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

Voltage-gated sodium (NaV) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine NaV channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (NaV1.1-NaV1.4, NaV1.6-NaV1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (NaV1.8 and NaV1.9) inhibited by millimolar concentrations, with NaV1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different NaV 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 NaV1.9-/- mice. In nerves from NaV1.8-/- mice, TTX-r C-CAPs could not be detected. These data indicate that NaV1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of NaV1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of NaV1.8 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 (NaV1.8). The functional prominence of NaV1.8 within the axonal compartment immediately proximal to its termination may affect strategies targeting pain of peripheral origin.

C-fiber recovery cycle supernormality depends on ion concentration and ion channel permeability.

Following each action potential, C-fiber nociceptors undergo cyclical changes in excitability, including a period of superexcitability, before recovering their basal excitability state. The increase in superexcitability during this recovery cycle depends upon their immediate firing history of the axon, but also determines the instantaneous firing frequency that encodes pain intensity. To explore the mechanistic underpinnings of the recovery cycle phenomenon a biophysical model of a C-fiber has been developed. The model represents the spatial extent of the axon including its passive properties as well as ion channels and the Na/K-ATPase ion pump. Ionic concentrations were represented inside and outside the membrane. The model was able to replicate the typical transitions in excitability from subnormal to supernormal observed empirically following a conducted action potential. In the model, supernormality depended on the degree of conduction slowing which in turn depends upon the frequency of stimulation, in accordance with experimental findings. In particular, we show that activity-dependent conduction slowing is produced by the accumulation of intraaxonal sodium. We further show that the supernormal phase results from a reduced potassium current Kdr as a result of accumulation of periaxonal potassium in concert with a reduced influx of sodium through Nav1.7 relative to Nav1.8 current. This theoretical prediction was supported by data from an in vitro preparation of small rat dorsal root ganglion somata showing a reduction in the magnitude of tetrodotoxin-sensitive relative to tetrodotoxin -resistant whole cell current. Furthermore, our studies provide support for the role of depolarization in supernormality, as previously suggested, but we suggest that the basic mechanism depends on changes in ionic concentrations inside and outside the axon. The understanding of the mechanisms underlying repetitive discharges in recovery cycles may provide insight into mechanisms of spontaneous activity, which recently has been shown to correlate to a perceived level of pain.

Anticancer drug oxaliplatin induces acute cooling-aggravated neuropathy via sodium channel subtype Na(V)1.6-resurgent and persistent current.

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.

Activity-dependent sensory signal processing in mechanically responsive slowly conducting meningeal afferents.

Activity-dependent processes in slowly conducting afferents have been shown to modulate conduction and receptive properties, but it is not known how the frequency of action potential firing determines the responses of such fibers to mechanical stimulation. We examined the responses of slowly conducting meningeal afferents to mechanical stimuli and the influence of preceding action potential activity. In hemisected rat heads with adhering cranial dura mater, recordings were made from meningeal nerves. Dural receptive fields of mechanically sensitive afferent fibers were stimulated with a custom-made electromechanostimulator. Sinusoidal mechanical stimuli of different stimulus durations and amplitudes were applied to produce either high-frequency (phasic) or low-frequency (tonic) discharges. Most fibers showed slowing of their axonal conduction velocity on electrically evoked activity at ≥2 Hz. In this state, the peak firing frequency of phasic responses to a 250-ms mechanical stimulus was significantly reduced compared with control. In contrast, the frequency of tonic responses induced by mechanical stimuli of >500 ms did not change. In a rare subtype of afferents, which showed conduction velocity speeding during activity, an increase in the phasic responses to mechanical stimuli was observed. Depending on the axonal properties of the afferent fibers, encoding of phasic components of mechanical stimuli is altered according to the immediate firing history. Preceding activity in mechanoreceptors slowing their conduction velocity seems to provide a form of low-pass filtering of action potential discharges predominantly reducing the phasic component. This may improve discrimination between harmless and potentially harmful mechanical stimuli in normal tissue.

