Late Subexcitability Research Articles

T38. Altered axonal excitability in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease characterized with prominent fasciculations. Fasciculations suggest motor nerve hyperexcitability and potentially result in a burden to motor neuron. Axonal excitability and nerve conduction studies were performed in 31 patients with SBMA and 140 with amyotrophic lateral sclerosis (ALS). These results were compared with those of 16 healthy males (HM). In SBMA, correlations between neurophysiological studies and clinical profiles were examined. Patient backgrounds in SBMA were as follows; mean age; 58 years, mean disease duration; 152 months, mean ALS functional rating scale (ALSFRS-R); 41, mean CAG repeat expansion; 46, mean compound muscle action potential (CMAP) amplitude on abductor pollicis brevis muscle was 6.0 mV. Compared with HM, SBMA patients had longer strength-duration time constant (SDTC), greater threshold change in depolarizing threshold electrotonus (TEd) and greater supernormality (all p < 0.05). ALS patients had similar findings and smaller late subexcitability (all p < 0.05). In SBMA, TEd (90–100 ms) was related to ALSFRS-R (R = 0.42, p < 0.05), and CAG repeat was related to CMAP amplitude (R = −0.52, p < 0.05) and superexcitability (R = −0.46, p < 0.05). Patients were divided into two groups according to CMAP amplitude (over and under 5 mV). Compared with HM, SBMA patients with over 5 mV had greater TEd (10–20 ms) and greater superexcitability (all p < 0.05). SBMA patients with under 5 mV had longer SDTC, greater TEd and greater superexcitability (all p < 0.05). ALS patients with over 5 mV had greater TEd (90–100 ms) and greater superexcitability (all p < 0.05). ALS patients with under 5 mV had longer SDTC, greater TEd, greater superexcitability and smaller subexcitability (all p < 0.05). A previous study investigated axonal excitabilities in 7 patients with SBMA and revealed increased sodium currents. Our findings suggest that SBMA patients have increased sodium and decreased fast potassium currents. Similar findings and decreased slow potassium currents were found in ALS. This difference may suggest that ALS patients have more hyperexcitable motor nerve. Moreover, our findings suggest that decreased fast potassium currents are related to CAG repeat expansion in SBMA. It is already reported that SBMA patients have disruption of axonal transport. CAG repeat expansion may bring about axonal hyperexcitability and result in a burden to motor neuron.

Modification of multiple ion channel functions in vivo by pharmacological inhibition: observation by threshold tracking and modeling.

Maintenance of axonal excitability relies on complex balance by multiple ion currents, but its evaluation is limited by in vitro single channel neurophysiological study on overall behavior. We sought to evaluate behaviors of multiple ion currents by pharmacological blockade. The threshold tracking technique was used to measure multiple excitability indices on tail sensory nerve of normal male mice before and after administration of either BaCl2 or ivabradine. Mathematical modeling was used to identify the interval changes of the channel parameters. After administration of BaCl2 and ivabradine, the following changes were present: greater threshold changes of both depolarizing and hyperpolarizing threshold electrotonus by both; additionally, reduced S2 accommodation, reduced late subexcitability and increased superexcitability by BaCl2, increased S3 accommodation by ivabradine. Mathematical modelling implied reduction of slow K+ conductance, along with reduction of H conductance (Ih) by BaCl2; and reduction of Ih while augmentation of K+ conductances by ivabradine. Pharmacological blockade of a selective ion channel may be compensated by other ion channels. Unintended effects by ion channel modification could be caused by secondary current alteration by multiple ion channels. J. Med. Invest. 64: 30-38, February, 2017.

Axonal Dysfunction Precedes Motor Neuronal Death in Amyotrophic Lateral Sclerosis.

