Organization and Plasticity of Sodium Channel Expression in the Mouse Olfactory and Vomeronasal Epithelia

Abstract

To understand the molecular basis of neuronal excitation in the mammalian olfactory system, we conducted a systematic analysis of the organization of voltage-gated sodium (Na_v) channel subunits in the main olfactory epithelium (MOE) and vomeronasal organ (VNO) of adult mice. We also analyzed changes in Na_v channel expression during development in these two systems and during regeneration of the MOE. Quantitative PCR shows that Na_v1.7 is the predominant isoform in both adult MOE and VNO. We detected pronounced immunoreactivity for Na_v1.7 and Na_v1.3 in axons of olfactory and vomeronasal sensory neurons (VSNs). Analysis of Na_v1.2 and Na_v1.6 revealed an unexpected subsystem-specific distribution. In the MOE, these Na_v channels are absent from olfactory sensory neurons (OSNs) but present in non-neuronal olfactory cell types. In the VNO, Na_v1.2 and Na_v1.6 are confined to VSNs, with Na_v1.2-immunoreactive somata solely present in the basal layer of the VNO. The subcellular localization of Na_v1.3 and Na_v1.7 in OSNs can change dramatically during periods of heightened plasticity in the MOE. During the first weeks of development and during regeneration of the olfactory epithelium following chemical lesion, expression of Na_v1.3 and Na_v1.7 is transiently enhanced in the somata of mature OSNs. Our results demonstrate a highly complex organization of Na_v channel expression in the mouse olfactory system, with specific commonalities but also differences between the MOE and the VNO. On the basis of their subcellular localization, Na_v1.3 and Na_v1.7 should play major roles in action potential propagation in both MOE and VNO, whereas Na_v1.2 and Na_v1.6 are specific to the function of VSNs. The plasticity of Na_v channel expression in OSNs during early development and recovery from injury could reflect important physiological requirements in a variety of activity-dependent mechanisms.

Keywords: development; olfactory epithelium; regeneration; voltage-gated sodium channels; vomeronasal organ.

Figure 1

Quantitative RT-PCR of different Na_v isoforms in main olfactory epithelium (MOE) and vomeronasal organ (VNO). Comparison of Na_v channel mRNA frequency in the (A) MOE and (B) VNO using quantitative RT-PCR. Na_v1.7 is the most abundant isoform in both subsystems and displays on average at least 5-fold higher copy numbers than other Na_v isoforms. The column diagrams show mean copy numbers ± SD from two to four independent experiments, each carried out as triplicates using total RNA of adult C57/B6 mice (Y-axis). Na_v channel isoforms (X-axis). (A) Copy numbers per ng total RNA in the MOE for Na_v1.1 (2.6 ± 0.72), Na_v1.2 (4.99 ± 0.62), Na_v1.3 (51.80 ± 7.75), Na_v1.5 (113.17 ± 27.35), Na_v1.6 (41.19 ± 10.65), Na_v1.7 (983.44 ± 221.34). (B) Copy numbers per ng total RNA in the VNO for Na_v1.1 (0.80 ± 0.42), Na_v1.2 (10.16 ± 2.10), Na_v1.3 (142.70 ± 29.05), Na_v1.5 (128.13 ± 25.56), Na_v1.6 (38.17 ± 5.39), Na_v1.7 (701.06 ± 203.50).