Oxaliplatin-induced Acute Neurotoxicity Recovers Between Repeat Infusion Cycles: An Axonal Excitability Repeated Multiple Measurements Study.

Background/Aim: Oxaliplatin, a platinum-based chemotherapy used in the treatment of colorectal cancer, induces acute neurotoxicity following infusion. The aim of this study was to establish whether alterations in axonal excitability develop progressively with higher cumulative doses and whether there is a recovery in motor axons after each cycle of treatment. Patients and Methods: Twenty consecutive patients with a colorectal cancer diagnosis, referred from the Oncology Department of Aretaieion Hospital of Athens, were enrolled in this study between October 2018 and May 2019. None of the participants had diabetes, alcohol abuse, known neuropathy or were previously treated with another neo-adjuvant therapy. Threshold Tracking techniques and Qtrac software were used for assessing axonal excitability in motor axons. Excitability recordings were undertaken before and immediately after the end of oxaliplatin infusion. Results: Statistically significant changes were found (p<0.01) in axonal excitability (relative refractory period, refractoriness at 2 ms and 2.5 ms, sub-excitability and super-excitability) before and after oxaliplatin infusion. No statistically significant changes (p>0.05) were found in threshold electrotonus and strength-duration parameters before and after oxaliplatin infusion. We also did not find statistically significant differences (p>0.05) between means of excitability parameters before infusion at each cycle. Conclusion: Our study confirms oxaliplatin-induced acute neurotoxicity following infusion and suggests that motor axons recover between repeat infusion cycles.

Activation of axonal Kv7 channels in human peripheral nerve by flupirtine but not placebo - therapeutic potential for peripheral neuropathies: results of a randomised controlled trial.

BACKGROUND: Flupirtine is an analgesic with muscle-relaxing properties that activates Kv7 potassium channels. Kv7 channels are expressed along myelinated and unmyelinated peripheral axons where their activation is expected to reduce axonal excitability and potentially contribute to flupirtine's clinical profile. TRIAL DESIGN: To investigate the electrical excitability of peripheral myelinated axons following orally administered flupirtine, in-vitro experiments on isolated peripheral nerve segments were combined with a randomised, double-blind, placebo-controlled, phase I clinical trial (RCT). METHODS: Threshold tracking was used to assess the electrical excitability of myelinated axons in isolated segments of human sural nerve in vitro and motoneurones to abductor pollicis brevis (APB) in situ in healthy subjects. In addition, the effect of flupirtine on ectopic action potential generation in myelinated axons was examined using ischemia of the lower arm. RESULTS: Flupirtine (3-30 µM) shortened the relative refractory period and increased post-conditioned superexcitability in human myelinated axons in vitro. Similarly, in healthy subjects the relative refractory period of motoneurones to APB was reduced 2 hours after oral flupirtine but not following placebo. Whether this effect was due to a direct action of flupirtine on peripheral axons or temperature could not be resolved. Flupirtine (200 mg p.o.) also reduced ectopic axonal activity induced by 10 minutes of lower arm ischemia. In particular, high frequency (ca. 200 Hz) components of EMG were reduced in the post-ischemic period. Finally, visual analogue scale ratings of sensations perceived during the post-ischemic period were reduced following flupirtine (200 mg p.o.). CONCLUSIONS: Clinical doses of flupirtine reduce the excitability of peripheral myelinated axons. TRIAL REGISTRATION: ClinicalTrials registration is NCT01450865.

Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures.

