Brain, Vol. 123, No. 12, 2542-2551,
December 2000
© 2000 Oxford University Press
Effects of membrane polarization and ischaemia on the excitability properties of human motor axons
Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London WC1N 3BG, UK
Correspondence to:
Professor H. Bostock, Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London WC1N 3BG, UK E-mail: H.Bostock{at}ion.ucl.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Multiple nerve excitability measurements have been proposed for clinical testing of nerve function, since excitability measures can provide evidence of altered axonal membrane properties and are complementary to conventional nerve conduction studies. An important determinant of excitability is membrane potential, and this study was undertaken to determine the changes in a range of excitability properties associated with alterations in membrane potential. Membrane potential was varied directly using DC polarizing currents and indirectly by ischaemia. The median nerve was stimulated at the wrist and the resultant compound muscle action potentials recorded from abductor pollicis brevis. Stimulus–response behaviour, strength–duration time constant (
SD), threshold electrotonus to 100-ms polarizing currents, a current–threshold relationship and the recovery of excitability following supramaximal activation were each followed in four normal subjects during the two manoeuvres, using a recently described protocol. Membrane depolarization and ischaemia produced an increase in axonal excitability, an increase in the slope of the current–threshold relationship, a `fanning in' of responses during threshold electrotonus, a decrease in super-excitability, and increases in both SD and the refractory period. Changes in the opposite direction occurred with membrane hyperpolarization and during the post-ischaemic period. One excitability parameter differentiated between the direct and indirect changes in membrane potential: late subexcitability was sensitive to polarizing currents but relatively insensitive to ischaemia, probably because of compensatory changes in extracellular potassium ions. These results should enable multiple excitability measurements to be used as a tool to identify changes in axonal membrane potential in neuropathy. excitability; membrane polarization; ischaemia
CMAP = compound muscle action potential; I/V = current/threshold relationship; RRP = relative refractory period; SD = strength–duration time constant
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
New techniques designed to study nerve excitability in vivo can provide an indirect measure of axonal membrane ion channel function (Bostock et al., 1998
). As these channels are voltage dependent, their function is related to the resting membrane potential of the axon, and to changes in membrane potential induced by applied currents or following an action potential. Recently, a convenient new method for measuring multiple parameters of nerve excitability was described (Kiernan et al., 2000). The method has now been incorporated into the diagnostic assessment of patients with peripheral nerve disease. This protocol measures stimulus–response behaviour, strength–duration time constant (SD), threshold electrotonus to 100-ms polarizing currents, a current–threshold relationship and the recovery of excitability following supramaximal activation. These excitability properties depend on membrane potential and on the function of different ion channels, but the relative sensitivity of the different excitability parameters in motor axons to membrane potential is not known, since they are not normally measured at the same time.
The present study was undertaken primarily to aid the interpretation of changes in excitability properties encountered clinically, by defining what changes could be explained by a change in membrane potential alone, and distinguishing these from changes due to altered ion channel function or altered ionic gradients. Changes in membrane potential have therefore been induced by passing small DC currents through the stimulating electrodes. A new equilibrium potential is quickly reached, allowing the excitability protocol to be re-run at a different membrane potential with minimal disturbance to ion channel function and ionic gradients. For comparison with these `pure' changes in membrane potential, we have also measured excitability properties during depolarization by ischaemia and during post-ischaemic hyperpolarization. Previous studies have shown that during brief periods of ischaemia axonal excitability increases due to membrane depolarization, caused by inhibition of the electrogenic sodium pump and extracellular K+ accumulation, whereas following ischaemia there is a period of reduced excitability, due to membrane hyperpolarization caused by hyperactivity of the electrogenic sodium pump (Bergmans, 1970; Bostock et al., 1991a, 1994; Mogyoros et al., 1997; Grosskreutz et al., 1999). The more complex changes in excitability that follow prolonged periods of ischaemia, and often result in ectopic discharges, have been avoided in this study, although we may note that these changes are also primarily caused by changes in sodium pump activity and the gradients of Na+ and K+ ions (Bostock et al., 1991b). The results of this study clearly define the relative sensitivity of different excitability parameters to changes in membrane potential, and also show that the changes in excitability during and after ischaemia can be distinguished from those due to changes in membrane potential alone.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Two separate sets of recordings were made, each on four healthy volunteers of both sexes (aged 24–54 years). All subjects gave informed consent and the study was approved by the Joint Research Ethics Committee of the National Hospital for Neurology and Neurosurgery, and the Institute of Neurology, London.
Studies were performed using a recently described protocol designed to measure a number of different nerve excitability parameters rapidly (Kiernan et al., 2000). Compound muscle action potentials (CMAPs) were recorded from thenar muscles using surface electrodes over abductor pollicis brevis, with the active electrode at the motor point and the reference on the proximal phalanx. The EMG signal was amplified (gain 1000, bandwidth 1.6–2 kHz) and digitized by computer (486 PC) with an analogue/digital board (DT2812, Data Translation Inc., Marlboro, Mass., USA.), using a sampling rate of 10 kHz. Stimulus waveforms generated by the computer were converted to current with a purpose-built isolated linear bipolar constant current stimulator (maximum output ±50 mA). The stimulus currents were applied via non-polarizable electrodes (Red Dot, 3M Health Care, D-46325 Borken, Germany), with the active electrode over the median nerve at the wrist and the reference electrode ~ 10 cm proximal over muscle. Stimulation and recording were controlled by new software, written in BASIC (QTRAC version 5.2, copyright Institute of Neurology, London, with multiple excitability protocol TRONDXM).
