Hugh Bostock

Threshold tracking techniques in the study of human peripheral nerve.

Conventional electrophysiological tests of nerve function focus on the number of conducting fibers and their conduction velocity. These tests are sensitive to the integrity of the myelin sheath, but provide little information about the axonal membrane. Threshold tracking techniques, in contrast, test nerve excitability, which depends on the membrane properties of the axons at the site of stimulation. These methods are sensitive to membrane potential, and to changes in membrane potential caused by activation of ion channels and electrogenic ion pumps, including those under the myelin sheath. This review describes the range of threshold tracking techniques that have been developed for the study of human nerves in vivo: resting threshold is compared with the threshold altered by a change in environment (e.g., ischemia), by a preceding single impulse (e.g., refractoriness, superexcitability) or impulse train, or by a subthreshold current (e.g., threshold electrotonus). Few clinical studies have been reported so far, mainly in diabetic neuropathy and motor neuron disease. Threshold measurements seem well suited for studies of metabolic and toxic neuropathies but insensitive to demyelination. Until suitable equipment becomes more widely available, their full potential is unlikely to be realized.

Multiple measures of axonal excitability: a new approach in clinical testing.

From measurements of nerve excitability and the changes in excitability produced by nerve impulses and conditioning currents, it is possible to infer information about the membrane potential and biophysical properties of peripheral axons. Such information cannot be obtained from conventional nerve conduction studies. This article describes a new method that enables several such measurements to be made on a motor nerve quickly and reproducibly, with minimal operator intervention. The protocol measures stimulus–response behavior using two stimulus durations (from which the distribution of strength–duration time constants can be estimated), threshold electrotonus to 100‐ms polarizing currents, a current–threshold relationship (indicating inward and outward rectification), and the recovery of excitability following supramaximal activation. The method was tested on 30 healthy volunteers, stimulating the median nerve at the wrist and recording from the abductor pollicis brevis. The results were comparable with previously published normal data, but the recordings took less than 10 min. The convenience and brevity of the new method make it appropriate for routine clinical use.

Action potentials and membrane currents in the human node of Ranvier

Action potentials and membrane currents were recorded in single human myelinated nerve fibres under current- and voltage-clamp conditions at room temperature. Nerve material was obtained from patients undergoing nerve graft operations. Successful recordings were made in 11 nerve fibres. In Ringers solution, large transient Na currents were recorded, which could be blocked completely with tetrodotoxin. Partial block of these currents with 3 nM tetrodotoxin was used to reduce the voltage-clamp error due to series resistance. Outward K currents were very small in intact nerve fibres, but had a large amplitude in fibres showing signs of paranodal demyelination. In isotonic KCl, the K current could be separated into three components: two fast components (Kf1 and Kf2) and one slow component (Ks). Time constants and steady-state activation and inactivation of Na permeability and of fast and slow K conductance were measured within the potential range of −145 mV to +115 mV. From these parameters, the corresponding rate constants were calculated and a mathematical model based on the Frankenhaeuser-Huxley equations was derived. Calculated action potentials closely matched those recorded. Single calculated action potentials were little affected by removing the fast or slow K conductance, but the slow K conductance was required to limit the repetitive response of the model to prolonged stimulating currents.

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 IKs. 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 μm) and linopirdine (IC50= 5.5 μm) completely inhibited IKs, as did TEA (IC50= 0.22 mm). The KCNQ channel opener retigabine (10 μm) shifted the activation curve to more negative membrane potentials by −24 mV, thereby increasing IKs. In isotonic KCl 50% of IKs 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 IKs were consistent with those of heterologously expressed homomeric KCNQ2 channels. The ability of XE991 to selectively block IKs was further exploited to study IKs 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−1i.p.) eliminated all nerve excitability functions previously attributed to IKs: 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 IKs current is mediated by KCNQ channels, which in large fibres of rat sciatic nerve appear to be KCNQ2 homomers.