Sensory acuity and motor dexterity deteriorate when human limbs cool down, but pain perception persists and cold-induced pain can become excruciating. Evolutionary pressure to enforce protective behaviour requires that damage-sensing neurons (nociceptors) continue to function at low temperatures. Here we show that this goal is achieved by endowing superficial endings of slowly conducting nociceptive fibres with the tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Na(v)1.8 (ref. 2). This channel is essential for sustained excitability of nociceptors when the skin is cooled. We show that cooling excitable membranes progressively enhances the voltage-dependent slow inactivation of tetrodotoxin-sensitive VGSCs. In contrast, the inactivation properties of Na(v)1.8 are entirely cold-resistant. Moreover, low temperatures decrease the activation threshold of the sodium currents and increase the membrane resistance, augmenting the voltage change caused by any membrane current. Thus, in the cold, Na(v)1.8 remains available as the sole electrical impulse generator in nociceptors that transmits nociceptive information to the central nervous system. Consistent with this concept is the observation that Na(v)1.8-null mutant mice show negligible responses to noxious cold and mechanical stimulation at low temperatures. Our data present strong evidence for a specialized role of Na(v)1.8 in nociceptors as the critical molecule for the perception of cold pain and pain in the cold.

Repetitive activity slows axonal conduction velocity and concomitantly increases mechanical activation threshold in single axons of the rat cranial dura.

The passage of an action potential along a peripheral axon modulates the conduction velocity of subsequent action potentials. In C-neurones with unmyelinated axons repetitive activity progressively slows axonal conduction velocity and in microneurographic recordings from healthy human subjects the magnitude of this slowing can be used to predict the receptive properties of individual axons. Recently, a reduction in the number of available voltage-gated sodium channels (Na(V)) through inactivation has been implicated as the predominant factor responsible for the slowing of axonal conduction. Since Na(V)s are also responsible for the initiation of action potentials in sensory nerve terminals, changes in their availability may be expected to affect activation threshold for sensory stimuli. To examine this proposal, dynamic mechanical stimuli were used to make precise estimates of activation threshold in single unmyelinated axons innervating the rat cranial dura mater. Decreases in axonal conduction velocity induced by repetitive electrical stimulation were paralleled by an increase in mechanical activation threshold. Application of TTX (10-20 nM) also slowed axonal conduction velocity in all 11 fibres examined and in 9 of these this resulted in a parallel increase in mechanical activation threshold. We interpret this as indicating that a reduction in available Na(V) number contributes to both axonal conduction velocity slowing and the observed parallel increase in mechanical activation threshold. The slowing of axonal conduction velocity observed during repetitive activity thus represents a form of accommodation, i.e. self inhibition, which is likely to be decisive in limiting peripheral input to the spinal dorsal horn and thereby regulating processes that could otherwise lead to central sensitization.

Sea-anemone toxin ATX-II elicits A-fiber-dependent pain and enhances resurgent and persistent sodium currents in large sensory neurons.

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 ß4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less ß4 mRNA than large sensory neurons. With the ß4-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 ß4-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.

The Kv7 potassium channel activator flupirtine affects clinical excitability parameters of myelinated axons in isolated rat sural nerve.

Flupirtine is an activator of Kv7 (KCNQ/M) potassium channels that has found clinical use as an analgesic with muscle relaxant properties. Kv7 potassium channels are expressed in axonal membranes and pharmacological activation of these channels may restore abnormal nerve excitability. We have examined the effect of flupirtine on the electrical excitability of myelinated axons in isolated segments of rat sural nerve. Axonal excitability was studied in vitro with the same parameters used by clinical neurophysiologists to assess peripheral nerve excitability in situ. Application of flupirtine in low micromolar concentrations resulted in an increase in threshold current, a reduction of refractoriness and an increase in post-spike superexcitability. These effects are consistent with an increase in Kv7 conductance and membrane hyperpolarization. Flupirtine also enhanced and prolonged the late, long-lasting period of axonal subexcitability that follows a short burst of action potentials. This effect was blocked by XE 991 (10 microM), an antagonist of Kv7 channels. In summary, flupirtine affects measures of excitability that are altered in the myelinated axons of patients with peripheral nerve disorders. This indicates that neuropathies with abnormal nerve excitability parameters corresponding to those affected by flupirtine may benefit from activation of axonal Kv7 potassium channels.

Slow depolarizing stimuli differentially activate mechanosensitive and silent C nociceptors in human and pig skin.

