Sodium Channel Nav1.8 Underlies TTX-Resistant Axonal Action Potential Conduction in Somatosensory C-Fibers of Distal Cutaneous Nerves

Abstract

Voltage-gated sodium (Na_V) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine Na_V channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (Na_V1.1-Na_V1.4, Na_V1.6-Na_V1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (Na_V1.8 and Na_V1.9) inhibited by millimolar concentrations, with Na_V1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different Na_V channel isoforms are distributed along the peripheral and central branches of their bifurcated axons. To determine the relative contribution of TTX-s and TTX-r channels to action potential conduction in different axonal compartments, we investigated the effects of TTX on C-fiber-mediated compound action potentials (C-CAPs) of proximal and distal peripheral nerve segments and dorsal roots from mice and pigtail monkeys (Macaca nemestrina). In the dorsal roots and proximal peripheral nerves of mice and nonhuman primates, TTX reduced the C-CAP amplitude to 16% of the baseline. In contrast, >30% of the C-CAP was resistant to TTX in distal peripheral branches of monkeys and WT and Na_V1.9^-/- mice. In nerves from Na_V1.8^-/- mice, TTX-r C-CAPs could not be detected. These data indicate that Na_V1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of Na_V1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of Na_V1.8 in distal axons may provide a useful target in the treatment of pain of peripheral origin.SIGNIFICANCE STATEMENT It is unclear whether individual sodium channel isoforms exert differential roles in action potential conduction along the axonal membrane of nociceptive, unmyelinated peripheral nerve fibers, but clarifying the role of sodium channel subtypes in different axonal segments may be useful for the development of novel analgesic strategies. Here, we provide evidence from mice and nonhuman primates that a substantial portion of the C-fiber compound action potential in distal peripheral nerves, but not proximal nerves or dorsal roots, is resistant to tetrodotoxin and that, in mice, this effect is mediated solely by voltage-gated sodium channel 1.8 (Na_V1.8). The functional prominence of Na_V1.8 within the axonal compartment immediately proximal to its termination may affect strategies targeting pain of peripheral origin.

Keywords: nociceptor; nonhuman primate; pain; sodium channels.

Figure 1.

Identification, isolation, and recording arrangement for the peripheral nerve segments in mouse. A, B, Photograph identifying the sural division of the sciatic nerve (A) and the distal reaches of nerves innervating the skin of the dorsal hindpaw (B). C, Organ bath to register compound extracellular signals. Isolated nerve segments were embedded in petroleum jelly at each end in glass stimulating and recording electrodes. D, Schematic arrangement of recording setup used to deliver electrical stimuli (left) and record ensuing CAP signal (right and inset). Peak-to-peak amplitude of the CAP signal (inset, blue double arrow) was determined within a variable time window (inset, red horizontal bars).

Figure 2.

Increased TTX-resistant conduction in distal peripheral nerves of WT mice. A–C. Specimen recordings from distal and proximal nerve and dorsal root, respectively. During superfusion with TTX (1 μm), latency of C-CAP increases and amplitude of C-CAP decreases. Washout leads to recovery of the C-CAP and lidocaine suppresses all neuronal conduction. D, Group data for both TTX (red circles) and TTX plus A803467 (5 μm, blue circles) differed significantly between neuronal compartments (p = 0.0169 and p = 0.008, respectively, Kruskal–Wallis ANOVA, followed by post hoc multiple comparisons). TTX and TTX plus A803467 data obtained from the same nerve sample are connected by lines. For incubation with TTX plus A803467, data were not displayed if TTX alone already completely inhibited C-CAP. On average, 24.7%, 7.1%, and 5.8% of the C-CAP remained under TTX in distal nerves (n = 13), proximal nerves (n = 16), and dorsal roots (n = 15), respectively. Incubation with TTX and A803467 further decreased the amplitude of the remaining C-CAP in distal nerve segments only (p < 0.05, Wilcoxon matched pairs, n = 11). Significant differences between C-CAP amplitudes of different nerve segments are indicated by red lines (TTX) and blue lines (TTX plus A803467) as detected by post hoc multiple comparisons. Black line indicates significantly smaller C-CAP under TTX plus A803467 compared with TTX alone in distal nerve segment. *p < 0.05.

Figure 3.

