Infusion of the chemotherapeutic agent oxaliplatin leads to an acute and a chronic form of peripheral neuropathy. Acute oxaliplatin neuropathy is characterized by sensory paresthesias and muscle cramps that are notably exacerbated by cooling. Painful dysesthesias are rarely reported for acute oxaliplatin neuropathy, whereas a common symptom of chronic oxaliplatin neuropathy is pain. Here we examine the role of the sodium channel isoform Na _V 1.6 in mediating the symptoms of acute oxaliplatin neuropathy. Compound and single-action potential recordings from human and mouse peripheral axons showed that cooling in the presence of oxaliplatin (30–100 μM; 90 min) induced bursts of action potentials in myelinated A, but not unmyelinated C-fibers. Whole-cell patch-clamp recordings from dissociated dorsal root ganglion (DRG) neurons revealed enhanced tetrodotoxin-sensitive resurgent and persistent current amplitudes in large, but not small, diameter DRG neurons when cooled (22 °C) in the presence of oxaliplatin. In DRG neurons and peripheral myelinated axons from Scn8a ^med/med mice, which lack functional Na _V 1.6, no effect of oxaliplatin and cooling was observed. Oxaliplatin significantly slows the rate of fast inactivation at negative potentials in heterologously expressed mNa _V 1.6r in ND7 cells, an effect consistent with prolonged Na _V open times and increased resurgent and persistent current in native DRG neurons. This finding suggests that Na _V 1.6 plays a central role in mediating acute cooling-exacerbated symptoms following oxaliplatin, and that enhanced resurgent and persistent sodium currents may provide a general mechanistic basis for cold-aggravated symptoms of neuropathy.

Background: Gain-of-function mutations of the nociceptive voltage-gated sodium channel Nav1.7 lead to inherited pain syndromes, such as paroxysmal extreme pain disorder (PEPD). One characteristic of these mutations is slowed fast-inactivation kinetics, which may give rise to resurgent sodium currents. It is long known that toxins from Anemonia sulcata, such as ATX-II, slow fast inactivation and skin contact for example during diving leads to various symptoms such as pain and itch. Here, we investigated if ATX-II induces resurgent currents in sensory neurons of the dorsal root ganglion (DRGs) and how this may translate into human sensations. Results: In large A-fiber related DRGs ATX-II (5 nM) enhances persistent and resurgent sodium currents, but failed to do so in small C-fiber linked DRGs when investigated using the whole-cell patch-clamp technique. Resurgent currents are thought to depend on the presence of the sodium channel β4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less β4 mRNA than large sensory neurons. With the β4-C-terminus peptide in the pipette solution, it was possible to evoke resurgent currents in small DRGs and in Nav1.7 or Nav1.6 expressing HEK293/N1E115 cells, which were enhanced by the presence of extracellular ATX-II. When injected into the skin of healthy volunteers, ATX-II induces painful and itch-like sensations which were abolished by mechanical nerve block. Increase in superficial blood flow of the skin, measured by Laser doppler imaging is limited to the injection site, so no axon reflex erythema as a correlate for C-fiber activation was detected. Conclusion: ATX-II enhances persistent and resurgent sodium currents in large diameter DRGs, whereas small DRGs depend on the addition of β4-peptide to the pipette recording solution for ATX-II to affect resurgent currents. Mechanical A-fiber blockade abolishes all ATX-II effects in human skin (e.g. painful and itch-like paraesthesias), suggesting that it mediates its effects mainly via activation of A-fibers.

Voltage-gated sodium (Na_V) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine Na_Vchannel 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_Vchannel 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 STATEMENTIt 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.

Low-frequency sinusoidal current applied to human skin evokes local axon reflex flare and burning pain, indicative of C-fibre activation. Because topical cooling works well as a local analgesic, we examined the effect of cooling on human pain ratings to sinusoidal and rectangular profiles of constant current stimulation. Unexpectedly, pain ratings increased upon cooling the skin from 32 to 18°C. To explore this paradoxical observation, the effects of cooling on C-fibre responses to stimulation with sinusoidal and rectangular current profiles were determined in ex vivo segments of mouse sural and pig saphenous nerve. As expected by thermodynamics, the absolute value of electrical charge required to activate C-fibre axons increased with cooling from 32°C to 20°C, irrespective of the stimulus profile used. However, for sinusoidal stimulus profiles, cooling enabled a more effective integration of low-intensity currents over tens of milliseconds resulting in a delayed initiation of action potentials. Our findings indicate that the paradoxical cooling–induced enhancement of electrically evoked pain in people can be explained by an enhancement of C-fibre responsiveness to slow depolarization at lower temperatures. This property may contribute to symptoms of enhanced cold sensitivity, especially cold allodynia, associated with many forms of neuropathic pain.

GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABA_AR. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to NKCC1 loading of axonal chloride. Activation of axonal GABA_AR stabilised C-fibre excitability during prolonged low frequency (2.5 Hz) firing. NKCC1 maintains intra-axonal chloride to provide feed-forward stabilisation of C-fibre excitability and thus support sustained firing. Abstract GABA_AR mediated depolarisation of DRG axonal projections in the spinal dorsal horn is implicated in pre-synaptic inhibition. Inhibition, in this case, is predicated on an elevated intra-axonal chloride concentration and a depolarizing GABA response. We report here that the peripheral axons of DRG neurons are also depolarised by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABA_AR agonists increased axonal excitability while GABA excitability responses were blocked by GABA_AR antagonists and were absent in mice lacking the GABA_AR β3 subunit selectively in DRG neurons (Advillin^Cre or sns^Cre). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5 - 2.5 Hz). However, during NKCC1 blockade electrical stimulation rate did not affect GABA response size, suggesting that NKCC1 regulation of axonal chloride is coupled to action potential firing. To examine this, activity-dependent conduction velocity slowing (ADS) was used to quantify C-fibre excitability loss during 2.5 Hz challenge. ADS was reduced by GABA_AR agonists and exacerbated by either GABA_AR antagonists, β3 deletion or NKCC1 blockade. This illustrates that activation of GABA_AR stabilises C-fibre excitability during sustained firing. We posit that NKCC1 acts in a feed-forward manner to maintain an elevated intra-axonal chloride in C-fibres during ongoing firing. The resulting chloride gradient can be utilised by GABA_AR to stabilise axonal excitability. The data imply that therapeutic strategies targeting axonal chloride regulation at peripheral loci of pain and itch may curtail aberrant firing in C-fibres. This article is protected by copyright. All rights reserved