Introduction

Amyotrophic lateral sclerosis (ALS) is a universally fatal neurodegenerative disorder that involves motor neurons in the spinal cord, brainstem and motor cortex.1 Ten per cent of all ALS cases are familial (FALS), in which two or more family members are clinically affected.2 Although eight genetic loci have been identified in FALS,2 mutations in the copper/zinc superoxide-dismutase-1 gene (SOD-1) gene, results in the typical ALS phenotype,3 termed ALS1.

From a clinical perspective, muscle cramps and fasciculations are an inevitable feature of both familial and sporadic forms of ALS, and are taken to reflect ectopic activity of motor axons.4–7 Widespread abnormalities in axonal ion channel function, including reduction of slow and fast K⁺ channel conductances combined with upregulation of persistent Na⁺ conductances,8–11 have been reported in sporadic ALS (SALS) and proposed as a likely mechanism underlying the generation of such ectopic activity.8 12 13 Whether such changes are intrinsic to the process of axonal degeneration, or develop as an epiphenomenon, remains to be clarified.

In addition to underlying the generation of ectopic axonal activity, abnormalities of axonal ion channel function, in particular upregulation of persistent Na⁺ conductances, has been associated with the process of axonal degeneration.14 Further, in SALS patients, cortical hyperexcitability was correlated with upregulation of persistent Na⁺ conductances,15 thereby suggesting that a ‘dying forward’ process, which proposes that corticomotorneurons drive anterior horn cell loss,16 underlies the neurodegeneration in ALS. The notion of a dying forward process as a potential mechanism of neurodegeneration in ALS was recently supported by findings that cortical hyperexcitability precedes the onset of FALS.17

The strength–duration time constant (τ_SD) is a measure of the rate at which the threshold current for a target potential declines as the stimulus duration increases18 19 and equates to chronaxie. Computer modelling of the behaviour of human motor axon suggests that τ_SD reflects persistent Na⁺ conductances which constitute approximately 1% of total Na⁺ current in motor axons.19 Specifically, increasing the fraction of the persistent Na⁺ current by means of membrane depolarisation prolongs τ_SD. Over recent years, techniques have been developed so that τ_SD can now be assessed in a clinical setting using a computerised threshold tacking protocol.20 Consequently, the present study used a novel combination of peripheral axonal and central threshold tracking techniques to assess whether upregulation of persistent Na⁺ conductances, as measured by τ_SD, was a feature of SOD-1 FALS, and whether this upregulation of persistent Na⁺ conductances was associated with the process of axonal degeneration and cortical hyperexcitability.

Methods

Studies were undertaken on 13 asymptomatic SOD-1 mutation carriers (three males: age range 25–50 years, mean age 40 years) and six clinically definite familial ALS patients as defined by the revised El Escorial criteria21 who expressed SOD-1 mutations (five males: age range 39–70 years, mean age 58 years). Families with three different SOD-1 mutations were studied, including: valine-to-glycine mutation in exon 5 at codon position 148 (V148G); isoleucine-to-threonine mutation in exon 4 at codon position 113 (I113T); glutamic acid-to-glycine mutation in exon 4 at codon position 100 (E100G). The pattern of inheritance was autosomal dominant, and all subjects were heterozygous for the SOD-1 mutation. For comparison, 45 SALS patients were studied (32 males, 13 females: age range 26–78 years, mean age 59 years). All ALS patients were clinically staged using the ALS-functional rating scale—revised (ALSFRS-R),22 Medical Research Council23 clinical grading of power and Trigg's hand function score.24 All subjects gave informed consent to the procedures, which were approved by the South East Sydney Area Health Service Human Research Ethics Committee.

In all studies, the median nerve was stimulated at the wrist, and the resultant compound muscle action potential (CMAP) was recorded from the abductor pollicis brevis using surface electrodes. Skin temperature was maintained at 32°C. Prior to excitability studies, CMAP amplitude and onset latency, F-wave latency and frequency were all measured. The neurophysiological index (NI) was derived according to a previously reported formula: NI=CMAP amplitude (mV) × F-wave frequency/Distal motor latency (ms), where F-wave frequency was expressed as the number of F responses recorded in 20.25

Axonal excitability

Test current pulses of 0.2 and 1 ms duration were applied at 0.8 s intervals and combined with either subthreshold polarising currents or suprathreshold conditioning stimuli. The CMAP amplitude was measured from baseline to negative peak, with tracking target set to 40% of supramaximal CMAP response.