A considerable portion of C-CAP in distal peripheral nerves of macaques is resistant to TTX. A, B, Specimen recording of C-CAPs in distal and proximal nerve, respectively. Superfusion with TTX (1 μm) increases C-CAP latency and decreases C-CAP amplitude. For the specimen from the distal nerve ∼33% of C-CAP remained under TTX, whereas ∼10% remained in the specimen recording from the proximal nerve. C, Group data for C-CAP from proximal and distal nerves. Normalized C-CAP amplitude under TTX was significantly larger in distal nerves (n = 27) than in proximal nerves (n = 31, p = 0.03, Mann–Whitney U test). Similarly, the C-CAP amplitude during incubation with TTX (1 μm) and A803467 (5 μm) was larger in distal nerves (n = 22) than in proximal segments (n = 26, p = 0.01, Mann–Whitney U test). Only in distal nerve segments did the C-CAP amplitude significantly decrease under incubation with TTX and A803467 compared with TTX alone (n = 22, p = 0.0041, Wilcoxon matched pairs). Red (blue) lines indicate significant differences for TTX (TTX plus A803467) between distal and proximal nerve segments. Black line indicates significant difference in C-CAP amplitude between TTX and TTX plus A803467 in distal nerve segments. *p < 0.05; **p < 0.01.

Figure 4.

Compared with proximal nerves, a significantly larger portion of C-CAP in distal nerves is TTX resistant in Na_V1.9 ^−/− animals. A, B, Specimen recordings from distal and proximal nerves of Na_V1.9^−/− mice, respectively. C, Group data. Significant differences between distal and proximal nerve segments are indicated by red (TTX, 1 μm) and blue lines (TTX plus A803467, 5 μm). For both TTX and TTX plus A803467, amplitudes of remaining C-CAPs were significantly larger in distal than proximal nerve segments (p < 0.05, Mann–Whitney U test; see text for details). *p < 0.05.

Figure 5.

Effect of temperature and TTX (500 nm) on axonal conduction in peripheral C-fibers of WT mice. A, B, Comparison of the absolute C-CAP amplitude in WT mice at 32°C (red bars) and 23°C (blue bars) for proximal sural (left), proximal saphenous (center), and distal (right) nerve segments before (A) and during TTX (500 nm; B). C, Amplitude ratio indicating the relative increase in amplitude upon cooling from 32°C to 23°C before (control) and in the presence of TTX (500 nm; gray shading) in proximal sural (left), proximal saphenous (center), and distal (right) nerve segments.

Figure 6.

Functional assessment of TTX-r C-fiber conduction in proximal and distal peripheral nerve segments from WT mice. A–C, Effect of cooling from 32°C (red bars) to 23°C (blue bars) on the normalized C-CAP amplitude of WT nerves before (control), in the presence of TTX (500 nm, gray shading), after washing, and in the presence of lidocaine (1 mm) for proximal sural (A), proximal saphenous (B), and distal (C) nerve segments of WT mice. Insets in A–C show representative examples of electrically evoked C-CAPs before (top, red trace), during TTX (500 nm; center, gray trace), and after washing (bottom, black trace). D, Individual TTX-r C-CAP amplitudes at 32°C (red circles) and 23°C (blue circles) for proximal sural (left), proximal saphenous (center), and distal (right) nerve segments.

Figure 7.

Functional assessment of TTX-r C-fiber conduction in proximal and distal peripheral nerve segments from Na_V1.8^−/− mice. A–C, Effect of cooling from 32°C (red bars) to 23°C (blue bars) on the normalized C-CAP amplitude of Na_V1.8^−/− nerves before (control), in the presence of TTX (500 nm, gray shading) and after washing for proximal sural (A), proximal saphenous (B), and distal (C) nerve segments. D, C-CAP amplitude in Na_V1.8^−/− mice for proximal sural (left), proximal saphenous (center), and distal (right) nerve segments at 32°C (red bars) and 23°C (blue bars). E, Comparison of C-CAP amplitude under control conditions between WT and Na_V1.8^−/− mice in proximal sural (left), proximal saphenous (center), and distal (right) nerve segments. F, Comparison of the AUC of the C-CAP signal under control conditions between WT and Na_V1.8^−/− mice in proximal sural (left), proximal saphenous (center), and distal (right) nerve segments.