Test current pulses of 0.2 or 1 ms were applied at 0.8 s intervals, and combined with suprathreshold conditioning stimuli or subthreshold polarizing currents as required. The amplitude of the CMAP was measured from baseline to negative peak. For all tracking studies, the target CMAP was set to 40% of maximum. Skin temperature was monitored close to the stimulation site and maintained >32°C, by wrapping the arm in a blanket if necessary.
Stimulus–response curves were recorded separately for test stimuli of durations 0.2 and 1 ms (Fig. 1A). The stimuli were increased in 6% steps, with two responses averaged for each step, until three averages were considered maximal. The stimulus–response data were used for several purposes. First, the 1-ms peak response was used to set the target submaximal response (40% of peak) for threshold tracking. Secondly, the slope of the 1-ms stimulus–response curve was used in conjunction with the tracking error (deviation from the target) to optimize the subsequent threshold tracking. Finally, the ratio between the 0.2 and 1 ms stimuli required to evoke the same response was used to estimate rheobase and the strength–duration time constants of axons of different threshold (Fig. 1D).
|
Prolonged subthreshold currents were used to alter the potential difference across the internodal as well as nodal axonal membrane. The changes in threshold associated with these electrotonic changes in membrane potential normally have a similar time course and are known as threshold electrotonus (Bostock et al., 1998
). In the present protocol, test stimuli of 1 ms duration were used to produce the target CMAP (40% of maximal). Threshold tracking was used to record the changes in threshold induced by 100-ms polarizing currents, set to 40% (depolarizing) and –40% (hyperpolarizing) of the control threshold current. The three stimulus combinations were tested in turn: test stimulus alone (to measure the control threshold current), test stimulus + depolarizing conditioning current, and test stimulus + hyperpolarizing conditioning current. Threshold was tested at 26 time points (maximum separation of 10 ms) before, during and after the 100-ms conditioning currents (Fig. 1E
). Each stimulus combination was repeated until three valid threshold estimates were recorded, as judged by the response being within 15% of the target response, or alternate responses being either side of the target.
The current/threshold relationship (I/V) (Fig. 1C) was tested with 1 ms pulses at the end of 200-ms polarizing currents, which were altered in a ramp fashion from +50% (depolarizing) to –100% (hyperpolarizing) of the control threshold in 10% steps. As with the conventional threshold electrotonus protocol, stimuli with conditioning currents were alternated with test stimuli alone, and each stimulus combination was repeated until three valid threshold estimates were obtained.
The final part of the protocol recorded the recovery of excitability following a supramaximal conditioning stimulus (Fig. 1F). These changes were recorded at 18 conditioning-test intervals, decreasing from 200 to 2 ms in approximately geometric progression. Three stimulus combinations were tested in turn: (i) unconditioned test stimulus (1 ms duration) tracking the control threshold; (ii) supramaximal conditioning stimulus (1 ms duration) alone; and (iii) conditioning + test stimuli. The response to (ii) was subtracted on-line from the response to (iii) before the test CMAP was measured, so that the conditioning maximal CMAP did not contaminate the measured response when the conditioning test interval was short. Each stimulus combination was repeated until four valid threshold estimates were obtained. From the recovery cycle three parameters were measured: (i) the relative refractory period (RRP), defined as the interstimulus interval at which threshold recovered to its control value (or, in the absence of superexcitability, the first threshold minimum); (ii) superexcitability measured as the greatest percentage reduction in threshold (mean of three adjacent values); and (iii) late subexcitability, measured as the greatest percentage increase in threshold following the superexcitable period (mean of three adjacent values).
The first series of studies investigated the effect of DC polarizing currents on nerve excitability. Graded depolarizing and hyperpolarizing currents were applied to the stimulating electrodes overlying the median nerve at the wrist, and added to the conditioning and test stimuli. Eleven recordings were made from each subject, with polarizing current amplitudes set consecutively to 0, 0.25, –0.25, 0.5, –0.5, 0.75, –0.75, 1.0, –1.0, 1.25, –1.25 mA. Excitability parameters were linearly related to current, except for the strongest currents (±1.25 mA), when thresholds in some cases became too high to complete the excitability tests. The data presented are therefore restricted to the first nine recordings in each subject. In a second series of studies, changes in axonal excitability were recorded during ischaemia and following its release. Ischaemia was induced by inflating a sphygmomanometer cuff around the upper arm to >200 mmHg, well above the systolic blood pressure in all subjects, and maintained at that level for 25 min. The excitability protocol was performed prior to ischaemia, following 5 and 15 min ischaemia and then repeated in the post-ischaemic period at both 5 and 20 min following release of the cuff. Some conduction block may have occurred by the end of the second ischaemic recording (i.e. after ~25 min of ischaemia), since there was a small decline (4–14%, mean 8.5%) in the amplitude of the response to supramaximal stimuli. Because of the steepness of the stimulus–response relationship, threshold measurements should have been affected less than the supramaximal responses, and no corrections for conduction block were made.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The full sequence of excitability measurements, as described in Methods, was recorded as a baseline in each subject. Mean data for all subjects are illustrated in Fig. 1
, plotted in a standard format, and superimposed on the means and 95% confidence limits obtained previously for normal subjects (Kiernan et al., 2000). All participants in the present study were found to be within normal limits for each of the individual excitability measures. The principle effects of altering membrane potential directly, by means of DC polarizing currents, and indirectly by ischaemia, are first illustrated separately (Figs 2 and 3), before the effects of both manoeuvres on different excitability parameters are compared with each other and with the normal inter-subject variability (Figs 4 and 5). The effects of small currents (up to ±0.5 mA) on 14 excitability parameters are summarized in Table 1. For weak currents the excitability changes are approximately linear, and a linear correlation analysis allows comparison of the sensitivity of different parameters to polarization in terms of the fractional change per milliampere polarizing current, i.e. the normalized slope of the regression line.