Wide-spread fasciculations are a characteristic feature in amyotrophic lateral sclerosis (ALS), suggesting motor axonal hyperexcitability. Previous excitability studies have shown increased nodal persistent sodium conductances and decreased potassium currents in motor axons of ALS patients, both of the changes inducing hyperexcitability. Altered axonal excitability potentially contributes to motor neuron death in ALS, but the relationship of the extent of motor neuronal death and abnormal excitability has not been fully elucidated. We performed multiple nerve excitability measurements in the median nerve at the wrist of 140 ALS patients and analyzed the relationship of compound muscle action potential (CMAP) amplitude (index of motor neuronal loss) and excitability indices, such as strength-duration time constant, threshold electrotonus, recovery cycle and current-threshold relationships. Compared to age-matched normal controls (n = 44), ALS patients (n = 140) had longer strength-duration time constant (SDTC: a measure of nodal persistent sodium current; p < 0.05), greater threshold changes in depolarizing threshold electrotonus (p < 0.05) and depolarizing current threshold relationship (i.e. less accommodation; (p < 0.05), greater superexcitability (a measure of fast potassium current; p < 0.05) and reduced late subexcitability (a measure of slow potassium current; p < 0.05), suggesting increased persistent sodium currents and decreased potassium currents. The reduced potassium currents were found even in the patient subgroups with normal CMAP (> 5mV). Regression analyses showed that SDTC (R = -0.22) and depolarizing threshold electrotonus (R = -0.22) increased with CMAP decline. These findings suggest that motor nerve hyperexcitability occurs in the early stage of the disease, and precedes motor neuronal loss in ALS. Modulation of altered ion channel function could be a treatment option for ALS.

Riluzole exerts transient modulating effects on cortical and axonal hyperexcitability in ALS

Riluzole is an established therapy for amyotrophic lateral sclerosis (ALS), although its effects are modest, prolonging survival by three months on average. While the neuroprotective effects of riluzole appear to be mediated by inhibition of glutaminergic transmission and antagonization of Na+ channel function, the duration of these effects remains to be elucidated. Consequently, the present study combined assessment of cortical and peripheral function to determine the longitudinal effects of riluzole in ALS patients. Assessment of cortical function by threshold tracking transcranial magnetic stimulation (TMS) combined with peripheral nerve function excitability studies were longitudinally undertaken on 19 sporadic ALS patients, with assessment occurring at baseline, four, eight, and 12 weeks post riluzole initiation. Baseline results were compared to 31 healthy controls. Results showed that, at baseline, cortical hyperexcitability was a feature of ALS as indicated by a marked reduction in averaged short interval intracortical inhibition [SICI] (3.6 ± 6.9%, p < 0.0001) and cortical silent period duration (p < 0.05) as well as an increase in motor evoked potential amplitude (p < 0.05). Riluzole therapy resulted in individual patient increase in SICI of 4.3% (p < 0.01) and 5.2% (p < 0.01) at four and eight weeks, respectively. At a peripheral level, riluzole therapy lead to a transient increase at four weeks in the relative refractory period (p < 0.05), superexcitability (p < 0.05) and late subexcitability (p < 0.05), all of which returned to baseline levels eight weeks after initiation of riluzole. In conclusion, the present study has established that riluzole exerts transient effects on cortical and axonal hyperexcitability, potentially accounting for the modest clinical effectiveness in ALS.

Impaired Axonal Na(+) Current by Hindlimb Unloading: Implication for Disuse Neuromuscular Atrophy.

This study aimed to characterize the excitability changes in peripheral motor axons caused by hindlimb unloading (HLU), which is a model of disuse neuromuscular atrophy. HLU was performed in normal 8-week-old male mice by fixing the proximal tail by a clip connected to the top of the animal's cage for 3 weeks. Axonal excitability studies were performed by stimulating the sciatic nerve at the ankle and recording the compound muscle action potential (CMAP) from the foot. The amplitudes of the motor responses of the unloading group were 51% of the control amplitudes [2.2 ± 1.3 mV (HLU) vs. 4.3 ± 1.2 mV (Control), P = 0.03]. Multiple axonal excitability analysis showed that the unloading group had a smaller strength-duration time constant (SDTC) and late subexcitability (recovery cycle) than the controls [0.075 ± 0.01 (HLU) vs. 0.12 ± 0.01 (Control), P < 0.01; 5.4 ± 1.0 (HLU) vs. 10.0 ± 1.3 % (Control), P = 0.01, respectively]. Three weeks after releasing from HLU, the SDTC became comparable to the control range. Using a modeling study, the observed differences in the waveforms could be explained by reduced persistent Na+ currents along with parameters related to current leakage. Quantification of RNA of a SCA1A gene coding a voltage-gated Na+ channel tended to be decreased in the sciatic nerve in HLU. The present study suggested that axonal ion currents are altered in vivo by HLU. It is still undetermined whether the dysfunctional axonal ion currents have any pathogenicity on neuromuscular atrophy or are the results of neural plasticity by atrophy.

