates the presence of Na_V1.2 in the parallel fibers traversing this part of the cerebellum. B1–B4, Antibody staining illustrating the decrease of Na_V1.1 channel expression in Purkinje neurons of HET mice. C1–C4, Antibody staining illustrating complete loss of Na_V1.1 expression in cerebellar Purkinje neurons of Na_V1.1 KO mice without detectable upregulation of Na_V1.2 or Na_V1.3 channels. Control sections with no primary antibody or with nonimmune primary antibody did not show detectable staining. For examples of control sections using the same antibodies and methods, see Yu et al. (2006), their supplemental information. Scale bar, 50 μm. M, Molecular layer; P, Purkinje cell layer; G, granule cell layer.

Figure 2.

Motor performance of WT, HET, and KO mice. A, B, Evaluation of motor coordination for P14 mice using a modified footprint test (see Materials and Methods). A, Top, Digital pictures illustrating consecutive footsteps of representative Na_V1.1 WT, HET, and KO mice. Bottom, Histograms showing the variability in stride length for each of these exemplar mice. B, Summary stride length and stride length variability of Na_V1.1 WT, HET, and KO mice (CV, coefficient of variation). C, D, Evaluation of motor reflex using the tail suspension test. C, Example pictures of mice performing the reaching test. D, Summary data showing the time spent reaching for the cage bars for Na_V1.1 WT, HET, and KO mice. E, Evaluation of motor coordination for P21 mice in a modified footprint test. Top, Representative digital pictures with marks showing placements of paws (gray circles for hindpaws; black for forepaws) as mice walked through the tunnel. Scale bars, 2 cm. Bottom, Summary of distance between ipsilateral forepaw and hindpaw position (FP–HP), stride length, and stride width for HET mice normalized versus WT mice. Error bars indicate SEM. *p < 0.02.

Figure 3.

Sodium currents in cerebellar Purkinje neurons of WT, HET, and KO mice. A, Current traces from representative cells evoked with a series of 50 ms depolarizations from a holding potential of −90 mV to potentials ranging from −80 to +30 mV in 5 mV increments. Inset, Diagram of stimulus protocol. Calibration: 1 ms, 2 nA. B, Left, Digital photograph of dissociated cerebellar Purkinje neurons. Scale bar, 50 μm. Right, Mean capacitance for cells of each genotype. C, Left, Current–voltage relationships for WT (filled circles), HET (filled squares), and KO (open triangles) mice. Right, Mean peak current densities. Error bars indicate SEM. *p < 0.001.

Figure 4.

Voltage dependence of activation and inactivation of whole-cell sodium current of Purkinje neurons from Na_V1.1 WT, HET, and KO mice. A, Representative current traces evoked in an isolated Purkinje neuron obtained using a family of 50 ms step depolarizations from −90 to 0 mV in 5 mV increments. For clarity, only the first 5 ms of the traces is illustrated. Inset, Stimulus protocol. Calibration: 0.5 ms, 2 nA. B, Mean conductance–voltage relationships for peak sodium current. C, Sodium channel inactivation. A 100 ms prepulse to a variable potential (5 mV increments) was followed by a 20 ms test pulse to 0 mV. Examples of complete current traces are illustrated above with the currents recorded during the test pulse expanded below. Calibration: top, 20 ms, 2 nA; bottom, 0.5 ms, 2 nA. D, Mean normalized inactivation curves. Peak normalized test pulse current is plotted as a function of prepulse potential. Error bars indicate SEM.

Figure 5.

Persistent sodium current in neurons of Na_V1.1 WT, HET, and KO mice. A, Top, representative current traces from a WT neuron evoked with a series of 50 ms depolarizations from a holding potential of −90 to −20 mV in 5 mV increments. For clarity, only the first five steps of the depolarization protocol are illustrated. Calibration: 10 ms, 1 nA. Bottom, Expanded view of the boxed region above illustrating the persistent sodium current. Calibration: 2 ms, 0.1 nA. B, Mean persistent sodium current versus voltage relationships for WT, HET, and KO neurons. C, Normalized persistent sodium current versus voltage relationships for WT, HET, and KO neurons. Error bars indicate SEM.

Figure 6.

Resurgent sodium current in Purkinje neurons of Na_V1.1 WT, HET, and KO mice. A, Representative resurgent sodium currents evoked using a prepulse to +30 mV for 10 ms, followed by 100 ms repolarizations to potentials ranging from −60 to +20 mV. The holding potential was −90 mV. Calibration: 20 ms, 1 nA. B, Top, Mean peak resurgent sodium current density versus voltage curves for WT, HET, and KO neurons. Bottom, Normalized resurgent sodium current versus voltage curves for WT, HET, and KO neurons. Error bars indicate SEM.

Figure 7.

Spontaneous firing of neurons from WT, HET, and KO mice. A, Examples of spontaneous firing recorded in current clamp. The dashed line in each trace represents −60 mV. Calibration: 20 mV, 50 ms. B, Mean action potential parameters. Firing rate, voltage at action potential peak, minimum voltage reached between action potentials, and mean threshold for action potential initiation (see Materials and Methods) are plotted for cells of each genotype. C, Effects of depolarization on spontaneous firing. Left, Examples of changes in firing activity in response to a series of 6 s current pulses that increased in 20 pA increments (top to bottom) applied to a spontaneously firing WT cell. Only a segment of the traces is shown to allow individual action potentials to be distinguished. Calibration: 50 mV, 50 ms. Right, Mean firing rate is plotted vs the amount of depolarizing current applied. D, Effects of long hyperpolarizations on spontaneous firing. Left, Firing activity of a representative WT neuron during application of 6 s hyperpolarizing currents. Calibration: 50 ms, 50 mV. Right, Mean percentage of Purkinje neurons remaining firing after injection of the indicated levels of hyperpolarizing current for WT, HET, and KO mice. Error bars indicate SEM. *p < 0.01.

Figure 8.

Spontaneous firing after partial blockade of sodium current with TTX. A, Representative examples of spontaneous action potential trains before, during, and after application of 10 nm TTX to a Purkinje neuron from a WT mouse. B, Top, Expanded and superimposed individual action potentials from recordings in A. Bottom, Mean action potential threshold in control and in the presence of TTX. Threshold was increased from −52.8 ± 0.4 to −48.5 ± 0.3 mV. The spontaneous firing rate was reduced from 65.5 ± 2.4 to 48.3 ± 5.0 Hz (p = 0.013) and peak voltage 26.6 ± 0.2 to 11.4 ± 0.2 mV (p = 0.02). Action potential minimum voltage did not change significantly in these experiments (−72.0 ± 0.7 mV in control solution vs −71.1 ± 0.4 mV in TTX; p = 0.083). Error bars indicate SEM. *p < 0.05.