Physiological maturation of photoreceptors depends on the voltage-gated sodium channel NaV1.6 (Scn8a)

Abstract

Voltage-gated sodium channels (VGSCs) ensure the saltatory propagation of action potentials along axons by acting as signal amplifiers at the nodes of Ranvier. In the retina, activity mediated by VGSCs is important for the refinement of the retinotectal map. Here, we conducted a full-field electroretinogram (ERG) study on mice null for the sodium channel NaV1.6. Interestingly, the light-activated hyperpolarization of photoreceptor cells (the a-wave) and the major "downstream" components of the ERG, the b-wave and the oscillatory potentials, are markedly reduced and delayed in these mice. The functional deficit was not associated with any morphological abnormality. We demonstrate that Scn8a is expressed in the ganglion and inner nuclear layers and at low levels in the outer nuclear layer beginning shortly before the observed ERG deficit. Together, our data reveal a previously unappreciated role for VGSCs in the physiological maturation of photoreceptors.

Figure 1.

Scn8a-null mice have a strongly reduced sensitivity to light. a, Representative ERG response in a P16 control littermate (ctrl LM) of Scn8a^dmu mice bred onto the DBA/2J background (left), a P16 Scn8a^dmu homozygous mouse (dmu) bred onto the DBA/2J background (middle), and a P12 wild-type DBA/2J mouse (right). The ERGs were elicited by flashes of white light of increasing intensities ranging from -3.0 (bottom) to 1.4 (top) log cd·s/m² (square box indicates flash onset and duration). Although ERG components, such as, from left to right, a pronounced a-wave, oscillatory potentials, and a b-wave, can be clearly distinguished in the P16 wild-type mouse, these components are strongly reduced and delayed in Scn8a^dmu mice. Oscillatory potentials can occasionally be seen, indicating that synaptic transmission can occur (arrowheads). The amplitude and general appearance of the Scn8a^dmu ERG is similar to that of the immature P12 wild-type ERG. b, Quantitative analysis of the amplitudes and latencies of the a-wave (absolute values) and b-wave relative to the flash onset from P16 wild-type DBA/2J mice (P16 WT; n = 18), normal littermates of Scn8a^dmu mice (P16 LM; n = 6), P12 wild-type DBA/2Jmice (P12WT; n=3), and P16 Scn8a^dmu mice (P16dmu; n = 6). Error bars represent ± SEM. P16 Scn8a^dmu mice are compared with their littermates using an unpaired Student's t test, and significance is assigned as ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001.

Figure 2.

Developmental time course of Scn8a expression in the retina. a-f, In situ hybridization of wild-type retinas during postnatal development with a digoxigenin-labeled Scn8a antisense probe reveals Scn8a expression in the ganglion cell and inner nuclear layers that begins between P7 and P10. The broad expression of Scn8a in the inner nuclear layer is corroborated by comparing with patched (g), a marker for Müller cell nuclei, which displays a more restricted expression pattern. h, Scn8a sense control. LN, Lens; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.

Figure 3.

Absence of Na_V1.6 does not affect retinal morphology. a, Hematoxylin and eosin staining of P16 control and P16 Scn8a^dmu (dmu) retinas does not reveal overt abnormalities. b, Quantification of the layer thickness. Error bars represent SEM. c, In Scn8a^dmu mice, GFAP is restricted to the end feet of Müller cells, indicating the absence of reactive gliosis. d-f, A survey of the Scn8a^dmu photoreceptor ultrastructure at P16 shows normal outer and inner segments with no signs of degeneration (d). e, f, In addition, in the outer plexiform layer, bipolar (B) and horizontal (H) dendritic processes invaginate rod spherules to form synaptic clefts that have the normal triad appearance with the electron-dense ribbon located between the horizontal cells. Scale bar: (in f) a, c, 60 μm; d, 3 μm; e, 0.5 μm; f, 0.2 μm. pe, Pigment epithelium; os, outer segment; is, inner segment; onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer; ofl, optic fiber layer.