The formalin test does not probe inflammatory pain but excitotoxicity in rodent skin.

The most widely used formalin test to screen antinociceptive drug candidates is still apostrophized as targeting inflammatory pain, in spite of strong opposing evidence published. In our rat skin-nerve preparation ex vivo, recording from all classes of sensory single-fibers (n = 32), 30 units were transiently excited by formaldehyde concentrations 1-100 mM applied to receptive fields (RFs) for 3 min, C and Aδ-fibers being more sensitive (1-30 mM) than Aß-fibers. From 30 mM on, ~1% of the concentration usually injected in vivo, all RFs were defunctionalized and conduction in an isolated sciatic nerve preparation was irreversibly blocked. Thus, formaldehyde, generated a state of 'anesthesia dolorosa' in the RFs in so far as after a quiescent interphase all fibers with unmyelinated terminals developed a second phase of vigorous discharge activity which correlated well in time course and magnitude with published pain-related behaviors. Sural nerve filament recordings in vivo confirmed that higher formalin concentrations (> 42 mM) have to be injected to the skin to induce this second phase of discharge. Patch-clamp and calcium-imaging confirmed TRPA1 as the primary transducer of formaldehyde (10 mM) effects on mouse sensory neurons. However, stimulated CGRP release from isolated skin of TRPA1+/+ and TRPA1-/- mice showed a convergence of the saturating concentration-response curves at 100 mM formaldehyde, which did not occur with nerve and trachea preparations. Finally, skin-nerve recordings from C and Aδ-fibers of TRPA1-/- mice revealed a massive reduction in formaldehyde (30 mM)-evoked discharge. However, the remaining activity was still biphasic, thus confirming additional unspecific excitotoxic actions of the fixative that diffuses along still excitable axons as previously published. The multiplicity of formaldehyde's actions requires extensive discussion and literature review, leading to a fundamental reevaluation of the formalin test.

Painful diabetic neuropathy leads to functional CaV3.2 expression and spontaneous activity in skin nociceptors of mice.

Painful diabetic neuropathy occurs in approximately 20% of diabetic patients with underlying pathomechanisms not fully understood. We evaluated the contribution of the CaV3.2 isoform of T-type calcium channel to hyperglycemia-induced changes in cutaneous sensory C-fiber functions and neuropeptide release employing the streptozotocin (STZ) diabetes model in congenic mouse strains including global knockouts (KOs). Hyperglycemia established for 3-5 weeks in male C57BL/6J mice led to major reorganizations in peripheral C-fiber functions. Unbiased electrophysiological screening of mechanosensitive single-fibers in isolated hairy hindpaw skin revealed a relative loss of (polymodal) heat sensing in favor of cold sensing. In healthy CaV3.2 KO mice both heat and cold sensitivity among the C-fibers seemed underrepresented in favor of exclusive mechanosensitivity, low-threshold in particular, which deficit became significant in the diabetic KOs. Diabetes also led to a marked increase in the incidence of spontaneous discharge activity among the C-fibers of wildtype mice, which was reduced by the specific CaV3.2 blocker TTA-P2 and largely absent in the KOs. Evaluation restricted to the peptidergic class of nerve fibers - measuring KCl-stimulated CGRP release - revealed a marked reduction in the sciatic nerve by TTA-P2 in healthy but not diabetic wildtypes, the latter showing CGRP release that was as much reduced as in healthy and, to the same extent, in diabetic CaV3.2 KOs. These data suggest that diabetes abrogates all CaV3.2 functionality in the peripheral nerve axons. In striking contrast, diabetes markedly increased the KCl-stimulated CGRP release from isolated hairy skin of wildtypes but not KO mice, and TTA-P2 reversed this increase, strongly suggesting a de novo expression of CaV3.2 in peptidergic cutaneous nerve endings which may contribute to the enhanced spontaneous activity. De-glycosylation by neuraminidase showed clear desensitizing effects, both in regard to spontaneous activity and stimulated CGRP release, but included actions independent of CaV3.2. However, as diabetes-enhanced glycosylation is decisive for intra-axonal trafficking, it may account for the substantial reorganizations of the CaV3.2 distribution. The results may strengthen the validation of CaV3.2 channel as a therapeutic target of treating painful diabetic neuropathy.

Axonal GABAA stabilizes excitability in unmyelinated sensory axons secondary to NKCC1 activity.

KEY POINTS: GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABAA receptor. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to Na-K-Cl cotransporter type 1 (NKCC1) loading of axonal chloride. Activation of axonal GABAA receptor stabilized C-fibre excitability during prolonged low frequency (2.5 Hz) firing. NKCC1 maintains intra-axonal chloride to provide feed-forward stabilization of C-fibre excitability and thus support sustained firing. ABSTRACT: GABAA receptor (GABAA R)-mediated depolarization of dorsal root ganglia (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. In the present study, we report that the peripheral axons of DRG neurons are also depolarized by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABAA R agonists increased axonal excitability, whereas GABA excitability responses were blocked by GABAA R antagonists and were absent in mice lacking the GABAA R ß3 subunit selectively in DRG neurons (AdvillinCre or snsCre ). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5-2.5 Hz). However, during Na-K-Cl cotransporter type 1 (NKCC1) blockade, the 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 (activity-dependent slowing; ADS) was used to quantify C-fibre excitability loss during a 2.5 Hz challenge. ADS was reduced by GABAA R agonists and exacerbated by either GABAA R antagonists, ß3 deletion or NKCC1 blockade. This illustrates that activation of GABAA R stabilizes 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 utilized by GABAA R to stabilize 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.