ABSTRACT: High-threshold mechanosensitive and mechanoinsensitive ("silent") nociceptors have similar electrical thresholds for transcutaneous sine wave stimulation at 4 Hz that selectively activates cutaneous C nociceptors in human skin. Their fundamentally different functions particularly in chronic pain warrant differential stimulation protocols. We used transcutaneously delivered slow depolarizing stimuli (half-sine, 500 ms duration, 0.01-1 mA) in humans to assess intensity-response relations for the induction of pain psychophysically and recorded activation of mechanosensitive and silent nociceptors in healthy volunteers by microneurography. Differential C-fiber activation was confirmed in single-fiber recordings in pig allowing for stimulation amplitudes up to 10 mA. Perception and pain thresholds to half-sine wave pulses were 0.06 ± 0.03 mA and 0.18 ± 0.1 mA, respectively, and caused pain in an amplitude-dependent manner (n = 24). When matched for pain intensity, only sine wave stimulation induced an instant widespread axon reflex erythema (n = 10). In human microneurography, half-sine stimulation activated mechanosensitive nociceptors (n = 13), but only one of 11 silent nociceptors. In pig skin, the amplitude-dependent activation of mechanosensitive nociceptors was confirmed (0.2-1 mA, n = 28), and activation thresholds for most silent nociceptors (n = 13) were found above 10 mA. Non-nociceptive low-threshold mechanosensitive C fibers (n = 14) displayed lower activation thresholds for half-sine wave stimuli with an amplitude-dependent discharge increase between 0.01 and 0.1 mA. We conclude that transcutaneous electrical stimulation with 500-ms half-sine wave pulses between 0.2 and 1 mA causes amplitude-dependent pain by preferential activation of mechanosensitive C nociceptors.

Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons.

Protein kinase type C-ε (PKCε) plays important roles in the sensitization of primary afferent nociceptors, such as ion channel phosphorylation, that in turn promotes mechanical hyperalgesia and pain chronification. In these neurons, PKCε is modulated through the local release of mediators by the surrounding Schwann cells (SCs). The progesterone metabolite allopregnanolone (ALLO) is endogenously synthesized by SCs, whereas it has proven to be a crucial mediator of neuron-glia interaction in peripheral nerve fibers. Biomolecular and pharmacological studies on rat primary SCs and dorsal root ganglia (DRG) neuronal cultures were aimed at investigating the hypothesis that ALLO modulates neuronal PKCε, playing a role in peripheral nociception. We found that SCs tonically release ALLO, which, in turn, autocrinally upregulated the synthesis of the growth factor brain-derived neurotrophic factor (BDNF). Subsequently, glial BDNF paracrinally activates PKCε via trkB in DRG sensory neurons. Herein, we report a novel mechanism of SCs-neuron cross-talk in the peripheral nervous system, highlighting a key role of ALLO and BDNF in nociceptor sensitization. These findings emphasize promising targets for inhibiting the development and chronification of neuropathic pain.

TTX-Resistant Sodium Channels Functionally Separate Silent From Polymodal C-nociceptors.

Pronounced activity-dependent slowing of conduction has been used to characterize mechano-insensitive, "silent" nociceptors and might be due to high expression of NaV1.8 and could, therefore, be characterized by their tetrodotoxin-resistance (TTX-r). Nociceptor-class specific differences in action potential characteristics were studied by: (i) in vitro calcium imaging in single porcine nerve growth factor (NGF)-responsive neurites; (ii) in vivo extracellular recordings in functionally identified porcine silent nociceptors; and (iii) in vitro patch-clamp recordings from murine silent nociceptors, genetically defined by nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) expression. Porcine TTX-r neurites (n = 26) in vitro had more than twice as high calcium transients per action potential as compared to TTX-s neurites (n = 18). In pig skin, silent nociceptors (n = 14) characterized by pronounced activity-dependent slowing of conduction were found to be TTX-r, whereas polymodal nociceptors were TTX-s (n = 12) and had only moderate slowing. Mechano-insensitive cold nociceptors were also TTX-r but showed less activity-dependent slowing than polymodal nociceptors. Action potentials in murine silent nociceptors differed from putative polymodal nociceptors by longer duration and higher peak amplitudes. Longer duration AP in silent murine nociceptors linked to increased sodium load would be compatible with a pronounced activity-dependent slowing in pig silent nociceptors and longer AP durations could be in line with increased calcium transients per action potential observed in vitro in TTX-resistant NGF responsive porcine neurites. Even though there is no direct link between slowing and TTX-resistant channels, the results indicate that axons of silent nociceptors not only differ in their receptive but also in their axonal properties.