7. Pathophysiological basis of motor neuronal hyperexcitability in amyotrophic lateral sclerosis

Objective Motor neuronal hyperexcitability potentially contributes to motor neuron death in amyotrophic lateral sclerosis (ALS). We aimed to reveal pathomechanisms of motor neuron hyperexcitability in ALS. Methods Threshold tracking transcranial magnetic stimulation and multiple excitability measurements were performed in 130 healthy controls (HC), 83 sporadic ALS (sALS) patients, 11 patients with superoxide dismutase-1 (SOD-1) mutations, 16 C9orf72 hexanucleotide repeat expansions and 2 FUS mutations. Results Compared to HC, in peripheral nerve excitability sALS had greater strength-duration time constant (p 0.05), depolarizing threshold electrotonus (p 0.0001) and superexcitability (p 0.05) and smaller late subexcitability (p 0.05). In cortical excitability, sALS had reduced short interval intracortical inhibition (p 0.0001). These indices were not significantly different in SOD-1 and C9orf72, compared to sALS. Conclusion These findings suggest both sALS and familial ALS have increased sodium currents, decreased potassium currents, rendering to peripheral nerve hyperexcitability. In addition, all ALS patients demonstrated decreased intracortical inhibition. Significance Motor neuronal hyperexcitability may be a common feature in both sALS and familial ALS associated with different pathological bases.

Nerve excitability and structural changes in myelinated axons from diabetic mice.

The mechanisms associated with nerve dysfunction and axonal loss in diabetes has not been fully clarified. Excitability and pathological aspects in nerves from diabetic mice were studied in order to explore the pathophysiology of diabetic neuropathy. Myelinated nerve fibres from the sciatic nerve of BKS.Cg-m (+/+) Lepr (db) /J mice were studied by registering the CMAP controlled by an automated threshold tracking method. The sciatic nerve was also studied pathologically. Diabetic mice displayed longer latencies, higher thresholds and lower amplitudes compared to controls and had a rightward shift in the stimulus response curves. Strength-duration time constant was lower in diabetic mice but not reaching statistical significance (p=0.09). Diabetics displayed an increase in accommodation, with a smaller change in excitability in threshold electrotonus. Refractoriness, mean superexcitability and late subexcitability were reduced in diabetic mice. Diabetic mice had a larger number of myelinated fibres compared to controls (p<0.05), but larger than 9 μm were virtually absent, accounting for near 7% in control animals. Db/db mice develop electrophysiological changes suggestive of membrane depolarization as the result of Na(+)/K(+) pump impairment. Loss of large myelinated fibres might also contribute to the nerve excitability profiles in this model.

Changes of the peripheral nerve excitability in vivo induced by the persistent Na+ current blocker ranolazine

Persistent Na+ current (Nap) in the peripheral axons play an important functional role in controlling the axonal excitability. Abnormal Nap is believed to contribute to neurodegeneration and neuropathic pain, and thus it is an attractive therapeutic target. To assess the behavior of selective Nap blockade, axonal excitability testing was performed in vivo in 10 normal male mice exposed to ranolazine by recording the tail sensory nerve action potentials (SNAPs). Twenty minutes after administering ranolazine i.p. (50mg/kg), the following changes were observed: lower SNAP amplitudes and the need for greater stimulus currents; greater threshold changes induced by long hyperpolarizing currents; reduced accommodation to long depolarizing current along with reduced late subexcitability; and reduced strength-duration time constant. These changes are explained by the suppression of Nap leading to greater threshold currents, partial block of transient Na+ current, and suppression of slow K+ currents. The suppressed slow K+ currents appear to limit the modification of the membrane excitability by ranolazine. This study confirms the utility of axonal excitability testing as a useful treatment biomarker in neurological conditions in which Nap function is being modified.