Changes in excitability of human motor axons underlying post-ischaemic fasciculations: evidence for two stable states.

1. We have investigated the origin of post‐ischaemic ectopic discharges in human nerve by recording changes in electrical excitability following periods of ischaemia (15‐20 min) sufficient to induce spontaneous motor fasciculations. The ulnar nerve was stimulated beneath a pressure cuff on the upper arm, and compound motor action potentials recorded from abductor digiti minimi. 2. On releasing the cuff after 15 min of ischaemia, thresholds to short current pulses increased in two distinct phases: a slow phase followed by a rapid rise to a peak threshold. The rapid rise was too fast to track (i.e. 100% threshold increase in less than 4 s), and was sometimes followed after 30‐40 s by an equally rapid fall. Small polarizing currents affected the timing of the rapid threshold increase, as if it was occurring at a particular membrane potential. 3. By recording complete stimulus‐response curves every few seconds, we found that the rapid threshold changes were associated with a bimodal distribution of thresholds. Most fibres were found in either a high‐threshold or low‐threshold state, and these two states converged over a period of about 10 min. 4. Spontaneous motor fasciculations were only recorded after the rapid rise in threshold and when the fibres existed in two threshold states. The spontaneous activity was not responsible for inducing the two states, since they could also be recorded in its absence. 5. A computer model of a human motor axon node and internode was constructed, incorporating channel types demonstrated in other axons, and channel densities adjusted to match the responses of human axons to depolarizing and hyperpolarizing current pulses. An increase in extracellular potassium concentration produced a region of negative slope conductance in the current‐voltage relationship of the model, and the appearance of two stable states with enhanced activity of the electrogenic sodium pump. 6. Transitions between the two stable states of the model could account qualitatively for the rapid threshold changes recorded from post‐ischaemic axons. In the model, spontaneous action potentials occurred following some transitions from the high potential state to the low potential state. We suggest that post‐ischaemic motor fasciculations in man also involve transitions between two equilibrium states, occurring in axons with high extracellular potassium and high electrogenic pump activity.

Slowly conducting afferents activated by innocuous low temperature in human skin

1 Microneurography was used to search for primary afferents responsive to innocuous low temperature in human nerves supplying the hairy skin of the hand or foot. Eighteen units were identified as cold‐specific units: they displayed a steady‐state discharge at skin temperatures in the range 28‐30 °C, they were sensitive to small changes in temperature, and they responded vigorously when a cool metal probe touched their receptive fields (RFs). They were insensitive to mechanical stimuli and sympathetic activation. Their RFs comprised one, or at most two, spots less than 5 mm in diameter. 2 Nine units were characterised in detail by a series of 10 s cooling and warming pulses from a holding temperature of 35 °C. The threshold temperature for activation by cooling was 29.4 ± 2.0 °C (mean ±s.d.). Adaptation of the responses to supra‐threshold cooling pulses was partial: mean peak and plateau firing rates were maximal on steps to 15 °C (35.9 and 19.9 impulses s−1, respectively). Three of these units also displayed a paradoxical response to warming, with a mean threshold of 42.3 °C. 3 Sixteen of the eighteen cold‐specific units were also studied by electrical stimulation of their RFs. They conducted in the velocity range 0.8‐3.0 m s−1. When stimulated at 2 Hz, their latency increased according to a characteristic time course, reaching a plateau within 3 min (mean slowing (±s.d.) 5.2 ± 1.1 %) and recovering quickly (50 % recovery in 17.8 ± 4.5 s). 4 To reconcile these findings with previous studies of reaction times and the effects of nerve compression on sensation, it is concluded that either human cold‐specific afferent fibres are incompletely myelinated ‘BC’ fibres, or else there are C as well as Aδ cold fibres, with the C fibre group contributing little to sensation.