|
|
|
|
|
Polarizing current and nerve excitability parameters
The most conspicuous effects of DC polarizing currents on nerve excitability are shown for a single subject in Fig. 2
. Progressive depolarizing currents increased the excitability of the axonal membrane, reducing the stimulus current required to evoke a particular response, whereas hyperpolarization reduced excitability. Depolarization therefore resulted in a shift to the left of the stimulus–response curve, and hyperpolarization a shift to the right, as indicated by the D and H arrowheads in Fig. 2A. There was also a slight change in the slope of the relationship (see also Fig. 4B), presumably because the axons most accessible to applied currents, and therefore with the lowest threshold to the 1-ms test stimuli, were also the axons most affected by the DC polarizing currents.
The current–threshold relationship reflects the rectifying properties of the axon, both nodal and internodal (Baker et al., 1987). At rest the steepening of the curve towards the top right results from outward rectification associated with activation of K+ channels (Fig. 1C). In contrast, the steepening of the curve toward the bottom left represents accommodation to the hyperpolarizing current due to inward rectification by the hyperpolarization-activated conductance, IH (Pape, 1996). With progressive increase in the intensity of the superimposed DC hyperpolarizing current, this steepening became more prominent, indicating increased activation of IH (Fig. 2B). Conversely, with depolarization, inward rectification was reduced and outward rectification increased.
Figure 2C shows that threshold electrotonus waveforms are particularly sensitive to changes in membrane potential, as has been demonstrated previously (Baker and Bostock, 1989; Bostock et al., 1998). Hyperpolarization reduces the conductance of the internodal membrane, so that the slow changes in internodal membrane potential during the prolonged currents are increased, and the electrotonus waveforms `fan out'. Conversely, a DC depolarizing current activates internodal K+ channels so that the conductance is increased and electrotonus becomes flatter. With strong depolarization, the threshold electrotonus responses to hyperpolarization almost mirror those of depolarization: the `fanning in' indicates accommodation, due to changes in activation of nodal slow K+ channels and in inactivation of sodium channels. In depolarized rat nerves, threshold electrotonus waveforms can be reduced to brief symmetrical spikes as excitability becomes dominated by sodium channel inactivation (Baker and Bostock, 1989; Bostock, 1995).
The recovery cycle of excitability records the changes in excitability following a single supramaximal conditioning stimulus (Gilliatt and Willison, 1963; Eisen et al., 1982; Kiernan et al., 1996) (Fig. 1F). The effects of polarization on the recovery cycle were dramatic (Fig. 2D). The refractory period, determined by inactivation of Na+ channels, was increased by depolarization and reduced by hyperpolarization. During the next phase of the recovery cycle, axons enter a superexcitable period produced by the depolarizing afterpotential. This is voltage dependent because it depends on spread of depolarization to the internodal axolemma (Barrett and Barrett, 1982), which is limited by internodal K+ channels, especially fast K+ channels in the paranodal region (Bostock et al., 1981; Waxman and Ritchie, 1993). Following the superexcitable period, the final phase of the recovery cycle is the late subexcitable period, due to activation of nodal voltage-dependent slow K+ channels (Baker et al., 1987; Stys and Waxman, 1994; Bostock, 1995; Kiernan et al., 1996). Hyperpolarization resulted in a reduction in the late subexcitable period, as the electrochemical gradient for K+ ions was reduced. It is important to note that although refractory period, superexcitability and late subexcitability depend on Na+ channel inactivation, depolarizing afterpotential and slow K+ channel activation, respectively, there is considerable overlap and interaction between these factors (see Discussion).
Comparison of the effects of small polarizing currents on different excitability parameters in Table 1 indicates that the most sensitive parameter is superexcitability, in that it is reduced by 82% per milliampere of polarizing current. However, because of its greater variability, superexcitability does not provide as reliable an index of membrane potential changes as the threshold electrotonus parameter TEd (90–100 ms), the threshold change after 90–100 ms of a standard depolarizing current (see Discussion).
Comparison with ischaemia and release from ischaemia
A comparable series of recordings made before (C), during (I1, I2) and after (PI) ischaemia is illustrated for a single subject (Fig. 3). It can be seen that ischaemia too produced marked changes in measures of axonal excitability, and that the changes during ischaemia were consistent with axonal depolarization: (i) the stimulus–response curve shifted to the left (Fig. 3A); (ii) the current–threshold relationship became steeper (Fig. 3B); (iii) there was a fanning inwards of threshold electrotonus curves (Fig. 3C); and (iv) refractoriness increased at the expense of superexcitability (Fig. 3D). However, while there was a leftward shift in the late subexcitable period, its amplitude was decreased in this subject in contrast to the increase seen with depolarization (Fig. 2D). During the post-ischaemic period, the trends in excitability reversed and the axon behaved as though hyperpolarized, although again the late subexcitable period behaved differently.