Clarifying distal axonal properties of the median nerve

Although length-dependent axonal excitability changes have been reported in the median nerve, the mechanisms underlying these changes remain to be further clarified. Axonal excitability studies were performed on median nerve at the palm and wrist in 20 healthy controls, with responses recorded over the abductor pollicis brevis. The strength-duration time constant was significantly shorter (palm: 0.35 ± 0.01 ms; wrist: 0.48 ± 0.03 ms; P < 0.001), whereas rheobase was significantly increased (palm: 2.90 ± 1.12 mA; wrist: 2.09 ± 1.11 mA; P < 0.05) at the palm. In addition, there was a significant increase in depolarizing threshold electrotonus at 90-100 ms (P < 0.001) and a reduction in S2 accommodation (P < 0.001) and late subexcitability (P < 0.001) at the palm. The changes in excitability were independent of factors influencing median nerve cross-sectional area. The present study reveals significant length dependent changes in median nerve excitability which may reflect differences in intrinsic membrane properties.

Protons regulate the excitability properties of rat myelinated sensory axons in vitro through block of persistent sodium currents

Little information is available on the pH sensitivity of the excitability properties of mammalian axons. Computer-assisted threshold tracking in humans has helped to define clinically relevant changes of nerve excitability in response to hyperventilation and ischaemia, but in vivo studies cannot directly differentiate between the impact of pH and other secondary factors. In this investigation, we applied an excitability testing protocol to a rat saphenous skin nerve in vitro preparation. Changes in extracellular pH were induced by altering pCO(2) in the perfusate, and excitability properties of large myelinated fibres were measured in the pH range from 6.9 to 8.1. The main effect of protons on nerve excitability was a near linear increase in threshold which was accompanied by a decrease in strength-duration time constant reflecting mainly a decrease in persistent sodium current. In the recovery cycle, late subexcitability following 7 conditioning stimuli was substantially reduced at acid pH, indicating a block of slow but not of fast potassium channels. Changes in threshold electrotonus were complex, reflecting the combined effects of pH on multiple channel types. These results provide the first systematic data on pH sensitivity of mammalian nerve excitability properties, and may help in the interpretation of abnormal clinical excitability measurements.

Examining the effects of age, sex, and body mass index on normative median motor nerve excitability measurements

Objectives The purpose of this study was to build a large reference database of excitability measures in normal subjects and to examine the effects of age, sex, and BMI. Methods One hundred and five healthy subjects had median motor nerve excitability testing performed at the wrist using the automated threshold-tracking program, QTRAC. Statistical linear regression was used to explore relationships between nerve excitability and the independent variables. Results The main effect of age is a reduced superexcitability. Lesser effects are flattening of the normalized stimulus response curve and reduction in threshold change following strong hyperpolarizing currents. Females have lower thresholds than males and small but significant differences in voltage-gated potassium channel (KCNQ) mediated properties (late subexcitability, accommodation half time, and threshold undershoot following depolarizing electrotonus), as well as a small increase in superexcitability. BMI has no influence on nerve excitability data and does not explain sex-related differences in threshold. Conclusions Age and sex have few and small effects on excitability parameters. Significance The expression of nodal KCNQ channels appears to be greater in females. Age-related increases in subexcitability may be attributable to changes in the muscle fibre and not the nerve.

Regional differences in ulnar nerve excitability may predispose to the development of entrapment neuropathy

Objective To assess whether there are differences in nerve excitability properties between proximal and distal stimulation sites in the ulnar nerve in healthy controls, which may provide information on whether alteration in ion channel function predisposes to the development of ulnar neuropathy at the elbow. Methods Nerve excitability studies were undertaken in 11 healthy controls. Studies were undertaken with stimulation of the ulnar nerve at the elbow and wrist. Recordings were obtained from abductor digiti minimi in both sets of studies. Results Recordings obtained following stimulation of the nerve at the elbow demonstrated significant differences to those obtained following stimulation of the nerve at the wrist. Specifically, there was a left shift in stimulus–response curves at the elbow compared to the wrist, with prolonged strength-duration time constant, and reduced rheobase ( P < 0.05). These changes were accompanied by increased refractoriness and reductions in superexcitability and late subexcitability ( P < 0.05). Conclusions The present findings may suggest relative depolarization of ulnar nerve axons at the elbow. Significance These changes may reflect regional differences in axonal Na +/K + pump function and thereby predispose the ulnar nerve to conduction failure and axonal degeneration when exposed to trauma.

