Excitability and the safety margin in human axons during hyperthermia.

Abstract Hyperthermia challenges the nervous system's ability to transmit action potentials faithfully. Neuromuscular diseases, particularly those involving demyelination have an impaired safety margin for action potential generation and propagation, and symptoms are commonly accentuated by increases in temperature. The aim of this study was to examine the mechanisms responsible for reduced excitability during hyperthermia. Additionally, we sought to determine if motor and sensory axons differ in their propensity for conduction block during hyperthermia. Recordings of axonal excitability were performed at normal temperatures and during focal hyperthermia for motor and sensory axons in six healthy subjects. There were clear changes in excitability during hyperthermia, with reduced superexcitability following an action potential, faster accommodation to long-lasting depolarization and reduced accommodation to hyperpolarization. A verified model of human motor and sensory axons was used to clarify the effects of hyperthermia. The hyperthermia-induced changes in excitability could be accounted for by increasing the modelled temperature by 6°C (and adjusting the maximum conductances and activation kinetics according to their Q10 values; producing a 2 mV hyperpolarization of resting membrane potential), further hyperpolarizing the voltage dependence of Ih (motor, 11 mV; sensory, 7 mV) and adding a small depolarizing current at the internode (motor, 20 pA; sensory, 30 pA). The modelling suggested that slow K(+) channels play a significant role in reducing axonal excitability during hyperthermia. The further hyperpolarization of the activation of Ih would limit its ability to counter the hyperpolarization produced by activity, thereby allowing conduction block to occur during hyperthermia.

The voltage dependence of I(h) in human myelinated axons.

HCN channels are responsible for I(h), a voltage-gated inwardly rectifying current activated by hyperpolarization. This current appears to be more active in human sensory axons than motor and may play a role in the determination of threshold. Differences in I(h) are likely to be responsible for the high variability in accommodation to hyperpolarization seen in different subjects. The aim of this study was to characterise this current in human axons, both motor and sensory. Recordings of multiple axonal excitability properties were performed in 10 subjects, with a focus on the changes in threshold evoked by longer and stronger hyperpolarizing currents than normally studied. The findings confirm that accommodation to hyperpolarization is greater in sensory than motor axons in all subjects, but the variability between subjects was greater than the modality difference. An existing model of motor axons was modified to take into account the behaviour seen with longer and stronger hyperpolarization, and a mathematical model of human sensory axons was developed based on the data collected. The differences in behaviour of sensory and motor axons and the differences between different subjects are best explained by modulation of the voltage dependence, along with a modest increase of expression of the underlying conductance of I(h). Accommodation to hyperpolarization for the mean sensory data is fitted well with a value of -94.2 mV for the mid-point of activation (V(0.5)) of I(h) as compared to -107.3 mV for the mean motor data. The variation in response to hyperpolarization between subjects is accounted for by varying this parameter for each modality (sensory: -89.2 to -104.2 mV; motor -87.3 to -127.3 mV). These voltage differences are within the range that has been described for physiological modulation of I(h) function. The presence of slowly activated I(h) isoforms on both motor and sensory axons was suggested by modelling a large internodal leak current and a masking of the Na(+)/K(+)-ATPase pump activity by a tonic depolarization. In addition to an increased activation of I(h), the modelling suggests that in sensory axons the nodal slow K(+) conductance is reduced, with consequent depolarization of resting membrane potential, and action potential of shorter duration.

Properties of low-threshold motor axons in the human median nerve.

This study investigated the excitability and accommodative properties of low-threshold human motor axons to test whether these motor axons have greater expression of the persistent Na(+) conductance, I(NaP). Computer-controlled threshold tracking was used to study 22 single motor units and the data were compared with compound motor potentials of various amplitudes recorded in the same experimental session. Detailed comparisons were made between the single units and compound potentials that were 40% or 5% of maximal amplitude, the former because this is the compound potential size used in most threshold tracking studies of axonal excitability, the latter because this is the compound potential most likely to be composed entirely of motor axons with low thresholds to electrical recruitment. Measurements were made of the strength-duration relationship, threshold electrotonus, current-voltage relationship, recovery cycle and latent addition. The findings did not support a difference in I(NaP). Instead they pointed to greater activity of the hyperpolarization-activated inwardly rectifying current (I(h)) as the basis for low threshold to electrical recruitment in human motor axons. Computer modelling confirmed this finding, with a doubling of the hyperpolarization-activated conductance proving the best single parameter adjustment to fit the experimental data. We suggest that the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel(s) expressed on human motor axons may be active at rest and contribute to resting membrane potential.