The magnitude and direction of the effects of polarizing currents and ischaemia on six pairs of excitability parameters are compared in Fig. 4 with the 95% confidence limits for these parameters in normal subjects from a previous study (see Fig. 4 in Kiernan et al., 2000). For simplicity, the data points plotted are restricted to the mean values for both control groups, and for the effects of 1-mA depolarizing (D) and hyperpolarizing (H) currents, and for recordings started 5 min after applying a pressure cuff (I) and 5 min after its release (PI). The recordings started 15 min after the start of ischaemia were less suitable for representing on these plots, since some recovery cycle parameters became unmeasurable, while the recordings started 20 min after the release of ischaemia were qualitatively similar to the control values.
In Fig. 4A, the values for SD correspond to those for a CMAP 40% of maximal (i.e. the normal target response). The corresponding rheobase was also calculated from the stimulus–response data using Weiss's law (Mogyoros et al., 1996), and was plotted on a logarithmic scale. It can be seen that depolarization and ischaemia produce an increase in SD, while hyperpolarization and post-ischaemia produce a corresponding reduction, in agreement with the proposal that SD is determined by the activation of persistent Na+ channels, as well as by the passive membrane time constant (Bostock and Rothwell, 1997). However, since all shifts in SD and rheobase remained within the range established for normal controls, these parameters cannot be regarded as particularly sensitive to membrane potential. The relative sensitivities of SD to polarization and ischaemia are shown in more detail in Fig. 5D. Only the reduction in SD following ischaemia reached statistical significance with four subjects. However, the more sensitive correlation analysis in Table 1 indicates significant effects of weak polarizing current on SD as well as rheobase.
In Fig. 4B, the peak CMAP values (corresponding to twice the 50% CMAP data in Figs 2A and 3A) are, as expected, unchanged by axonal polarization or ischaemia. However, the normalized stimulus–response slopes (derived from data in Fig. 1B as described in Methods) show systematic changes with polarization but not with ischaemia. Depolarization reduced and hyperpolarization increased the slope of the stimulus–response curves. This is understandable on the assumption that the motor axons all have similar properties, and the differences in threshold reflect different ease of access of the applied currents rather than (or in addition to) differences in fibre diameter: low threshold axons will therefore be depolarized more by the depolarizing current than high threshold axons, increasing the range of thresholds, while the converse occurs with hyperpolarizing currents.
The relationship between the minimal and resting current–threshold slopes is similar for both membrane polarization and ischaemia (Fig. 4C). The resting slope, which is closely related to the input conductance of the axons, is very sensitive to changes in membrane potential, whether caused directly by polarizing currents or indirectly by ischaemia, since the main conductances responsible for the resting potential are voltage-dependent potassium channels (Grafe et al., 1994). Either 5 min of ischaemia or 1 mA of depolarizing current is sufficient to approximately double the current–threshold slope, taking it well outside the range of normal values. The minimum current–threshold slope occurs on moderate hyperpolarization, when the voltage-dependent K+ channels open at rest are largely deactivated and the hyperpolarization-activated IH channels are only partially activated. This minimum slope, corresponding to the minimum membrane conductance, was less affected by changes in membrane potential, at least those caused by small polarizing currents (see Table 1), since the primary effect of polarization on the I/V relationship is to displace the origin along the current axis.
Earlier studies demonstrated the inverse relationship of the RRP with change in temperature (Kiernan et al., 2000). Over a constant temperature, the voltage dependence of this relationship can also be demonstrated (Fig. 4D). RRP was derived from the recovery cycle data as the first intercept between the recovery curves and the x-axis. Membrane depolarization produced a substantial prolongation of the RRP, as did ischaemia, and these changes were highly significant with only four subjects (Fig. 5A). At least two factors contribute to the prolonged RRP: the increase in steady-state level of Na2+ channel inactivation with membrane depolarization, and the reduction in depolarizing after-potential and superexcitability. Figure 4D also indicates that 5 min of ischaemia produces a greater increase in RRP than 1 mA of depolarizing current, although these treatments have similar effects on the I/V curves and threshold electrotonus. This observation is in agreement with the conclusion of Grosskreutz and colleagues that ischaemia has a greater effect on refractoriness in human cutaneous afferents than can be explained simply by the change in membrane potential (Grosskreutz et al., 2000). Accumulation of some metabolite, such as H+ ions could be responsible, and the persistence of this metabolite during the post-ischaemic period could account for the lack of a significant reduction in RRP (Fig. 5A), despite other evidence of membrane hyperpolarization.
In studies on patients using threshold electrotonus, the early depolarizing response (TEd averaged between 10 and 20 ms after the start of a 40% depolarizing current) and the late hyperpolarizing response (TEh from 90 to 100 ms after the start of a 40% hyperpolarizing current) have been found to be altered in disease (Bostock et al., 1995; Horn et al., 1996). These two parameters are plotted in Fig. 4E. They are well correlated across normal subjects, as indicated by the eccentricity of the ellipse, and our new data suggest that much of this co-variation may be related to differences in resting membrane potential. Both the directly and indirectly induced changes in membrane potential in the four subjects caused marked changes in these electrotonus parameters in the direction of the long axis of the ellipse. The changes with membrane polarization and during ischaemia were statistically significant with only four subjects (Fig. 5E and F). It is important to add that, although these two electrotonus parameters vary similarly with membrane potential, they may be altered differentially by disease, presumably because they depend on different ion channels (Horn et al., 1996).