Acute tetrodotoxin-induced neurotoxicity after ingestion of puffer fish

This study documents the effects of puffer‐fish poisoning on peripheral nerve. Excitability measurements investigated membrane properties of sensory and motor axons in four patients. The median nerve was stimulated at the wrist, with compound muscle potentials recorded from abductor pollicis brevis and compound sensory potentials from digit 2. Stimulus–responses, strength–duration time constant (τSD), threshold electrotonus, and current–threshold relations were recorded. The urine of each patient tested positive for tetrodotoxin. Compared with controls, axons were of higher threshold, compound muscle action potentials and compound sensory nerve action potentials were reduced in amplitude, latency was prolonged, and τSD was reduced. In recovery cycles, refractoriness, superexcitability, and late subexcitability were decreased. Threshold electrotonus of motor axons exhibited distinctive abnormalities with less threshold decline than normal on depolarization and greater threshold increase on hyperpolarization (p < 0.0005 for each patient). The changes in excitability were reproduced in a mathematical model by reducing sodium (Na+) permeabilities by a factor of two. This study confirms that the neurotoxic effects of puffer‐fish poisoning can be explained by tetrodotoxin blockade of Na+ channels. It demonstrates the ability of noninvasive nerve excitability studies to detect Na+ channel blockade in vivo and also the utility of mathematical modeling to aid interpretation of altered excitability properties in disease. Ann Neurol 2005;57:339–348

Clinical evaluation of excitability measures in sensory nerve.

A recently described method for recording multiple excitability parameters of human motor nerves has been adapted to the study of sensory nerves. The protocol measures stimulus–response behavior using two stimulus durations (from which the distribution of strength–duration time constants is estimated), threshold electrotonus to 100 ms polarizing currents, a current–threshold relationship (indicating inward and outward rectification), and the recovery of excitability following supramaximal activation. The method was tested on 50 healthy volunteers, stimulating the median nerve at the wrist and recording the antidromic compound sensory nerve action potential (SNAP) from digit 2. The excitability measurements were similar, where comparisons were possible, with published sensory nerve data, and confirmed differences from motor nerves, particularly in strength–duration behavior and recovery cycle, likely to reflect functional differences between sensory and motor nerves. Although slower than for motor nerves, the sensory nerve recordings were sufficiently quick (16 to 18 min) to allow them to be included in routine clinical studies. We propose that this method, which provides quite different and complementary information about nerve function to conventional conduction studies, provides a useful new approach for exploring the pathophysiology of sensory neuropathies.

Differences in membrane properties of axonal and demyelinating Guillain-Barré syndromes.

Guillain‐Barré syndrome is classified into acute motor axonal neuropathy (AMAN) and acute inflammatory demyelinating polyneuropathy (AIDP) by electrodiagnostic and pathological criteria. In AMAN, the immune attack appears directed against the axolemma and nodes of Ranvier. Threshold tracking was used to measure indices of axonal excitability (refractoriness, supernormality, and threshold electrotonus) for median nerve axons at the wrist of patients with AMAN (n = 10) and AIDP (n = 8). Refractoriness (the increase in threshold current during the relative refractory period) was greatly increased in AMAN patients, but the abruptness of the threshold increases at short interstimulus intervals indicated conduction failure distal to the stimulation (ie, an increased refractory period of transmission). During the 4 week period from onset, the high refractoriness returned toward normal, and the amplitude of the compound muscle action potential increased, consistent with improvement in the safety margin for impulse transmission in the distal nerve. In contrast, refractoriness was normal in AIDP, even though there was marked prolongation of distal latencies. Supernormality and threshold electrotonus were normal in both groups of patients, suggesting that, at the wrist, membrane potential was normal and pathology was relatively minor. These results support the view that the predominantly distal targets of immune attack are different for AMAN and AIDP. Possible mechanisms for the reduced safety factor in AMAN are discussed.

Hyperexcitable C nociceptors in fibromyalgia

To test the hypothesis that peripheral C nociceptor function may be abnormal in fibromyalgia and that C nociceptor dysfunction may contribute to the symptoms reported by these patients.