Threshold behaviour of human axons explored using subthreshold perturbations to membrane potential.

The present study explores the threshold behaviour of human axons and the mechanisms contributing to this behaviour. The changes in excitability of cutaneous afferents in the median nerve at the wrist were recorded to a long-lasting subthreshold conditioning stimulus, with a waveform designed to maximize the contribution of currents active in the just-subthreshold region. The conditioning stimulus produced a decrease in threshold that developed over 3-5 ms following the end of the depolarization and then decayed slowly, in a pattern similar to the recovery of axonal excitability following a discharge. To ensure that the conditioning stimulus did not activate low-threshold axons, similar recordings were then made from single motor axons in the ulnar nerve at the elbow. The findings were comparable, and behaviour with the same pattern and time course could be reproduced by subthreshold stimuli in a model of the human axon. In motor axons, subthreshold depolarizing stimuli, 1 ms long, produced a similar increase in excitability, but the late hyperpolarizing deflection was less prominent. This behaviour was again reproduced by the model axon and could be explained by the passive properties of the nodal membrane and conventional Na+ and K+ currents. The modelling studies emphasized the importance of leak current through the Barrett-Barrett resistance, even in the subthreshold region, and suggested a significant contribution of K+ currents to the threshold behaviour of axons. While the gating of slow K+ channels is slow, the resultant current may not be slow if there are substantial changes in membrane potential. By extrapolation, we suggest that, when human axons discharge, nodal slow K+ currents will be activated sufficiently early to contribute to the early changes in excitability following the action potential.

Inflections in threshold electrotonus to depolarizing currents in sensory axons.

Threshold electrotonus involves tracking the changes in axonal excitability produced by subthreshold polarizing currents and is the only technique that allows insight into the function of internodal conductances in human subjects in vivo. There is often an abrupt transient reversal of the threshold change as excitability increases in response to conditioning depolarizing currents (S1 phase). In recordings from motor axons, it has been recently demonstrated that this notch or inflection is due to activation of low-threshold axons. We report that a notch is frequently seen in sensory recordings (in 33 of 50 healthy subjects) using the standard threshold electrotonus protocol. When large, the notch can distort subsequent phases of threshold electrotonus and could complicate quantitative measurements and modeling studies.

Outwardly rectifying deflections in threshold electrotonus due to K+ conductances.

A transient decrease in excitability occurs regularly during the S1 phase of threshold electrotonus to depolarizing conditioning stimuli for sensory and, less frequently, motor axons. This has been attributed to the outwardly rectifying action of fast K(+) channels, at least in patients with demyelinating diseases. This study investigates the genesis of this notch in healthy axons. Threshold electrotonus was recorded for sensory and motor axons in the median nerve at the wrist in response to test stimuli of different width. The notch occurred more frequently the briefer the test stimulus, and more frequently in sensory studies. In studies on motor axons, the notch decreased in latency and increased in amplitude as the conditioning stimulus increased or the limb was cooled. Low-threshold axons displayed profound changes in strength-duration time constant even though the threshold electrotonus curves contained no detectable notch. When a 1.0 ms current was added to subthreshold conditioning stimuli to trigger EMG, the notch varied with the timing and intensity of the brief current pulse. This study finds no evidence for an outwardly rectifying deflection due to K(+) channels, other than the slow accommodation attributable to slow K(+) currents. In normal motor axons, a depolarization-induced notch during the S1 phase of threshold electrotonus is the result of the conditioning stimulus exceeding threshold for some axons. The notch is more apparent in sensory axons probably because of the lower slope of the stimulus-response curve and their longer strength-duration time constant rather than a difference in K(+) conductances. This may also explain the notch in demyelinating diseases.

Augmentation of the contraction force of human thenar muscles by and during brief discharge trains.

We investigated the influence of the history of activity on the contractile properties of abductor pollicis brevis (APB) to define how the forces produced by individual stimuli change within a stimulus train, with a view to clarifying the optimal discharge frequency for force production in brief trains. Supramaximal electrical stimuli were delivered to the median nerve at the wrist singly or in trains of 2-5 at various interstimulus intervals (ISIs). The force and electromyographic (EMG) responses to trains of n stimuli were defined by online subtraction of the responses to n - 1 stimuli. The force attributable to the nth stimulus was normalized to that produced by a single stimulus. The contraction force produced by 2 stimuli exceeded the force expected with linear summation of 2 single twitches by 30-40% at ISIs of 2-100 ms. Increasing the number of stimuli resulted in less augmentation of the force produced by the last stimulus in the train for ISIs up to 20 ms, but greater augmentation for ISIs of 50-100 ms. At ISIs of less than 10 ms, the time to peak force produced by the last stimulus in a 5-pulse train was delayed by approximately 100 ms, the peak force produced by that stimulus was less than that produced by a single stimulus, and it occurred on the falling phase of the overall contraction. These properties are best explained by the catchlike property of muscle. This implies that the augmentation of contraction force due to this property can increase throughout a stimulus train, and is not restricted to the doublet discharges that have conventionally been studied. We conclude that, with brief discharge trains, maximal forces occur at ISIs of 56-75 ms, intervals that are longer than those conventionally associated with the catchlike property. Discharge rates of 15-20 HZ appear to be optimal for force generation by APB during steady contractions.