Superexcitability is plotted against late subexcitability in Fig. 4F, and the statistical significance of these changes is indicated in Fig. 5B and C. These figures show that there are differential effects of membrane polarization and ischaemia on the late subexcitable period. Whereas both depolarization and ischaemia abolish superexcitability, the late subexcitability is increased by depolarizing currents but on average unchanged by ischaemia. Possible reasons for this striking discrepancy, which contrasts with the broadly similar effects of depolarization and ischaemia on the other excitability parameters, are discussed below.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study has compared the effects of membrane polarization and ischaemia on various excitability properties of human motor axons. The main aim of the study was to document the changes induced by membrane polarization, to help interpretation of changes in excitability parameters found in neuropathies. The modest polarizing currents employed were expected to produce relatively pure changes in membrane potential, uncomplicated by changes in ion concentrations or densities of ion channels, and indeed all the changes in excitability parameters we have observed with polarizing currents are understandable in terms of expected changes in activation of voltage-dependent ion channels alone. Of course, such `pure' changes in membrane potential could not easily arise in a neuropathy. For a nerve to be abnormally depolarized in the absence of an externally applied depolarizing current, there would have to be a defect in electrogenic pumping, a reduction in the ratio [K+]in/[K+]out, a lack of potassium channels, or some other changes in membrane properties to bring about depolarization. One example of how depolarization could arise in a neuropathy would be by inhibition of the sodium pump, which we have modelled here by inducing ischaemia with a pressure cuff. The results have both confirmed previous evidence that the main excitability changes induced can be attributed primarily to changes in membrane potential, and also produced a new finding, that subexcitability does not change as expected for a change in membrane potential alone.
Differences in late subexcitability
The long-lasting phase of subexcitability that follows the refractory and superexcitable periods in the recovery of excitability following a single impulse is due to activation of voltage-dependent slow K+ channels (GKs) at the nodes of Ranvier (Baker et al., 1987; Stys and Waxman, 1994; Bostock, 1995). These channels take tens of milliseconds to activate or deactivate at the resting potential, but activation is much faster at the levels of depolarization reached during the action potential. Consequently a single action potential causes a step increase in activation which then normally outlasts the depolarizing afterpotential to produce the late hyperpolarization and hence subexcitability (Schwarz et al., 1995). If an axon conducts a brief train of impulses, this late subexcitable period becomes accentuated, producing the H1 phase of activity-dependent hypoexcitability (Bergmans, 1970; Taylor et al., 1992; Miller et al., 1996). Slow K+ channels are activated by depolarization, but it has been estimated that 35% are open at rest, so that they contribute to the resting membrane potential (Bostock et al., 1991b; Schwarz et al., 1995) and resting input conductance (Grafe et al., 1994). Since late subexcitability depends on hyperpolarization by current through slow K+ channels (IKs) exceeding or outlasting the depolarizing afterpotential, changes in subexcitability can reflect changes in IKs or the depolarizing afterpotential or both. A further complication is that the depolarizing afterpotential contributes to prolonged activation of IKs, so that a large depolarizing afterpotential tends to be followed by a longer lasting period of late subexcitability. Both types of interaction between superexcitability and subexcitability are in evidence in Figs 2D and 3D. Given this background information, how can we account for the different changes in late subexcitability on membrane polarization and during or after ischaemia?
Nodal slow K+ current (IKs) depends not only on the activation of GKs, but also on the electrochemical gradient for K+ currents, i.e. the difference between the membrane potential and the potassium equilibrium potential (Em–EK). The simplest explanation for the difference in IKs between ischaemic and `pure' membrane depolarization is that the changes in EK are different, specifically that ischaemic depolarization is in part due to extracellular K+ accumulation and depolarization of EK. Similarly, in the post-ischaemic state, the extracellular K+ concentration outside the nodal membrane is likely to undershoot its resting value because of the hyperactivity of the sodium pump (Bostock and Grafe, 1985), so that the electrochemical gradient for K+ ions is maintained despite the hyperpolarization. Thus, the principle difference between the excitability changes occurring during and after ischaemia from those occurring during membrane depolarization and hyperpolarization is entirely consistent with the accepted mechanisms of ischaemic potential changes.
What is the best index of axonal membrane potential?
In this study, the parameter showing the highest sensitivity to applied currents, and therefore to changes in membrane potential, was superexcitability (Table 1), in that superexcitability changed on average by 82% of its resting value per milliampere of applied current. Almost as sensitive were the resting current–threshold slope (71% increase), hyperpolarizing threshold electrotonus at 90–100 ms (71%), and subexcitability (70% increase). However, as an index of small changes in membrane potential, the most accurate single parameter should be depolarizing electrotonus at 90–100 ms. There is normally little variation in this parameter between subjects, or between recordings from the same subject on different occasions, and it has the highest correlation with polarizing current (r = –0.92). The sensitivities of excitability parameters to applied currents can be converted to approximate sensitivities to changes in membrane potential, on the assumption that a mean (1 ms) resting threshold current of 3.14 mA would cause a passive membrane depolarization of 10–15 mV. A sensitivity of 82% per milliampere for superexcitability therefore corresponds roughly to a sensitivity of 21% per millivolt, whereas threshold itself, which changes by almost exactly 1 mA per milliampere of polarizing current, has a sensitivity corresponding to ~8% per millivolt.