Utility of recovery cycle with two conditioning pulses for detection of impaired axonal slow potassium current in ALS

Objective Slow potassium current ( I Ks) is important in controlling nerve excitability and its impairment is known in various neurological diseases, including amyotrophic lateral sclerosis (ALS). I Ks gives rise to the late subexcitability phase of the recovery cycle, which can be amplified by the use of multiple conditioning pulses. The clinical utility of this technique has not previously been explored. Methods Nerve excitability tests, including recovery cycles with single and double conditioning pulses 4 ms apart (RC and RC2, respectively) were performed in patients with ALS and control subjects. Late subexcitability values obtained by RC and RC2 were compared in both groups. Results RC2 was well tolerated in all the subjects. The threshold changes in late subexcitability by RC2 were greater than those by RC in both groups (mean (%): RC, 16.0/13.3; RC2, 34.9/29.4 (Control/ALS)). The ALS group showed lower threshold changes than controls by both methods. Statistical analysis between the ALS and control groups provided smaller P value by RC2 ( P = 0.018) than by RC ( P = 0.046). Also, RC2 provided non-significant, but slightly more distinguishing non-parametric rank analysis and greater Area Under the Curve (AUC) by Receiver Operating Characteristic (ROC). RC2 produced more identifiable single peak for late subexcitability than RC in an ALS patient whose late subexcitability was decreased. Conclusions Two conditioning stimuli provide greater threshold change for late subexcitability and possibly clearer identification of a peak threshold change than conventional recovery cycle. The findings obtained by this new protocol reinforce the previously reported impairment of I Ks in ALS. Significance Amplification of I Ks by double conditioning pulses is applicable in humans and may help elucidating its clinical significance in pathophysiology in neurological diseases.

Effects of experimental focal compression on excitability of human median motor axons

To characterize the effect of focal compression by threshold tracking and other excitability measures of human median motor axons. We conducted a sequence of excitability studies using a software written in BASIC (QTRAC version 4.0, (c)Institute of Neurology, London, UK, with multiple excitability protocol TRONDXM 2) in 24 healthy subjects, stimulating the median nerve at the wrist and recording compound muscle action potentials from the abductor pollicis brevis. Constant, localized compression was applied at the wrist by mechanically lowering a probe attached to a disk electrode, which also served as the stimulating cathode. Compared with the pre-compression values, measurements during compression showed a shift of threshold electrotonus waveforms toward the baseline (fanning-in), steeper current-threshold relationships, increased strength-duration time constants, prolonged relative refractory periods and reduced levels of superexcitability, but no alteration in late subexcitability. These excitability changes indicating depolarization reversed to hyperpolarization immediately after release of compression. The nerve compression altered none of the excitability measures when recorded 2 cm distally from the pressure probe. Mild nerve compression produces a very localized axonal depolarization at the compression site followed by hyperpolarization upon release of compression, as expected from focal ischemia. The current results imply that the sharply-localized conduction abnormalities demonstrated electrophysiologically in peripheral nerve entrapment syndromes and compression myelopathies may, in part, result from compression-induced focal nerve ischemia.

