GABA increases electrical excitability in a subset of human unmyelinated peripheral axons

A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established

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

GABA_A receptors mediate responses to GABA in human C-fibres.

<p>Increases in the electrical excitability index of unmyelinated axons in human sural nerve fascicles following bath application of GABA (10–30 µM) are blocked by prior application of the GABA_A receptor antagonists bicuculline (20 µM, grey bar A), picrotoxin (10 µM, grey bar B) and gabazine (10 µM, grey bar C). In contrast to both bicuculline and gabazine, the blocking effect of picrotoxin is not reversed upon wash-out (B). The pooled effect of each compound on the change in excitability index following bath application of GABA (10 µM) is shown in panel D. A significant reduction in the response to GABA (10 µM) was observed in the presence of gabazine (p<0.05, Student's paired t-test) and bicuculline (p<0.05, Student's paired t-test). Owing to the limited availability of human nerve fascicles a statistical comparison was not made for three fascicles exposed to picrotoxin.</p

Two types of C-fibre response profile for individual human sural nerve fascicles.

<p>Individual fascicles were designated as either Type A (panel A) or Type B (panel B) on the basis of changes in electrical excitability and the latency to half-maximum of the compound C-fibre action potential observed during stimulation at 2 Hz (grey shading). Three features can be used to differentiate the two C-fibre response profiles. Firstly, at low frequencies of stimulation (0.33 Hz), the compound C-fibre response in Type A fascicles is typically sub-excitable (positive excitability index) whereas Type B fascicles are super-excitable (negative excitability index). Secondly, during stimulation at higher frequencies (2 Hz), the compound C-fibre response in Type A fascicles exhibits a monotonic decrease in excitability index and a slowing of conduction latency. For Type B responses, repetitive stimulation initially reduces the excitability index and slows conduction before these changes partially reverse and conduction latency and excitability index both approach a plateau. Finally, during stimulation at 2 Hz, Type A C-fibre responses typically show a reversal from sub- to super-excitability whereas the super-excitability characteristic of Type B responses simply increases in magnitude.</p

Higher rates of electrical stimulation render human C-fibres less excitable but enhance responses to GABA.

<p>The magnitude of the excitability index increase in response to GABA increases with the rate of electrical stimulation. An increase in the rate of electrical stimulation reduces the excitability index of C-fibres (A & B). The absolute magnitude of stimulus rate-induced decreases in excitability index varies (B). The reduction in excitability index produced by increased electrical stimulation rate always increases the magnitude of the change in excitability index observed in response to bath application of GABA or muscimol (10–30 µM, A & C).</p

Only a sub-population of human C-fibres respond to GABA.

<p>The magnitude of GABA (10–30 µM) evoked increases in excitability index correlate with parameters of electrical excitability. The compound C-fibre response in Type A fascicles (A) is typically sub-excitable (i.e. positive excitability index) at low rates of stimulation and shows a pronounced change in excitability index upon increasing the frequency of repetitive stimulation (open circles, C). In addition, C-fibre responses in Type A fascicles exhibit a large change in excitability index during bath application of GABA (30 µM, open circles, D). In contrast, C-fibre responses electrophysiologically classified as Type B (B) are typically super-excitable at low stimulus frequencies, show a modest change in excitability upon repetitive stimulation at 2 Hz (encircled crosses, C) and typically respond poorly or not at all to GABA (30 µM, encircled crosses, D). The filled markers in panels C and D represent fascicles for which a classification based upon the C-fibre response profile to repetitive electrical stimulation at 2 Hz was not determined.</p

GABA activation of human C-fibres is concentration dependent.

<p>The excitability index was determined for C-fibres in human sural nerve fascicles during bath application of GABA (0.1–100 µM). A time window restricted the domain over which the amplitude of the electrically-evoked compound C-fibre action potential was determined (A, grey bars). Excitability index was calculated from the ratio of the current required to evoke an unconditioned C-fibre response of 40% maximum amplitude to that required to evoke a conditioned 40% response, i.e. 30 ms after a supra-maximal conditioning stimulus (40% cond., grey). Negative values of excitability index indicate that more current is required to evoke an unconditioned 40% C-fibre response. Following the addition of GABA (0.1–30 µM, 90 s application) to the bathing solution the excitability index increases, i.e. becomes more positive (B), and the magnitude of this change increases as the concentration of GABA in the perfusing solution increases (B & C). The EC₅₀ determined from a sigmoid fit to normalised excitability index on GABA concentration was 6.88±0.01 µM.</p

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

<p>Abstract</p> <p>Background</p> <p>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 <it>Anemonia sulcata</it>, 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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p