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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. GABAA receptor (GABAAR)‐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. GABAAR agonists increased axonal excitability, whereas GABA excitability responses were blocked by GABAAR antagonists and were absent in mice lacking the GABAAR β3 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 GABAAR agonists and exacerbated by either GABAAR antagonists, β3 deletion or NKCC1 blockade. This illustrates that activation of GABAAR 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 GABAAR 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. 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 FROM AUTHOR]

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°C; 7.7 ± 9.6 spikes in a 20 s 32- 48°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. [ABSTRACT FROM AUTHOR]

Painful diabetic neuropathy occurs in approximately 20% of diabetic patients with underlying pathomechanisms not fully understood. We evaluated the contribution of the Ca V 3.2 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 Ca V 3.2 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 Ca V 3.2 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 Ca V 3.2 KOs. These data suggest that diabetes abrogates all Ca V 3.2 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 Ca V 3.2 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 Ca V 3.2. However, as diabetes-enhanced glycosylation is decisive for intra-axonal trafficking, it may account for the substantial reorganizations of the Ca V 3.2 distribution. The results may strengthen the validation of Ca V 3.2 channel as a therapeutic target of treating painful diabetic neuropathy., (Copyright © 2021 Elsevier Inc. All rights reserved.)