This protocol details methods to identify and record from cutaneous primary afferent axons in an isolated mammalian skin–saphenous nerve preparation. The method is based on extracellular recordings of propagated action potentials from single-fiber receptive fields. Cutaneous nerve endings show graded sensitivities to various stimulus modalities that are quantified by adequate and controlled stimulation of the superfused skin with heat, cold, touch, constant punctate pressure or chemicals. Responses recorded from single-fibers are comparable with those obtained in previous in vivo experiments on the same species. We describe the components and the setting-up of the basic equipment of a skin–nerve recording station (few days), the preparation of the skin and the adherent saphenous nerve in the mouse (15–45 min) and the isolation and recording of neurons (approximately 1–3 h per recording). In addition, stimulation techniques, protocols to achieve single-fiber recordings, issues of data acquisition and action potential discrimination are discussed in detail.

In primate C-fiber polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically-sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, a MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically-insensitive C-fibers (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin responsive neurons in primate DRG. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibers likely corresponding to murine MrgprD- expressing, non-peptidergic nociceptive afferents.

Nerve growth factor (NGF) is involved in the long-term sensitization of nociceptive processing linked to chronic pain. Functional and structural ("sprouting") changes can contribute. Thus, humans report long-lasting hyperalgesia to mechanical and electrical stimulation after intradermal NGF injection and NGF-induced sprouting has been reported to underlie cancer bone pain and visceral pain. Using a human-like animal model we investigated the relationship between the structure and function of unmyelinated porcine nociceptors 3 weeks after intradermal NGF treatment. Axonal and sensory characteristics were studied by in vivo single-fiber electrophysiology and immunohistochemistry. C fibers recorded extracellularly were classified based on mechanical response and activity-dependent slowing (ADS) of conduction velocity. Intraepidermal nerve fiber (IENF) densities were assessed by immunohistochemistry in pigs and in human volunteers using the same NGF model. NGF increased conduction velocity and reduced ADS and propagation failure in mechano-insensitive nociceptors. The proportion of mechano-sensitive C nociceptors within NGF-treated skin areas increased from 45.1% (control) to 71% and their median mechanical thresholds decreased from 40 to 20 mN. After NGF application, the mechanical receptive fields of nociceptors increased from 25 to 43 mm(2). At the structural level, however, IENF density was not increased by NGF. In conclusion, intradermal NGF induces long-lasting axonal and mechanical sensitization in porcine C nociceptors that corresponds to hyperalgesia observed in humans. Sensitization is not accompanied by increased IENF density, suggesting that NGF-induced hyperalgesia might not depend on changes in nerve fiber density but could be linked to the recruitment of previously silent nociceptors.

Nerve growth factor (NGF) induces acute sensitization of nociceptive sensory endings and long-lasting hyperalgesia. NGF modulation of sodium channel expression might contribute to neurotrophin-induced hyperalgesia. Here, we investigated NGF-evoked changes of the activity-dependent slowing of conduction in porcine C-fibers. Animals received intradermal injections of NGF (2 μg or 8 μg) or saline in both hind limbs. Extracellular recordings from the saphenous nerves were performed 1 week later. Based on sensory thresholds and electrically induced activity-dependent slowing (ADS) of axonal conduction, C-fibers were classified as mechano-sensitive afferents, mechano-insensitive afferents, cold nociceptors, and sympathetic efferents. NGF (2 μg) increased conduction velocity in C-fibers from 1.0±0.05 m/s to 1.2±0.07 m/s. In mechano-insensitive afferents, NGF (8 μg) reduced activity-dependent slowing of conduction, from 5.3±0.2% to 3.2±0.5% (0.125-0.5 Hz stimulation) and from 28.5±1.3% to 20.9±1.9% (2 Hz stimulation), such that ADS no longer differentiated between mechano-sensitive and mechano-insensitive fibers. Accordingly, the number of fibers with pronounced ADS decreased but more units with pronounced ADS were mechano-sensitive. Spontaneously active C-fibers were increased above the control level (1%) by NGF 8 μg (8%). The results demonstrate that NGF changes the functional axonal characteristics of mechano-insensitive C-fibers and enhances spontaneous activity thereby possibly contributing to hyperalgesia.

Voltage-gated sodium (Na) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine Na channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (Na1.1-Na1.4, Na1.6-Na1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (Na1.8 and Na1.9) inhibited by millimolar concentrations, with Na1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different Na 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 (). 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 Na1.9 mice. In nerves from Na1.8 mice, TTX-r C-CAPs could not be detected. These data indicate that Na1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of Na1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of Na1.8 in distal axons may provide a useful target in the treatment of pain of peripheral origin. 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 (Na1.8). The functional prominence of Na1.8 within the axonal compartment immediately proximal to its termination may affect strategies targeting pain of peripheral origin.