To commence the protocol, stimulus-response (SR) curves were recorded separately for stimuli of 0.2 and 1 ms duration. Stimuli were increased in 4% steps, with two responses averaged at each step until three averages were considered maximal. The ratio between the SR curves for two different stimulus durations that produced the same CMAP response were used to calculate the strength–duration time constant (τ_SD) of motor axons at different thresholds using the Weiss formula.26 27

Threshold electrotonus was measured using prolonged subthreshold polarising currents of 100 ms duration, set to +40% (depolarising) and −40% (hyperpolarising) of controlled threshold current.27 28 Threshold was tested at 26 time points before, during and after the 100 ms polarising pulse. The stimulus combinations were repeated until three valid estimates were recorded within 15% of target response.20 Changes in membrane threshold to subthreshold depolarising and hyperpolarising currents were recorded at the following time points: 10–20 ms, depolarizing threshold electrotonus (TEd) (10–20 ms); 40–60 ms, TEd (40–60 ms); and 90–100 ms, TEd (90–100 ms); 10–20 ms, hyperpolarizing threshold electrotonus (TEh) (10–20 ms) and 90–100 ms, TEh (90–100 ms).

A current–threshold relationship (I/V) was obtained by tracking the changes in threshold of 1 ms test pulses that occurred following subthreshold polarising currents of 200 ms duration which were altered in ramp fashion from +50% (depolarising) to −100% (hyperpolarising) of controlled threshold in 10% steps. The following parameters were recorded: (1) resting I/V slope, calculated from polarising currents between +10% and −10%; and (2) hyperpolarising I/V slope, calculated from polarising current between 0–100%.

The recovery of axonal membrane excitability following a supramaximal conditioning stimulus, the recovery cycle, was also recorded. Eighteen conditioning-test stimulus intervals were studied, decreasing from 200 to 2 ms according to a previously described technique.20 The following parameters were measured: (1) relative refractory period (ms), defined as the first intercept at which the recovery curve crosses the x-axis; (2) superexcitability (%), defined as the largest reduction in threshold, peaking at a conditioning-test interval of <10 ms; (3) late subexcitability (%), defined as the largest increase in threshold following the superexcitability period after 10 ms.20

In the same sitting, cortical excitability was assessed in FALS patients by applying TMS to the motor cortex by means of a 90 mm circular coil. A threshold tracking paradigm was applied as previously reported.29 Cortical excitability findings in the present cohort of asymptomatic SOD-1 subjects, FALS and SALS patients have been in part reported previously.15 17

Recordings of motor-evoked potentials and CMAPs were amplified and filtered (3 Hz–3 kHz) using a GRASS ICP511 A.C. amplifier (Grass-Telefactor, Astro-Med, West Warwick, Rhode Island) and sampled at 10 kHz using a 12-bit data acquisition card (National Instruments PCI-MIO-16E-4). Data acquisition and stimulation delivery were controlled by QTRACS software version 0.9.7. Stimulus waveforms were converted to current with a purpose-built isolated linear bipolar constant-current stimulator.

Statistical analysis

The values of axonal excitability at rest were compared with normal control data obtained from 30 subjects (21 men; nine women aged 24–58 years).20 Further, cortical excitability in FALS patients was compared with control data obtained from 55 subjects (28 men; aged 23–73 years, mean: 46 years). Single comparisons in excitability parameters were analysed using the Student t test. Since measures of excitability vary with age and temperature, all individual measurements were compensated for age, temperature and sex before statistical analysis, using the relations found in control subjects.20 30 A probability (p) value of <0.05 was considered statistically significant. Results are expressed as mean±SEM.

Results

The clinical and genetic features for 13 asymptomatic SOD-1 mutation carriers, five clinically affected FALS patients and 45 SALS patients are summarised in table 1. Physical examination in all the asymptomatic SOD-1 mutation carriers was normal. The CMAP amplitude (symptomatic FALS 6.3±1.3 mV; SALS 6.0±0.4 mV; controls 10.0±0.4 mV, p<0.05) and NI (symptomatic FALS 1.1±0.4; SALS 0.7±0.1; controls 2.5±0.1, p<0.01) were significantly reduced in symptomatic FALS and SALS patients. There were no differences in the CMAP amplitude (asymptomatic SOD-1 mutation carriers, 11.1±0.7 mV; controls 10.3±0.5 mV) and NI (asymptomatic SOD-1 mutation carriers, 2.6±0.1; controls 2.6±0.2) between asymptomatic SOD-1 mutation carriers and normal controls.