Figure 2

Analysis of Na_v channel expression in MOE of adult mice. Confocal images showing immunoreactivity (red) for (A) Na_v1.2, (B) Na_v1.3, (C) Na_v1.6, and (D) Na_v1.7. We used coronal MOE cryosections (12 μm) of adult OMP-GFP mice (left, overviews; right, magnifications). Endogenous GFP (green) is located to mature, OMP⁺ OSNs as shown in the magnifications at the right. (A) Na_v1.2 staining is restricted to microvillar cells (arrowheads) but absent from OSNs or axon bundles (dotted circles) identified by OMP-GFP labeling. (B) Robust Na_v1.3 staining is present in axon bundles (arrows, left) that colocalize with OMP-GFP (dotted circles, right). (C) Na_v1.6 labeling of the MOE surface (left, arrow heads) corresponds to sustentacular cells (right). OSNs and axon bundles (dotted line) lack Na_v1.6 staining. (D) Na_v1.7 immunoreactivity is profound in axon bundles, and occasionally found in microvillar cells (asterisk). (E,F,H,I) Blocking peptide control experiments lack immunoreactivity. (G) Na_v1.3 immunoreactivity is present in cNa_v1.7^−/− mice. (J) Na_v1.7 immunoreactivity is absent in cNa_v1.7^−/− mice. Images for each Na_v channel immunostaining are representatives of n ≥ 3 mice and n = 20 sections per mouse. Scale bars overviews 100 μm, and magnifications 20 μm.

Figure 3

Analysis of Na_v channel expression in VNO of adult and early postnatal mice. (A–H) Confocal images showing immunoreactivity (red) observed for (A) Na_v1.2, (B) Na_v1.3, (C) Na_v1.6, and (D) Na_v1.7 in 12 μm coronal VNO cryosections of adult mice (left, overviews; right, magnifications). (A) Na_v1.2 staining is present in VSN knobs (arrowheads), dendrites and somata of basal (b) VSNs but absent in apical (a) VSNs (overview left). The magnification at the right shows colocalization of Na_v1.2 with V2R2 (green). (B) Na_v1.3 staining is moderate in VSN knobs (arrowheads), dendrites and somata and robust in axon bundles as identified in OMP-GFP mice (green signal). (C) Na_v1.6 is strong in knobs (arrowheads) and somata (bracket). The magnification and colocalization with OMP-GFP shows a decline in Na_v1.6 intensity from basal to apical. (D) Na_v1.7 staining is prominent in VSN knobs (arrowheads) and axon bundles (arrows). Peptide control experiments (E,F,G) lack immunoreactivity. (H) Na_v1.7 staining (top) is absent in axon bundles (arrows) of cNa_v1.7^−/− mice, visualized by OMP staining (bottom). (I-L) Immunoreactivity (red) for (I) Na_v1.2, (J) Na_v1.3, (K) Na_v1.6, and (L) Na_v1.7 at postnatal (P) day 2, P7, and P14. (I) Onset of Na_v1.2 in P2 VSNs (asterisks). At P7 and P14, basal (b) confinement of Na_v1.2 somata and labeled knobs (arrowheads; a). (J) Na_v1.3 is visible in single somata (asterisks) and axon bundles (arrows) at P7, and shows diffuse staining of the apical VNO (arrowheads) starting at P2. (K) Onset of Na_v1.6 in somata (asterisks) at P2 with increasing numbers at P7 and P14 throughout the VNO (brackets). (L) Na_v1.7 in single VSN somata (asterisks) and axon bundles (arrows) starts at P2. The knob area is substantially stained from P7 onwards. Images are representatives of n ≥ 3 adult mice (n = 20 sections per mouse) and n = 2 juvenile mice each at P2, P7, and P14 (n ≥ 10 sections per mouse). Scale bars overviews (A–H, left) and (E–H) 100 μm; magnifications (A–D) and (I–L) 20 μm.

Figure 4

Subcellular localization of Na_v1.7 and Na_v1.3 staining during mouse MOE development. (A,B) Coronal tissue sections showing the dorsal aspect of the left nasal cavity (septum to the left) of a postnatal day 7 (P7) mouse. A strip of strong immunoreactivity is visible for (A) Na_v1.7 and (B) Na_v1.3 in the most apical MOE layer (arrowheads) and in axon bundles (arrows). (C,D) Higher magnifications of the MOE derived at different mouse ages stained with antibodies for (C) Na_v1.7 and (D) Na_v1.3. The confocal images show strongly stained OSN somata solely in the apical MOE. Somatic OSN staining is visible at embryonic day 18 (E18), P2, and P7, declines at about P14 and is nearly diminished at P21. Axon bundles stain early on (arrows) and increase in size with age in relation to epithelial thickness. Images are representatives of (n ≥ 2) mice at each age with n ≥ 10 sections per mouse. Scale bars (A,B) 200 μm, (C,D) 20 μm.