The comparative sensitivities of different excitability parameters to polarizing currents in Table 1 appear to conflict with the comparative `voltage dependencies' reported previously for human cutaneous afferents by Burke et al. (1998). They found that a depolarizing current that reduced threshold by 50% produced changes in `refractoriness', `supernormality' and SD corresponding to +300, –150 and +75% of their resting values, respectively. However, `refractoriness', measured as the relative increase in threshold 2 ms after a supramaximal conditioning stimulus, cannot be compared with the relative refractory period. Also, in the earlier study the polarizing currents were not applied continuously, but started 10 ms before each test stimulus, so that there was insufficient time for the nerve (especially internodal membrane and slow K+ channels) to reach a steady state. Because of these methodological differences it is not possible to conclude that motor and sensory fibres differ in the relative sensitivities of different excitability parameters to changes in membrane potential.
Although single excitability parameters, such as supernormality or depolarizing threshold electrotonus at 90–100 ms, can provide a reliable index of membrane potential in normal motor axons, this cannot be assumed when testing diseased nerves. All the excitability parameters depend on factors, in addition to membrane potential, that could be altered in neuropathy. The strength of the multiple excitability protocol used in this study is that information from parameters that depend particularly on membrane potential and nodal sodium channels (e.g. SD, RRP) can be collated with information from parameters that depend more on membrane potential and internodal potassium channels (e.g. resting I/V slope). The best index of axonal membrane potential that can be obtained non-invasively is therefore likely to be some combination of different excitability parameters.
Relevance to clinical studies
Excitability studies are now being used to provide functional information about neurological disease. Thus, strength–duration time constants have been reported to be increased in some patients with acquired neuromyotonia and in amyotrophic lateral sclerosis (Mogyoros et al., 1998; Maddison et al., 1999), altered threshold electrotonus responses have been described in amyotrophic lateral sclerosis and diabetic neuropathy (Bostock et al., 1995; Horn et al., 1996), and reduced superexcitability has been reported in multiple sclerosis (Eisen et al., 1982; Shefner et al., 1992). With the probable exception of a reduction in IH in diabetic neuropathy, the interpretation of the abnormalities described in terms of membrane properties has been uncertain, but in most cases an alteration in membrane potential is probably involved. The data in the present study, defining the changes in a range of excitability properties that occur with changes in membrane potential, have provided the first clear map to guide the interpretation of these axonal abnormalities.
|
Acknowledgments |
|---|
Dr Kiernan was supported by a CJ Martin/RG Menzies Fellowship from The National Health and Medical Research Council of Australia.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Baker M, Bostock H. Depolarization changes the mechanism of accommodation in rat and human motor axons. J Physiol (Lond) 1989; 411: 545–61.
Barrett EF, Barrett JN. Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotential. J Physiol (Lond) 1982; 323: 117–44.
Baker M, Bostock H, Grafe P, Martius P. Function and distribution of three types of rectifying channel in rat spinal root myelinated axons. J Physiol (Lond) 1987; 383: 45–67.
Bergmans J. The physiology of single human nerve fibres. Louvain (Belgium): Vander; 1970.
Bostock H. Mechanisms of accommodation and adaptation in myelinated axons. In: Waxman SG, Kocsis JD, Stys PK, editors. The axon. New York: Oxford University Press; 1995. p. 311–27.
Bostock H, Grafe P. Activity-dependent excitability changes in normal and demyelinated rat spinal root axons. J Physiol (Lond) 1985; 365: 229–57.
Bostock H, Rothwell JC. Latent addition in motor and sensory fibres of human peripheral nerve. J Physiol (Lond) 1997; 498: 277–94.
Bostock H, Sears TA, Sherratt RM. The effects of 4-aminopyridine and tetraethylammonium ions on normal and demyelinated mammalian nerve fibres. J Physiol (Lond) 1981; 313: 301–15.
Bostock H, Baker M, Grafe P, Reid G. Changes in excitability and accommodation of human motor axons following brief periods of ischaemia. J Physiol (Lond) 1991a; 441: 513–35.
Bostock H, Baker M, Reid G. Changes in excitability of human motor axons underlying post-ischaemic fasciculations: evidence for two stable states. J Physiol (Lond) 1991b; 441: 537–57.
Bostock H, Burke D, Hales JP. Differences in behaviour of sensory and motor axons following release of ischaemia. Brain 1994; 117: 225–34.
Bostock H, Sharief MK, Reid G, Murray NM. Axonal ion channel dysfunction in amyotrophic lateral sclerosis. Brain 1995; 118: 217–25.
Bostock H, Cikurel K, Burke D. Threshold tracking techniques in the study of human peripheral nerve. [Review]. Muscle Nerve 1998; 21: 137–58.[Web of Science][Medline]
Burke D, Mogyoros I, Vagg R, Kiernan MC. Quantitative description of the voltage dependence of axonal excitability in human cutaneous afferents. Brain 1998; 121: 1975–83.
Eisen A, Paty D, Hoirch M. Altered supernormality in multiple sclerosis peripheral nerve. Muscle Nerve 1982; 5: 411–4.[Web of Science][Medline]
Gilliatt RW, Willison RG. The refractory and supernormal periods of the human median nerve. J Neurol Neurosurg Psychiatry 1963; 26: 136–47.