Strength–duration time constant

The strength–duration time constant reflects persistent Na⁺ conductances at the node of Ranvier.19 To estimate τ_SD, nine axonal populations were studied from SR curves, starting from axons contributing to CMAP responses between 5 and 15% up to the maximum of 85–95%. As illustrated in figure 1A, the τ_SD was significantly longer in both symptomatic FALS (figure 1A) and SALS patients when plotted against their CMAP responses compared with controls (figure 1B). In addition, mean τ_SD was significantly increased in FALS (0.55±0.10 ms, p<0.05, figure 2) and SALS patients (0.52±0.02 ms, p<0.01, figure 2) when compared with controls. Although the numbers were small, the increase in τ_SD was a uniform finding, present across the different SOD mutations.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Strength–duration time constant (τ_SD), reflecting nodal persistent Na⁺ channel conductances: distribution of τ_SD in nine different populations of axons contributing to the compound muscle action potential (CMAP) response, starting from axons that contribute 5–15% of maximal response and increasing in 10% increments up to a maximum of 85–95%. (A) Strength–duration time constant was significantly increased in familial amyotrophic lateral sclerosis patients (clear circles) compared with controls (filled circles). (B) Strength–duration time constant was also significantly increased in sporadic amyotrophic lateral sclerosis patients (clear triangles) compared with controls (filled circles) for each of the populations.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Mean data illustrating that the strength–duration time constant (SDTC) was significantly increased in both familial amyotrophic lateral sclerosis patients (FALS) and sporadic amyotrophic lateral sclerosis (SALS) patients when compared with asymptomatic superoxide dismutase-1 (SOD-1) gene mutation carriers and controls (*p<0.05).

There were no significant differences in mean τ_SD between asymptomatic SOD-1 mutation carriers (0.37±0.02) and controls (0.41±0.02, p=31, figure 2). Axonal τ_SD ranged from 0.3 to 0.48 ms in asymptomatic SOD-1 mutation carriers, such that τ_SD overlapped with FALS patients in five asymptomatic SOD-1 mutation carriers.

Threshold electrotonus

Threshold electrotonus is the only technique that provides insight into both nodal and internodal membrane conductance. Previously, two types of abnormalities of threshold electrotonus have been described in SALS, namely the type I abnormality, in which there is a greater change in response to a subthreshold depolarising pulse, and the type II abnormality, in which there is a sudden decrease in membrane excitability marked by an abrupt increase in threshold.31 In the present study, neither the type I or type II abnormalities were evident in FALS patients or asymptomatic SOD-1 mutation carriers for the type I abnormality. However, the type I abnormality was evident in 31% of SALS patients, while the type II abnormality was not evident. Analysis of mean data revealed that there were no significant differences of threshold electrotonus between FALS patients, asymptomatic SOD-1 mutation carriers and controls (figure 3A, B). In SALS patients, there were greater threshold changes to both depolarising and hyperpolarising subthreshold conditioning pulses similar to the ‘fanned out’ response evident with membrane hyperpolarisation (figure 3C, D).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

Threshold electrotonus refers to changes in membrane excitability in response to long duration polarising currents. Threshold reduction with depolarisation is represented in an upward direction and hyperpolarisation in a downward direction. Threshold electrotonus was not significantly different in (A) familial amyotrophic lateral sclerosis patients (ALS) (filled circles) and (B) asymptomatic superoxide dismutase-1 carriers (filled diamonds) when compared with controls (open circles). In sporadic amyotrophic lateral sclerosis (SALS) patients, group data revealed a significant increase in threshold to (C) subthreshold depolarising currents at 90–100 ms, TEd (90–100 ms), and (D) subthreshold hyperpolarising currents at 90–100 ms, TEh (90–100 ms), producing a ‘fanned out’ response. *p<0.05; ***p<0.001.

I/V relationship

The I/V relationship estimates rectifying properties of nodal and internodal segments of the axon.27 32 The I/V gradient during depolarising subthreshold currents reflects conduction through outward rectifying K⁺ channels, while the I/V gradient during hyperpolarising subthreshold currents reflects inwardly rectifying conductances activated by hyperpolarisation.20 The hyperpolarising I/V gradient was not significantly different between the groups (FALS, 0.32±0.01; SALS, 0.38±0.01; asymptomatic SPD-1 mutation carriers, 0.35±0.01; controls, 0.37±0.01). Further, the resting I/V gradient was similar across the groups (FALS, 0.60±0.02; SALS, 0.56±0.02; asymptomatic SOD-1 mutation carriers, 0.59±0.02; controls, 0.62±0.02).