Action potential initiation in the peripheral terminals of cold-sensitive neurones innervating the guinea-pig cornea.

The site at which action potentials initiate within the terminal region of unmyelinated sensory axons has not been resolved. Combining recordings of nerve terminal impulses (NTIs) and collision analysis, the site of action potential initiation in guinea-pig corneal cold receptors was determined. For most receptors (77%), initiation mapped to a point in the time domain that was closer to the nerve terminal than to the site of electrical stimulation at the back of the eye. Guinea-pig corneal cold receptors are Adelta-neurones that lose their myelin sheath at the point where they enter the cornea, and therefore their axons conduct more slowly within the cornea. Allowing for this inhomogeneity in conduction speed, the resulting spatial estimates of action potential initiation sites correlated with changes in NTI shape predicted by simulation of action potentials initiating within a nerve terminal. In some receptors, more than one NTI shape was observed. Simulations of NTI shape suggest that the origin of differing NTI shapes result from action potentials initiating at different, spatially discrete, locations within the nerve terminal. Importantly, the relative incidence of NTI shapes resulting from action potential initiation close to the nerve termination increased during warming when nerve activity decreased, indicating that the favoured site of action potential initiation shifts toward the nerve terminal when it hyperpolarizes. This finding can be explained by a hyperpolarization-induced relief of Na(+) channel inactivation in the nerve terminal. The results provide direct evidence that the molecular entities responsible for stimulus transduction and action potential initiation reside in parallel with one another in the unmyelinated nerve terminals of cold receptors.

Photoactivation of olfactory sensory neurons does not affect action potential conduction in individual trigeminal sensory axons innervating the rodent nasal cavity.

Olfactory and trigeminal chemosensory systems reside in parallel within the mammalian nose. Psychophysical studies in people indicate that these two systems interact at a perceptual level. Trigeminal sensations of pungency mask odour perception, while olfactory stimuli can influence trigeminal signal processing tasks such as odour localization. While imaging studies indicate overlap in limbic and cortical somatosensory areas activated by nasal trigeminal and olfactory stimuli, there is also potential cross-talk at the level of the olfactory epithelium, the olfactory bulb and trigeminal brainstem. Here we explored the influence of olfactory and trigeminal signaling in the nasal cavity. A forced choice water consumption paradigm was used to ascertain whether trigeminal and olfactory stimuli could influence behaviour in mice. Mice avoided water sources surrounded by both volatile TRPV1 (cyclohexanone) and TRPA1 (allyl isothiocyanate) irritants and the aversion to cyclohexanone was mitigated when combined with a pure odorant (rose fragrance, phenylethyl alcohol, PEA). To determine whether olfactory-trigeminal interactions within the nose could potentially account for this behavioural effect we recorded from single trigeminal sensory axons innervating the nasal respiratory and olfactory epithelium using an isolated in vitro preparation. To circumvent non-specific effects of chemical stimuli, optical stimulation was used to excite olfactory sensory neurons in mice expressing channel-rhodopsin (ChR2) under the olfactory marker protein (OMP) promoter. Photoactivation of olfactory sensory neurons produced no modulation of axonal action potential conduction in individual trigeminal axons. Similarly, no evidence was found for collateral branching of trigeminal axon that might serve as a conduit for cross-talk between the olfactory and respiratory epithelium and olfactory dura mater. Using direct assessment of action potential activity in trigeminal axons we observed neither paracrine nor axon reflex mediated cross-talk between olfactory and trigeminal sensory systems in the rodent nasal cavity. Our current results suggest that olfactory sensory neurons exert minimal influence on trigeminal signals within the nasal cavity.

