The sodium channel Scn8a is the major contributor to the postnatal developmental increase of sodium current density in spinal motoneurons

Abstract

Sodium currents were recorded from motoneurons that were isolated from mice at postnatal days 0-8 (P0-P8) and maintained in culture for 12-24 hr. Motoneurons from normal mice exhibited a more than threefold increase in peak sodium current density from P0 to P8. For mice lacking a functional Scn8a sodium channel gene, motoneuronal sodium current density was comparable at P0 to that of normal mice but failed to increase from P0 to P8. The absence of Scn8a sodium channels is associated with the phenotype "motor end plate disease," which is characterized by a progressive neuromuscular failure and is fatal by 3-4 postnatal weeks. Thus, it appears that the development and function of mature motoneurons depends on the postnatal induction of Scn8a expression.

Fig. 1.

Postnatal increase of sodium current density in normal mouse motoneurons. A, Representative sodium currents in motoneurons isolated from normal mice at P0, P4, and P8 and cultured overnight. Here (and in Fig. 4), the illustrated currents were elicited by step depolarizations ranging from −40 to +10 mV at 10 mV intervals, extracellular sodium concentration was 35 mm, and the holding potential was −80 mV. B, Average current–voltage relationships for motoneurons obtained from normal mice at P0–P1 (n = 45), P4 (n= 7), and P7–P8 (n = 12). Error bars indicate ± SEM. C, Peak sodium current density as a function of postnatal age. The data point plotted midway between P7 and P8 was obtained from P7 and P8 motoneurons (n = 28, 17, 16, 9, 8, and 12 at P0, P1, P2, P3, P4, and P7–P8, respectively).

Fig. 2.

Expression of Scn8a in mouse spinal cord. Twenty-microgram aliquots of total RNA were assayed by ribonuclease protection using a 511 bp Scn8a specific riboprobe. The predicted size of the protected fragment is 381 bp.Left panel, All samples were run on the same gel, which was dried and exposed to film for 1.5 hr (probe) or 5 hr (samples plus probe). Right panel, The concentration and integrity of the RNA samples were compared by staining with ethidium bromide after electrophoresis of 1 μg aliquots on a 1.0% agarose gel.

Fig. 3.

Determination of Scn8a genotypes by PCR. Genomic DNA was prepared from offspring of the cross (tg/+ × tg/+) and amplified using two pairs of primers, as described in Materials and Methods. Amplification of the wild-type Scn8a gene generated a 103 bp product; amplification of the transgene generated a 244 bp product. The inferred genotypes, based on presence or absence of each product, are shown above each lane. A minor 280 bp product was occasionally amplified from wild-type DNA by the transgene primers (arrowhead). The positions of molecular weight markers (1 kb ladder; BRL, Bethesda, MD) are shown at the leftin base pairs.

Fig. 4.

Lack of postnatal increase in sodium current density in med^tg motoneurons. A, Representative sodium currents in motoneurons isolated frommed^tg mice at P0, P3, and P7. Note that the currents from the P7 motoneuron are displayed at a 10-fold higher gain.B, C, D, Average current–voltage relationships for wild-type andmed^tg motoneurons at P0–P1, P2–P3, and P7–P8. In B, C, and D,n = 4, 7, and 11 for the wild-type motoneurons, andn = 6, 9, and 11 for themed^tg motoneurons, respectively. Each of the data points is based on currents measured in at least two separate motoneuron cultures.

Fig. 5.

Calcium currents in motoneurons from a P0 (top traces) or a P6 (bottom traces)med^tg mouse. Test pulses were to potentials of −20 to +20 mV, in 10 mV increments. Peak calcium current densities at +10 mV were as follows: P0, 3.6 and 5.4 pA/pF; P6, 10.9 and 9.9 pA/pF.

Fig. 6.

Peak sodium current density as a function of postnatal age in motoneurons from P0–P1, P2–P3, and P7–P8 animals that were wild-type (shaded symbols), heterozygous (filled symbols), or med^tg(open symbols). The numbers of cells for the three age groups were 4, 7, and 11 (wild-type), 4, 7, and 10 (heterozygous), and 6, 9, and 11 (med^tg), respectively. Cells identified as motoneurons by DiI labeling are indicated withcircles, and cells not identified by DiI labeling are indicated by triangles.