Expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to central preterminal branches and terminals in the dorsal horn

Abstract

Background: Sodium channel Nav1.7 has emerged as a target of considerable interest in pain research, since loss-of-function mutations in SCN9A, the gene that encodes Nav1.7, are associated with a syndrome of congenital insensitivity to pain, gain-of-function mutations are linked to the debiliting chronic pain conditions erythromelalgia and paroxysmal extreme pain disorder, and upregulated expression of Nav1.7 accompanies pain in diabetes and inflammation. Since Nav1.7 has been implicated as playing a critical role in pain pathways, we examined by immunocytochemical methods the expression and distribution of Nav1.7 in rat dorsal root ganglia neurons, from peripheral terminals in the skin to central terminals in the spinal cord dorsal horn.

Results: Nav1.7 is robustly expressed within the somata of peptidergic and non-peptidergic DRG neurons, and along the peripherally- and centrally-directed C-fibers of these cells. Nav1.7 is also expressed at nodes of Ranvier in a subpopulation of Aδ-fibers within sciatic nerve and dorsal root. The peripheral terminals of DRG neurons within skin, intraepidermal nerve fibers (IENF), exhibit robust Nav1.7 immunolabeling. The central projections of DRG neurons in the superficial lamina of spinal cord dorsal horn also display Nav1.7 immunoreactivity which extends to presynaptic terminals.

Conclusions: The expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to preterminal central branches and terminals in the dorsal horn. These data support a major contribution for Nav1.7 in pain pathways, including action potential electrogenesis, conduction along axonal trunks and depolarization/invasion of presynaptic axons. The findings presented here may be important for pharmaceutical development, where target engagement in the right compartment is essential.

Figure 1

Expression of Nav1.7 in DRG neurons. A. DRG sections were reacted to antibodies against peripherin, neurofilament 200 (NF) and Nav1.7. Peripherin-positive (green) neurons are generally of small diameter (<30 μm) and most exhibit colocalization (yellow) with Nav1.7 (red). A few small peripherin-negative neurons display robust Nav1.7 immunolabeling (arrows). Neurofilament 200 (NF)-positive (blue) neurons are generally larger than peripherin-positive cells and most do not display colocalization with Nav1.7. A few smaller NF-positive cells exhibit Nav1.7 immunolabeling (magenta). B. DRG sections were reacted with IB4-488 and antibodies to CGRP and Nav1.7. Virtually all IB4-positive (green) neurons display colocalization (yellow) with Nav1.7. Similarly, nearly all CGRP-positive (blue) neurons exhibit colocalization (magenta) with Nav1.7 (red). Few DRG neurons (arrow) display colocalization of IB4, CGRP and Nav1.7.

Figure 2

Expresion of Nav1.7 in sciatic nerve. A. Numerous peripherin-positive (green) unmyelinated fibers are immunolabeled in sciatic nerve and these fibers exhibit extensive colocalization (yellow) with Nav1.7 (red). Inset. At increased magnification, peripherin-positive (green) fiber displays colocalization (yellow) with Nav1.7. B. Nodal regions in sciatic nerve were identified by paranodal caspr (green) labeling. Nav1.7 (red) immunolabeling at a node is displayed by a small diameter (<1 μm) myelinated fiber. Not all small diameter myelinated fibers exhibit Nav1.7 labeling at nodes.

Figure 3

Expression of Nav1.7 in glabrous skin. PGP9.5 (green) intraepidermal nerve fibers (IENF) branch from nerve bundles (arrowheads) at dermis/epidermis boundary (dotted line) and ascend in the epidermis. PGP9.5-positive IENF exhibit Nav1.7 (red) immunolabeling. Inset. Both IENF extending to stratum granulosum (more superficial) and to the stratum spinosum exhibit Nav1.7 labeling.

Figure 4

Expression of Nav1.7 in dorsal and ventral roots. A., B. Sections of dorsal (A) and ventral (B) roots were reacted with antibodies to peripherin, neurofilament 200 (NF) and Nav1.7. Numerous peripherin-positive (green) fibers are present in dorsal root but few in ventral root. There is extensive colocalization (yellow) of peripherin (green) and Nav1.7 in dorsal root fibers. Ventral roots do not exhibit detectable Nav1.7 labeling. C., D. Both dorsal (C) and ventral (D) roots display caspr paranodal labeling (green). Nav1.7 (red) immunolabeling is displayed by small diameter fiber in dorsal root but not in ventral root. Inset. Increased magnification of nodal region demonstrates that Nav1.7 labeling is confined to node.

Figure 5

Nav1.7 expression in spinal cord dorsal horn. Sections of spinal cord were labeled for IB4 (green), CGRP (blue) and Nav1.7 (red). A. IB4 labeling is prominent in lamina IIi , while CGRP immunoreactivity is localized to lamina I and IIo. Robust Nav1.7 immunolabeling is present within lamina I and II, and exhibits co-localization with IB4 (yellow) and with CGRP (magenta). There is limited overlap of IB4 and CGRP in the superficial lamina. B. Increased magnification of IB4, CGRP and Nav1.7 labeling in superficial lamina of dorsal horn.

Figure 6

Nav1.7 is expressed in pre-synaptic fibers in dorsal horn. A. Synaptophysin (green), a marker of synapses, exhibits prominent labeling in the dorsal horn. Nav1.7 (red) and synaptophysin display extensive colocalization in the superfical layers of the dorsal horn. Inset. Increased magnification of superfical lamina of dorsal horn demonstrates colocalization of synaptophysin and Nav1.7. B. NeuN (green) immunolabels nuclei and cell bodies of neurons in dorsal horn. Post-synaptic neurons in the superficial lamina exhibit a lack Nav1.7 (red) labeling, which is localized in extracellular regions to the post-synaptic neurons. Right inset: increased magnification demonstrating lack of Nav1.7 immunoreactivity (red) within NeuN-labeled (green) dorsal horn neuronal cell bodies. Left inset: NeuN-labeled motor neurons (green) in ventral horn lack Nav1.7 immunolabeling (red).