Conduction velocity is regulated by sodium channel inactivation in unmyelinated axons innervating the rat cranial meninges.

Axonal conduction velocity varies according to the level of preceding impulse activity. In unmyelinated axons this typically results in a slowing of conduction velocity and a parallel increase in threshold. It is currently held that Na(+)-K(+)-ATPase-dependent axonal hyperpolarization is responsible for this slowing but this has long been equivocal. We therefore examined conduction velocity changes during repetitive activation of single unmyelinated axons innervating the rat cranial meninges. In direct contradiction to the currently accepted postulate, Na(+)-K(+)-ATPase blockade actually enhanced activity-induced conduction velocity slowing, while the degree of velocity slowing was curtailed in the presence of lidocaine (10-300 microm) and carbamazepine (30-500 microm) but not tetrodotoxin (TTX, 10-80 nm). This suggests that a change in the number of available sodium channels is the most prominent factor responsible for activity-induced changes in conduction velocity in unmyelinated axons. At moderate stimulus frequencies, axonal conduction velocity is determined by an interaction between residual sodium channel inactivation following each impulse and the retrieval of channels from inactivation by a concomitant Na(+)-K(+)-ATPase-mediated hyperpolarization. Since the process is primarily dependent upon sodium channel availability, tracking conduction velocity provides a means of accessing relative changes in the excitability of nociceptive neurons.

NaV1.7 and pain: contribution of peripheral nerves.

The sodium channel NaV1.7 contributes to action potential (AP) generation and propagation. Loss-of-function mutations in patients lead to congenital indifference to pain, though it remains unclear where on the way from sensory terminals to central nervous system the signalling is disrupted. We confirm that conditional deletion of NaV1.7 in advillin-expressing sensory neurons leads to impaired heat and mechanical nociception in behavioural tests. With single-fiber recordings from isolated skin, we found (1) a significantly lower prevalence of heat responsiveness to normally mechanosensitive C-fibers, although (2) the rare heat responses seemed quite vigorous, and (3) heat-induced calcitonin gene-related peptide release was normal. In biophysical respects, although electrical excitability, rheobase, and chronaxy were normal, (4) axonal conduction velocity was 20% slower than in congenic wild-type mice (5) and when challenged with double pulses (<100 milliseconds interval), the second AP showed more pronounced latency increase (6). On prolonged electrical stimulation at 2 Hz, (7) activity-dependent slowing of nerve fiber conduction was markedly less, and (8) was less likely to result in conduction failure of the mutant single fibers. Finally, recording of compound APs from the whole saphenous nerve confirmed slower conduction and less activity-dependent slowing as well as the functional absence of a large subpopulation of C-fibers (9) in conditional NaV1.7 knockouts. In conclusion, the clear deficits in somatic primary afferent functions shown in our study may be complemented by previously reported synaptic dysfunction and opioidergic inhibition, together accounting for the complete insensitivity to pain in the human mutants lacking NaV1.7.

Abnormal function of C-fibers in patients with diabetic neuropathy.

The mechanisms underlying the development of painful and nonpainful neuropathy associated with diabetes mellitus are unclear. We have obtained microneurographic recordings from unmyelinated fibers in eight patients with diabetes mellitus, five with painful neuropathy, and three with neuropathy without pain. All eight patients had large-fiber neuropathy, and seven patients had pathological thermal thresholds in their feet, indicating the involvement of small-caliber nerve fibers. A total of 163 C-fibers were recorded at knee level from the common peroneal nerve in the patients (36-67 years old), and these were compared with 77 C-fibers from healthy controls (41-64 years old). The ratio of mechano-responsive to mechano-insensitive nociceptors was approximately 2:1 in the healthy controls, whereas in the patients, it was 1:2. In patients, a fairly large percentage of characterized fibers (12.5% in nonpainful and 18.9% in painful neuropathy) resembled mechano-responsive nociceptors that had lost their mechanical and heat responsiveness. Such fibers were rarely encountered in age-matched controls (3.2%). Afferent fibers with spontaneous activity or mechanical sensitization were found in both patient groups. We conclude that small-fiber neuropathy in diabetes affects receptive properties of nociceptors that leads to an impairment of mechano-responsive nociceptors.