Changes in human sensory axonal excitability induced by an ischaemic insult

Objective To identify the sensitivity and the patterns of change in sensory excitability that accompany an ischaemic insult. Methods Sensory excitability studies were undertaken in 10 subjects (mean age 36), and monitored throughout ischaemia and following its release. Ischaemia was induced using a sphygmomanometer inflated to 200 mm/Hg above the elbow. Results During ischaemia there was reduction in threshold ( P < 0.001), associated with a significant increase in refractoriness (106 ± 6.62%; P < 0.001), reduction in superexcitability (30.4 ± 0.42%; P < 0.001), and ‘fanning in’ of threshold electrotonus, all indicative of axonal depolarization. Paraesthesiae were minimal during ischaemia, but became severe on release, at which stage numbness was prominent. Late subexcitability in sensory axons was completely abolished by a relatively shorter period of ischaemia than previously observed in motor axons. Conclusions The present study has successfully developed a template for changes in sensory axonal excitability parameters that accompany ischaemia, and established their relative sensitivity to an ischaemic change. Further, it is proposed that the inhibition of the Na +/K + pump, in the setting of increased persistent Na + currents and abolition of late subexcitability may underlie the development of paraesthesiae during ischaemia. Significance Changes in axonal excitability induced by ischaemia may serve as a tool to identify and interpret changes in axonal membrane potential recorded in neuropathic patients.

Retigabine reduces the excitability of unmyelinated peripheral human axons

Enhancement of membrane K + conductance may reduce the abnormal excitability of primary afferent nociceptive neurons in neuropathic pain. It has been shown that retigabine, a novel anticonvulsant, activates Kv7 (KCNQ/M) channels in the axonal/nodal membrane of peripheral myelinated axons. In this study, we have tested the effects of retigabine on excitability parameters of C-type nerve fibers in isolated fascicles of human sural nerve. Application of retigabine (3–10 μM) produced an increase in membrane threshold. This effect was pronounced in depolarized axons and small in hyperpolarized axons. This finding indicates that retigabine produces a membrane hyperpolarization which is limited by the K + equilibrium potential. The retigabine-induced reduction in excitability was accompanied by modifications of the post-spike recovery cycle. Most notable is the development of a late subexcitability at 250–400 ms following a short burst of action potentials. All effects of retigabine were blocked in the presence of XE991 (10 μM). The data show that Kv7 channels are present on axons of unmyelinated, including nociceptive, peripheral human nerve fibers. It is likely that activation of these channels by retigabine may reduce the ectopic generation of action potentials in neuropathic pain.

A rat in vitro model for the measurement of multiple excitability properties of cutaneous axons

Objective To establish an in vitro model for measurement of the excitability properties of cutaneous sensory axons. Methods We used a saphenous skin–nerve preparation from adult rat in combination with computerized threshold tracking. We measured strength–duration time constant, the recovery of excitability after a supramaximal stimulus and the accommodation to conditioning subthreshold polarizing stimuli (threshold electrotonus, current–threshold relationship) and compared these with previously published recordings from sensory axons in human median nerve. Results Threshold electrotonus and the amplitude of superexcitability were indistinguishable between human median nerve in vivo and rat saphenous nerve in vitro, but several excitability parameters were significantly different in the rat: strength–duration time constant was significantly shorter (0.19 ± 0.01 vs. 0.53 ± 0.02 ms); the refractory period was shorter (1.9 ± 1.1 ms vs. 3.5 ± 1.0 ms) and late subexcitability was smaller (6.3 ± 0.3% vs. 11.3 ± 0.5%); thirdly, during recording of current–threshold relationship, rat nerves displayed more inward rectification to strong hyperpolarizing currents. Parameters were stable over more than 3 h. Conclusions Excitability changes of sensory Aβ-fibres can be reliably studied in the rat in vitro and are qualitatively similar to humans. Significance This rat model will facilitate pharmacological studies of nerve excitability and work on models of neuropathy.