Grafe P, Bostock H, Schneider U. The effects of hyperglycaemic hypoxia on rectification in rat dorsal root axons. J Physiol (Lond) 1994; 480: 297–307.
Grosskreutz J, Lin C, Mogyoros I, Burke D. Changes in excitability indices of cutaneous afferents produced by ischaemia in human subjects. J Physiol (Lond) 1999; 518: 301–14.
Grosskreutz J, Lin C, Mogyoros I, Burke D. Ischaemic changes in refractoriness of human cutaneous afferents under threshold-clamp conditions. J Physiol (Lond) 2000; 523: 807–15.
Horn S, Quasthoff S, Grafe P, Bostock H, Renner R, Schrank B. Abnormal axonal inward rectification in diabetic neuropathy. Muscle Nerve 1996; 19: 1268–75.[Web of Science][Medline]
Kiernan MC, Mogyoros I, Burke D. Differences in the recovery of excitability in sensory and motor axons of human median nerve. Brain 1996; 119: 1099–105.
Kiernan MC, Burke D, Andersen KV, Bostock H. Multiple measures of axonal excitability: a new approach in clinical testing. Muscle Nerve 2000; 23: 399–409.[Web of Science][Medline]
Maddison P, Newsom-Davis J, Mills KR. Strength-duration properties of peripheral nerve in acquired neuromyotonia. Muscle Nerve 1999; 22: 823–30.[Web of Science][Medline]
Miller TA, Kiernan MC, Mogyoros I, Burke D. Activity-dependent changes in impulse conduction in a focal nerve lesion. Brain 1996; 119: 429–37.
Mogyoros I, Kiernan MC, Burke D. Strength-duration properties of human peripheral nerve. Brain 1996; 119: 439–47.
Mogyoros I, Kiernan MC, Burke D, Bostock H. Excitability changes in human sensory and motor axons during hyperventilation and ischaemia. Brain 1997; 120: 317–25.
Mogyoros I, Kiernan MC, Burke D, Bostock H. Strength-duration properties of sensory and motor axons in amyotrophic lateral sclerosis. Brain 1998; 121: 851–9.
Pape HC. Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. [Review]. Annu Rev Physiol 1996; 58: 299–327.[Web of Science][Medline]
Schwarz JR, Reid G, Bostock H. Action potentials and membrane currents in the human node of Ranvier. Pflügers Arch 1995; 430: 283–92.
Shefner JM, Carter JL, Krarup C. Peripheral sensory abnormalities in patients with multiple sclerosis. Muscle Nerve 1992; 15: 73–6.[Web of Science][Medline]
Stys PK, Waxman SG. Activity-dependent modulation of excitability: implications for axonal physiology and pathophysiology [editorial]. [Review]. Muscle Nerve 1994; 17: 969–74.[Web of Science][Medline]
Taylor JL, Burke D, Heywood J. Physiological evidence for a slow K+ conductance in human cutaneous afferents J Physiol (Lond) 1992; 453: 575–89.
Waxman SG, Ritchie JM. Molecular dissection of the myelinated axon. [Review]. Ann Neurol 1993; 33: 121–36.[Web of Science][Medline]
Received May 10, 2000. Revised July 12, 2000. Accepted July 27, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. B. Park, C. S.-Y. Lin, A. V. Krishnan, D. Goldstein, M. L. Friedlander, and M. C. Kiernan Oxaliplatin-induced neurotoxicity: changes in axonal excitability precede development of neuropathy Brain, October 1, 2009; 132(10): 2712 - 2723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moldovan, S. Alvarez, and C. Krarup Motor axon excitability during Wallerian degeneration Brain, February 1, 2009; 132(2): 511 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ng, J. Howells, J. D. Pollard, and D. Burke Up-regulation of slow K+ channels in peripheral motor axons: a transcriptional channelopathy in multiple sclerosis Brain, November 1, 2008; 131(11): 3062 - 3071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S.-Y. Lin, A. V. Krishnan, M.-J. Lee, A. S. Zagami, H.-L. You, C.-C. Yang, H. Bostock, and M. C. Kiernan Nerve function and dysfunction in acute intermittent porphyria Brain, September 1, 2008; 131(9): 2510 - 2519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Krishnan, C. S.-Y. Lin, and M. C. Kiernan Activity-dependent excitability changes suggest Na+/K+ pump dysfunction in diabetic neuropathy Brain, May 1, 2008; 131(5): 1209 - 1216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Vucic and M. C. Kiernan Pathophysiologic insights into motor axonal function in Kennedy disease Neurology, November 6, 2007; 69(19): 1828 - 1835. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Trevillion, J. Howells, and D. Burke Outwardly rectifying deflections in threshold electrotonus due to K+ conductances J. Physiol., April 15, 2007; 580(2): 685 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S.-Y. Lin, V. G. Macefield, M. Elam, B. Gunnar Wallin, S. Engel, and M. C. Kiernan Axonal changes in spinal cord injured patients distal to the site of injury Brain, April 1, 2007; 130(4): 985 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. Z'Graggen, C. S. Y. Lin, R. S. Howard, R. J. Beale, and H. Bostock Nerve excitability changes in critical illness polyneuropathy Brain, September 1, 2006; 129(9): 2461 - 2470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Krishnan, R. K. S. Phoon, B. A. Pussell, J. A. Charlesworth, H. Bostock, and M. C. Kiernan Ischaemia induces paradoxical changes in axonal excitability in end-stage kidney disease Brain, June 1, 2006; 129(6): 1585 - 1592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A V Krishnan, R K S Phoon, B A Pussell, J A Charlesworth, and M C Kiernan Sensory nerve excitability and neuropathy in end stage kidney disease