Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals

Abstract

Background: Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals.

Results: Small diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro.

Conclusions: NCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

Figure 1

Na⁺/Ca²⁺ exchanger isoforms in rat dorsal root ganglion. NCX1 is not detectable in DRG neurons (PGP 9.5 positive), but exhibits robust expression in the ensheathing satellite cells. NCX2 is highly expressed in DRG neurons with small diameters. Low levels of NCX3 are present in most neurons irrespective of cell body diameter. NCX1, 2, and 3: red; PGP 9.5: green; merge: yellow. Scale bar, 50 μm.

Figure 2

NCX expression in free nerve endings within epidermis. PGP 9.5-positive free nerve endings exhibit NCX2, but not NCX1 and NCX3 immunolabelling. NCX2 labeling extends from the dermal boundary to the tip of the nerve terminal. NCX: red; PGP 9.5: green; merge: yellow. Scale bar, 10 μm.

Figure 3

NCX2 expression in dermal nerve bundles and epidermal nerve endings. Small bundles of axons in the dermis subjacent to the epidermis exhibit NCX2 immunolabeling. Virtually all PGP 9.5-positive free nerve endings in the epidermis display NCX2 immunoreactivity. Dashed line indicates the border between epidermis (top) and dermis (bottom). NCX2: red; PGP 9.5: green; merge: yellow. Scale bar, 20 μm.

Figure 4

NCX2 expression in DRG neurons in vitro. A. NCX2 (green) immunoreactivity in the cell body and neurites of a peripherin-positive (red) neuron. Merged image (yellow) demonstrates co-localization of NCX2 and peripherin. B. High magnification image showing NCX2 (yellow = merge of NCX2 and PGP 9.5 signals) present at a neurite tip (indicated with arrow, left image). Scale bars, 50 μm (left); 10 μm (right).

Figure 5

Sodium channel expression in nerve bundles within the dermis. The small bundles of fibers within the dermis subjacent to the epidermis exhibit Na_V1.6, Na_V1.7, Na_V1.8 and Na_V1.9 immunolabeling. Na_V1.1 immunolabeling is not detectable within the small nerve bundles. Sodium channels: red; PGP 9.5: green; merge: yellow. Scale bar, 10 μm.

Figure 6

Sodium channel expression within free nerve endings in epidermis. PGP 9.5-positive (green) free nerve endings exhibit Na_V1.6, Na_V1.7, Na_V1,8 and Na_V1.9 immunolabeling (red). Na_V1.1 is not detectable in epidermal nerve fibers. Scale bar, 10 μm.

Figure 7

Co-localization of Na_V1.8 and Na_V1.7 in epidermal nerve endings. Na_V1.8 (red) and Na_V1.7 (green) are co-localized (yellow) in dermal nerve bundles subadjacent to the epidermis and in single intra-epidermal nerve terminals. Scale bar, 5 μm.

Figure 8

Sodium channel expression in DRG neurons in vitro. Left panels: Na_V1.6-EGFP, Na_V1.7-EGFP, Na_V1,8 and Na_V1.9 immunolabeling is displayed within DRG cell bodies and neurites. Na_V1.1 is detectable at low levels in the cell body and only in neurites close to the cell body. Right panel: High magnification of neurite endings: tips of free nerve terminals exhibit Na_V1.6-EGFP, Na_V1.7-EGFP, Na_V1.8 and Na_V1.9 immunolabeling. Na_V1.1 immunostaining is not detectable in neuritic nerve ending. Sodium channels: green; PGP 9.5: red; merge: yellow. Insets: Peripherin-positive (green) neurite tips exhibit immunolabeling (red) with antibodies directed against Na_V1.6 and Na_V1.7. Scale bar, 50 μm (left), 10 μm (right).