Nerve excitability changes in critical illness polyneuropathy

Patients in intensive care units frequently suffer muscle weakness and atrophy due to critical illness polyneuropathy (CIP), an axonal neuropathy associated with systemic inflammatory response syndrome and multiple organ failure. CIP is a frequent and serious complication of intensive care that delays weaning from mechanical ventilation and increases mortality. The pathogenesis of CIP is not well understood and no specific therapy is available. The aim of this project was to use nerve excitability testing to investigate the changes in axonal membrane properties occurring in CIP. Ten patients (aged 37-76 years; 7 males, 3 females) were studied with electrophysiologically proven CIP. The median nerve was stimulated at the wrist and compound action potentials were recorded from abductor pollicis brevis muscle. Strength-duration time constant, threshold electrotonus, current-threshold relationship and recovery cycle (refractoriness, superexcitability and late subexcitability) were recorded using a recently described protocol. In eight patients a follow-up investigation was performed. All patients underwent clinical examination and laboratory investigations. Compared with age-matched normal controls (20 subjects; aged 38-79 years; 7 males, 13 females), CIP patients exhibited reduced superexcitability at 7 ms, from -22.3 +/- 1.6% to -7.6 +/- 3.1% (mean +/- SE, P approximately 0.0001) and increased accommodation to depolarizing (P < 0.01) and hyperpolarizing currents (P < 0.01), indicating membrane depolarization. Superexcitability was reduced both in patients with renal failure and without renal failure. In the former, superexcitability correlated with serum potassium (R = 0.88), and late subexcitability was also reduced (as also occurs owing to hyperkalaemia in patients with chronic renal failure). In patients without renal failure, late subexcitability was normal, and the signs of membrane depolarization correlated with raised serum bicarbonate and base excess, indicating compensated respiratory acidosis. It is inferred that motor axons in these CIP patients are depolarized, in part because of raised extracellular potassium, and in part because of hypoperfusion. The chronic membrane depolarization may contribute to the development of neuropathy.

KCNQ channels mediate IKs, a slow K+ current regulating excitability in the rat node of Ranvier.

Mutations that reduce the function of KCNQ2 channels cause neuronal hyperexcitability, manifested as epileptic seizures and myokymia. These channels are present in nodes of Ranvier in rat brain and nerve and have been proposed to mediate the slow nodal potassium current I(Ks). We have used immunocytochemistry, electrophysiology and pharmacology to test this hypothesis and to determine the contribution of KCNQ channels to nerve excitability in the rat. When myelinated nerve fibres of the sciatic nerve were examined by immunofluorescence microscopy using antibodies against KCNQ2 and KCNQ3, all nodes showed strong immunoreactivity for KCNQ2. The nodes of about half the small and intermediate sized fibres showed labelling for both KCNQ2 and KCNQ3, but nodes of large fibres were labelled by KCNQ2 antibodies only. In voltage-clamp experiments using large myelinated fibres, the selective KCNQ channel blockers XE991 (IC50 = 2.2 microm) and linopirdine (IC50 = 5.5 microm) completely inhibited I(Ks), as did TEA (IC50 = 0.22 mm). The KCNQ channel opener retigabine (10 microm) shifted the activation curve to more negative membrane potentials by -24 mV, thereby increasing I(Ks). In isotonic KCl 50% of I(Ks) was activated at -62 mV. The activation curve shifted to more positive potentials as [K+]o was reduced, so that the pharmacological and biophysical properties of I(Ks) were consistent with those of heterologously expressed homomeric KCNQ2 channels. The ability of XE991 to selectively block I(Ks) was further exploited to study I(Ks) function in vivo. In anaesthetized rats, the excitability of tail motor axons was indicated by the stimulus current required to elicit a 40% of maximal compound muscle action potential. XE991 (2.5 mg kg(-1) i.p.) eliminated all nerve excitability functions previously attributed to I(Ks): accommodation to 100 ms subthreshold depolarizing currents, the post-depolarization undershoot in excitability, and the late subexcitability after a single impulse or short trains of impulses. Due to reduced spike-frequency adaptation after XE991 treatment, 100 ms suprathreshold current injections generated long trains of action potentials. We conclude that the nodal I(Ks) current is mediated by KCNQ channels, which in large fibres of rat sciatic nerve appear to be KCNQ2 homomers.

Evidence for motor axon depolarization in Fabry disease

To investigate the pathophysiology of neuropathy in Fabry disease, multiple nerve excitability properties of median motor axons in 20 patients with this disorder but without hyperkalemia were compared with 35 age-matched normal controls. In the patients, depolarizing threshold electrotonus was reduced (P < 0.0001) and superexcitability was reduced (P < 0.001), but late subexcitability was normal. These findings indicate that the axons were mildly depolarized, probably due to ischemia, and are consistent with the hypothesis that poor nerve perfusion in Fabry disease contributes to axonal damage.