J. Neurol. Neurosurg. Psychiatry, April 1, 2006; 77(4): 548 - 551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kanai, S. Kuwabara, S. Misawa, N. Tamura, K. Ogawara, M. Nakata, S. Sawai, T. Hattori, and H. Bostock Altered axonal excitability properties in amyotrophic lateral sclerosis: impaired potassium channel function related to disease stage Brain, April 1, 2006; 129(4): 953 - 962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Krishnan, J. G. Colebatch, and M. C. Kiernan Hypokalemic weakness in hyperaldosteronism: Activity-dependent conduction block Neurology, October 25, 2005; 65(8): 1309 - 1312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G Ardolino, B Bossi, S Barbieri, and A Priori Non-synaptic mechanisms underlie the after-effects of cathodal transcutaneous direct current stimulation of the human brain J. Physiol., October 15, 2005; 568(2): 653 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Krishnan, R. K. S. Phoon, B. A. Pussell, J. A. Charlesworth, H. Bostock, and M. C. Kiernan Altered motor nerve excitability in end-stage kidney disease Brain, September 1, 2005; 128(9): 2164 - 2174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Priori, B. Bossi, G. Ardolino, L. Bertolasi, M. Carpo, E. Nobile-Orazio, and S. Barbieri Pathophysiological heterogeneity of conduction blocks in multifocal motor neuropathy Brain, July 1, 2005; 128(7): 1642 - 1648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Krishnan and M. C. Kiernan Altered nerve excitability properties in established diabetic neuropathy Brain, May 1, 2005; 128(5): 1178 - 1187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moldovan and C. Krarup Mechanisms of hyperpolarization in regenerated mature motor axons in cat J. Physiol., November 1, 2004; 560(3): 807 - 819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moldovan and C. Krarup Persistent abnormalities of membrane excitability in regenerated mature motor axons in cat J. Physiol., November 1, 2004; 560(3): 795 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Hales, C. S.-Y. Lin, and H. Bostock Variations in excitability of single human motor axons, related to stochastic properties of nodal sodium channels J. Physiol., September 15, 2004; 559(3): 953 - 964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Kiernan, C. S.-Y. Lin, and D. Burke Differences in activity-dependent hyperpolarization in human sensory and motor axons J. Physiol., July 1, 2004; 558(1): 341 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Bostock, R. J. L. Walters, K. V. Andersen, N. M. F. Murray, D. Taube, and M. C. Kiernan Has potassium been prematurely discarded as a contributing factor to the development of uraemic neuropathy? Nephrol. Dial. Transplant., May 1, 2004; 19(5): 1054 - 1057. [Full Text] [PDF]
|
|
|
|
|
|
H. Nodera, H. Bostock, S. Kuwabara, T. Sakamoto, K. Asanuma, S. Jia-Ying, K. Ogawara, N. Hattori, M. Hirayama, G. Sobue, et al. Nerve excitability properties in Charcot-Marie-Tooth disease type 1A Brain, January 1, 2004; 127(1): 203 - 211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Bostock, M. Campero, J. Serra, and J. Ochoa Velocity recovery cycles of C fibres innervating human skin J. Physiol., December 1, 2003; 553(2): 649 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kanai, S. Kuwabara, K. Arai, J.-Y. Sung, K. Ogawara, and T. Hattori Muscle cramp in Machado-Joseph disease: Altered motor axonal excitability properties and mexiletine treatment Brain, April 1, 2003; 126(4): 965 - 973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cappelen-Smith, C. S.-Y. Lin, S. Kuwabara, and D. Burke Conduction block during and after ischaemia in chronic inflammatory demyelinating polyneuropathy Brain, August 1, 2002; 125(8): 1850 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H L Chan, C. S-Y Lin, E. Pierrot-Deseilligny, and D. Burke Excitability changes in human peripheral nerve axons in a paradigm mimicking paired-pulse transcranial magnetic stimulation J. Physiol., August 1, 2002; 542(3): 951 - 961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S-Y Lin, S. Kuwabara, C. Cappelen-Smith, and D. Burke Responses of human sensory and motor axons to the release of ischaemia and to hyperpolarizing currents J. Physiol., June 15, 2002; 541(3): 1025 - 1039. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Kiernan, R. J. L. Walters, K. V. Andersen, D. Taube, N. M. F. Murray, and H. Bostock Nerve excitability changes in chronic renal failure indicate membrane depolarization due to hyperkalaemia Brain, June 1, 2002; 125(6): 1366 - 1378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Kiernan, J.-M. Guglielmi, R. Kaji, N. M. F. Murray, and H. Bostock Evidence for axonal membrane hyperpolarization in multifocal motor neuropathy with conduction block Brain, March 1, 2002; 125(3): 664 - 675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cappelen-Smith, S. Kuwabara, C. S.-Y. Lin, I. Mogyoros, and D. Burke Membrane properties in chronic inflammatory demyelinating polyneuropathy Brain, December 1, 2001; 124(12): 2439 - 2447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Kiernan, K. Cikurel, and H. Bostock Effects of temperature on the excitability properties of human motor axons Brain, April 1, 2001; 124(4): 816 - 825. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||