Assessment of cortical excitability using threshold tracking techniques.

Conventional paired-pulse transcranial magnetic stimulation (TMS) techniques of assessing cortical excitability are limited by fluctuations in the motor evoked potential (MEP) amplitude. The aim of the present study was to determine the feasibility of threshold tracking TMS for assessing cortical excitability in a clinical setting and to establish normative data. Studies were undertaken in 26 healthy controls, tracking the MEP response from abductor pollicis brevis. Short-interval intracortical inhibition (SICI) occurred up to an interstimulus interval (ISI) of 7-10 ms, with two distinct peaks evident, at ISIs of < or =1 and 3 ms, followed by intracortical facilitation to an ISI of 30 ms. Long-interval intracortical inhibition (LICI) occurred at ISIs of 50-300 ms, peaking at 150 ms. The present study has confirmed the effectiveness of the threshold tracking TMS technique in reliably and reproducibly measuring cortical excitability. Simultaneous assessment of upper and lower motor neuronal function with threshold tracking techniques may help to determine the site of disease onset and patterns of progression in neurodegenerative diseases.

After-effects of near-threshold stimulation in single human motor axons.

Subthreshold electrical stimuli can generate a long-lasting increase in axonal excitability, superficially resembling the phase of superexcitability that follows a conditioning nerve impulse. This phenomenon of 'subthreshold superexcitability' has been investigated in single motor axons in six healthy human subjects, by tracking the excitability changes produced by conditioning stimuli of different amplitudes and waveforms. Near-threshold 1 ms stimuli caused a mean decrease in threshold at 5 ms of 22.1 +/- 6.0% (mean +/-s.d.) if excitation occurred, or 6.9 +/- 2.6% if excitation did not occur. The subthreshold superexcitability was maximal at an interval of about 5 ms, and fell to zero at 30 ms. It appeared to be made up of two components: a passive component linearly related to conditioning stimulus amplitude, and a non-linear active component. The active component appeared when conditioning stimuli exceeded 60% of threshold, and accounted for a maximal threshold decrease of 2.6 +/- 1.3%. The passive component was directly proportional to stimulus charge, when conditioning stimulus duration was varied between 0.2 and 2 ms, and could be eliminated by using triphasic stimuli with zero net charge. This change in stimulus waveform had little effect on the active component of subthreshold superexcitability or on the 'suprathreshold superexcitability' that followed excitation. It is concluded that subthreshold superexcitability in human motor axons is mainly due to the passive electrotonic effects of the stimulating current, but this is supplemented by an active component (about 12% of suprathreshold superexcitability), due to a local response of voltage-dependent sodium channels.

Axonal excitability measured by tracking twitch contraction force.

The present study addressed whether the excitability of motor axons could be documented by tracking a target submaximal contraction force rather than a target submaximal compound muscle action potential (CMAP). In 10 subjects, multiple excitability measures were recorded using the Trond protocol, tracking twitch contraction force and the CMAP in response to stimulation of the median nerve at the wrist and twitch force to stimulation at the motor point. With stimulation at the wrist, the findings were virtually identical with force tracking and CMAP tracking for indices dependent on unconditioned thresholds (stimulus-response curves; strength-duration properties) and when the conditioning stimulus was subthreshold (threshold electrotonus; current-threshold relationship). However, when the conditioning stimulus was supramaximal, as in recovery cycle studies, thresholds for the target force were lower in all subjects than for the target CMAP. There was variability between different subjects in the extent of this offset. However, force tracking can still be used to follow changes in refractoriness and supernormality when membrane potential changes during an experiment. The excitability indices differed with motor point stimulation, but it is argued that this could be due to the geographic dispersion of motor axons at the motor point in addition to or instead of differences in biophysical properties of the stimulated nodes. Thus, tracking twitch contraction force is a potentially valuable alternative to tracking the CMAP, but is more complicated and the results need to be interpreted with caution.