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.

Photosensitization of TRPA1 and TRPV1 by 7-dehydrocholesterol: implications for the Smith-Lemli-Opitz syndrome.

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.

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.

Use dependence of peripheral nociceptive conduction in the absence of tetrodotoxin-resistant sodium channel subtypes.

KEY POINTS: This study examines conduction in peripheral nerves and its use dependence in tetrodotoxin-resistant (TTXr) sodium channel (Nav 1.8, Nav 1.9) knockout and wildtype animals. We observed use-dependent decreases of single fibre and compound action potential amplitude in peripheral mouse C-fibres (wildtype). This matches the previously published hypothesis that increased Na/K-pump activity is not the underlying mechanism for use-dependent changes of neural conduction. Knocking out TTXr sodium channels influences use-dependent changes of conductive properties (action potential amplitude, latency, conduction safety) in the order Nav 1.8 KO > Nav 1.9KO > wildtype. This is most likely explained by different subsets of presumably (relatively) Nav 1.7-rich conducting fibres in knockout animals as compared to wildtypes, in combination with reduced per-pulse sodium influx. ABSTRACT: Use dependency of peripheral nerves, especially of nociceptors, correlates with receptive properties. Slow inactivation of voltage-gated sodium channels has been discussed to be the underlying mechanism - pointing to a receptive class-related difference of sodium channel equipment. Using electrophysiological recordings of single unmyelinated cutaneous fibres and their compound action potential (AP), we evaluated use-dependent changes in mouse peripheral nerves, and the contribution of the tetrodotoxin-resistant (TTXr) sodium channels Nav 1.8 and Nav 1.9 to these changes. Nerve fibres were electrically stimulated using single or double pulses at 2 Hz. Use-dependent changes of latency, AP amplitude, and duration as well as the fibres' ability to follow the stimulus were evaluated. AP amplitudes substantially diminished in used fibres from C57BL/6 but increased in Nav 1.8 knockout (KO) mice, with Nav 1.9 KO in between. Activity-induced latency slowing was in contrast the most pronounced in Nav 1.8 KOs and the least in wildtype mice. The genotype was also predictive of how long fibres could follow the double pulsed stimulus with wildtype fibres blocking first and Nav 1.8 KO fibres enduring the longest. In contrast, changes in spike duration were less pronounced and displayed no significant tendency. Thus, all major measures of peripheral nerve accommodation (amplitude, latency and durability) depended on genotype. All use-dependent changes appeared in the order NaV 1.8 KO > NaV 1.9 KO > wildtype, which is most likely explained by the relative contribution of Nav 1.7 varying in the same order and the amounts of per-pulse sodium influx expected in the opposite order.

Sensory transduction in peripheral nerve axons elicits ectopic action potentials.

Sensory properties of unmyelinated axons in the isolated rat sciatic nerve have been revealed previously by measuring stimulated neuropeptide release in response to noxious stimuli. In addition, axonal sensitization by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel transient receptor potential vanilloid receptor-1. It was unclear whether this responsiveness is accompanied by ectopic generation of action potentials, which may play a crucial role in painful neuropathies. We explored this hypothesis using the isolated mouse skin-nerve preparation. This method enabled us to directly compare the sensory properties of axons in the peripheral nerve with their characterized cutaneous terminals in the receptive field using propagated action potentials as an index of axonal activation. Single-fiber recordings from 51 mechanosensitive mouse C-fibers revealed that a majority of the polymodal nociceptors responded with an encoding discharge rate to graded heating of the cutaneous receptive field (n = 38) as well as of the saphenous nerve carrying the fiber under investigation (n = 25; 66%). Axonal heat responses paralleled those of the receptive fields with regard to thresholds and discharge rates (41.5 +/- 4.3 degrees C; 7.7 +/- 9.6 spikes in a 20 s 32-48 degrees C ranged stimulation). In contrast, axonal mechanosensitivity was poor and noxious cold sensitivity more rarely encountered. In conclusion, peripheral nerve axons exhibit sensory transduction capacities similar to their nociceptive terminals in the skin with respect to noxious heat, although not to mechanical and cold sensitivity. This may become a source of ectopic discharge and pain if axonal heat threshold drops to body temperature, as may be the case during inflammation-like processes in peripheral nerves.

Calcitonin gene-related peptide release from intact isolated dorsal root and trigeminal ganglia.

Neuropeptides like calcitonin gene-related peptide (CGRP) and substance P are found in significant proportions of primary afferent neurons. Release of these neuropeptides as well as prostaglandin E(2) is an approved index for the activation of these primary afferents. Previous studies have used cultures of enzyme-treated and mechanically dissociated primary afferent neurons, fresh tissue slices or cubes. In the present study we demonstrate CGRP and prostaglandin E(2) release from intact isolated dorsal root and trigeminal ganglia. Stimulation with noxious heat, low pH, inflammatory mediators and high potassium concentration increased CGRP release. In conclusion, neuropeptide release from intact isolated ganglia is a reliable method to study the responsiveness of sensory neurons in situ in comparison with neuronal cell cultures.