Reduced excitability and impaired nociception in peripheral unmyelinated fibers from Nav1.9-null mice.

The upregulation of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 has previously been associated with inflammatory hyperalgesia. Na1.9 knockout (KO) mice, however, did not seem insensitive in conventional tests of acute nociception. Using electrophysiological, neurochemical, and behavioral techniques, we now show NaV1.9-null mice exhibit impaired mechanical and thermal sensory capacities and reduced electrical excitability of nociceptors. In single-fiber recordings from isolated skin, the electrical threshold of NaV1.9 KO C fibers was elevated by 55% and the median von Frey threshold was 32 mN in contrast to 8 mN in wild types (WTs). The prevalence of C mechano-heat-sensitive (CMH) fibers was only 25.6% in NaV1.9 KO animals compared to 75.8% in the WT group, and the heat threshold of these CMH fibers was 40.4°C in the control vs 44°C in the KO group. Compound action potential recordings from isolated sciatic nerve segments of NaV1.9 KO mice revealed lower activity-induced slowing of conduction velocity upon noxious heat stimulation: 8% vs 30% in WTs. Heat-induced calcitonin gene-related peptide release from the skin was less in the KO than in the WT group. The reduced noxious heat sensitivity was finally confirmed with the Hargreaves test using 2 rates of radiant heating of the plantar hind paws. In conclusion, NaV1.9 presumably contributes to acute thermal and mechanical nociception in mice, most likely through increasing the excitability but probably also by amplifying receptor potentials irrespective of the stimulus modality.

The effects of polarizing current on nerve terminal impulses recorded from polymodal and cold receptors in the guinea-pig cornea.

It was reported recently that action potentials actively invade the sensory nerve terminals of corneal polymodal receptors, whereas corneal cold receptor nerve terminals are passively invaded (Brock, J.A., S. Pianova, and C. Belmonte. 2001. J. Physiol. 533:493-501). The present study investigated whether this functional difference between these two types of receptor was due to an absence of voltage-activated Na(+) conductances in cold receptor nerve terminals. To address this question, the study examined the effects of polarizing current on the configuration of nerve terminal impulses recorded extracellularly from single polymodal and cold receptors in guinea-pig cornea isolated in vitro. Polarizing currents were applied through the recording electrode. In both receptor types, hyperpolarizing current (+ve) increased the negative amplitude of nerve terminal impulses. In contrast, depolarizing current (-ve) was without effect on polymodal receptor nerve terminal impulses but increased the positive amplitude of cold receptor nerve terminal impulses. The hyperpolarization-induced increase in the negative amplitude of nerve terminal impulses represents a net increase in inward current. In both types of receptor, this increase in inward current was reduced by local application of low Na(+) solution and blocked by lidocaine (10 mM). In addition, tetrodotoxin (1 microM) slowed but did not reduce the hyperpolarization-induced increase in the negative amplitude of polymodal and cold nerve terminal impulses. The depolarization-induced increase in the positive amplitude of cold receptor nerve terminal impulses represents a net increase in outward current. This change was reduced both by lidocaine (10 mM) and the combined application of tetraethylammomium (20 mM) and 4-aminopyridine (1 mM). The interpretation is that both polymodal and cold receptor nerve terminals possess high densities of tetrodotoxin-resistant Na(+) channels. This finding suggests that in cold receptors, under normal conditions, the Na(+) conductances are rendered inactive because the nerve terminal region is relatively depolarized.