Figure 5

Somatic expression of Na_v1.3 and Na_v1.7 occurs in mature OSNs. Colocalization of Na_v1.3, GAP43, and Na_v1.7 in the olfactory epithelium of a P7 OMP-GFP mouse (endogenous fluorescence, green). (A) Single fluorescence images for Na_v1.3 (red), OMP-GFP (green), and GAP43 (blue). (B) Magnified merge of the images in (A) shows that Na_v1.3 colocalizes with OMP-GFP (arrows) in mature OSN somata located in the apical layer (dotted line). Occasionally, triple-labeled OSNs, positive for Na_v1.3, OMP-GFP, and GAP43 were detected (asterisk). (C) Colocalization of Na_v1.3 (red) and Na_v1.7 (blue) in OMP-GFP⁺ OSNs (asterisks) intermingle with singly, OMP-GFP labeled OSNs (arrowhead). Images are representatives of (n = 2) mice with n ≥ 10 sections per mouse. Scale bars, 20 μm.

Figure 6

Recovery of Na_v1.3 and Na_v1.7 expression in OSN somata during regeneration from chemical ablation. (A–C) Coronal MOE sections of the anterior left nasal cavity (septum to the left) of OMP-GFP mice. (A) Untreated control mice display OMP-GFP labeling of OSNs throughout the MOE (arrowheads). Inset magnification exemplifies regular thickness of axon bundles (arrows) and the MOE (double arrow, 80 μm). (B) Severely damaged MOE 2 weeks post-lesion. Small patch of unlesioned MOE at the septal wall (asterisk). The inset magnification depicts the reduced thickness of axon bundles (arrows) and the MOE (double arrow, 35 μm) 2 weeks post-lesion. Few newly generated OMP-GFP positive OSNs are visible (arrowheads). (C) The MOE has largely recovered 8 weeks post-lesion (arrowheads). The inset magnification shows that the thickness of axon bundles (arrows) and the MOE (double arrow, 70 μm) is increased. Basal membrane (dotted line). (D) Immunoreactivity for Na_v1.3 and Na_v1.7 in the intact, unlesioned MOE shows strong labeling of axon bundles (asterisks). (E) Eight weeks post-lesion, tissue stretches with heavy immunolabeling for Na_v1.3 and Na_v1.7 in OSN somata (arrowheads) reside side-by-side with areas devoid of any somatic immunoreactivity (arrows). This pattern likely coincides with the different levels of initial damage yielding various levels of MOE regeneration. (F,G) Magnifications of the MOE at 6, 8, and 10 weeks post-lesion showing somatic staining for (F) Na_v1.3 (red) and (G) Na_v1.7 (red) colocalizing with OMP-GFP (green). Images are representatives of (n = 2) mice at each recovery time point with n ≥ 20 sections per mouse. Scale bars (A–C) 200 μm, (D,E) 50 μm (F,G) 20 μm.

Figure 7

Summary scheme depicting the cellular and subcellular distribution of various Na_v channel isoforms in mouse MOE (A) and VNO (B). The drawings at the left illustrate the results obtained in this study and refer to the table at the right. Expression of Na_v1.5 (asterisk) in OSN dendritic knobs (Frenz et al., 2014) has been included for completeness. The different Na_v channel subtypes are color-coded as indicated. The extent of immunoreactivity was categorized as + (present), − (absent), or (+) (close to detection threshold). MV, microvillar cells; OSN, olfactory sensory neuron; SUS, sustentacular cells; VSN, vomeronasal sensory neuron; a, apical VNO layer; b, basal VNO layer.