Recovery cycle of excitability

The recovery cycle reflects changes in membrane excitability in response to a supramaximal-conditioning stimulus.33 The initial phase of the recovery cycle is the refractory period and reflects recovery of transient Na⁺ channels from inactivation. The duration of the relative refractory period was not significantly different when compared with controls (FALS, 3.4±1.1 ms; SALS, 3.1±1.0 ms; asymptomatic SOD-1 mutation carriers, 3.2±1.1 ms; controls, 3.15±1.0 ms). Superexcitability, a period of increased axonal excitability due to a depolarising afterpotential spreading to the internodal axolemma,34 was significantly increased in SALS patients compared with controls (ALS, −27.9±1.4%; controls, −24.4±1.5%, p<0.05). However, superexcitability was not significantly different in FALS and asymptomatic SOD-1 mutation carriers, when compared with controls (FALS, −25.1±2.6%; asymptomatic SOD-1 mutation carriers, −24.3±1.3%; controls, −24.4±1.5%, figure 3B). The final phase of the recovery cycle, referred to as the late subexcitability period, reflects conduction of slow K⁺ channels.28 33 There were no significant differences in late subexcitability across the groups (FALS, 16.3±2.2%; SALS, 13.6±0.7%; asymptomatic SOD-1 mutation carriers, 17.1±1.4%; controls, 13.87±0.96%).

Cortical excitability and the processes of neurodegeneration

To clarify whether the upregulation of persistent Na⁺ conductances was intrinsic to the processes of neurodegeneration, changes in cortical excitability in FALS patients, in part reported previously,17 were correlated to the peripheral axonal abnormalities. Peak SICI, defined as the threshold required to produce and maintain a target response of 0.2 mV29 was significantly reduced in FALS patients (FALS, −2.7±1.3%; controls 13.7±1.3%, p<0.0001). In addition, averaged SICI, from ISI 1 to 7 ms, was also significantly reduced in FALS patients (FALS, −2.7±0.6%; controls, 9.1±1.1%, p<0.0001).

Correlation with clinical parameters and disease duration

Combining measures of axonal excitability, clinical assessment and disease severity, it was evident that τ_SD in FALS patients correlated with the CMAP amplitude (r=−0.4) and NI (r=−0.4), thereby suggesting that the increase in τ_SD was a marker of axonal loss. Further, τ_SD correlated with SICI (r=−0.8). Taken together, these findings may suggest that cortical hyperexcitability, as measured by reduction in SICI, underlies the increase in τ_SD and thereby axonal degeneration in FALS.

Discussion

The present study has established an increase in the strength duration time constant in FALS patients, while axonal excitability was normal in asymptomatic SOD-1 mutation carriers The increase in the strength duration time constant correlated with markers of peripheral disease burden, the CMAP amplitude and NI as well as SICI, a marker of cortical hyperexcitability. Interestingly, there were no significant changes in threshold electrotonus, recovery cycle and I/V in FALS patients. Taken together, these findings suggest that the increase in τ_SD may be associated with the process of axonal degeneration, and that cortical hyperexcitability may be associated with this increase in τ_SD in FALS.

The strength–duration time constant is a measure of the rate at which the threshold current declines as stimulus duration increases, and reflects the behaviour of persistent Na⁺ conductances in the axonal membrane.18 19 As reported previously, τ_SD was prolonged in SALS patients.8–11 35 In addition, τ_SD was also prolonged in FALS patients, thereby suggesting that upregulation of persistent Na⁺ conductances is a pathophysiological mechanism in FALS. Such a finding would be consistent with recent studies reporting upregulation of persistent Na⁺ channel conductances in the transgenic SOD-1 mouse model.36–39 Specifically, using patch-clamp techniques, upregulation of persistent Na⁺ conductances was reported in cortical and spinal motor neurons in the G93A and G85R SOD-1 transgenic mouse models.36–38 Further, this upregulation of persistent Na⁺ conductances was inhibited by riluzole, resulting in reduction of repetitive firing and thereby motoneuronal excitability.

Although mean τ_SD was not increased in asymptomatic SOD-1 mutation carriers, in five subjects there was an overlap in τ_SD values relative to FALS patients. This finding may suggest that ALS is not of acute onset, and that prolongation of τ_SD, and thereby upregulation of persistent Na⁺ conductances, may develop with time prior to disease onset. Further, the changes documented in the present series are consistent with studies reporting that upregulation of persistent Na⁺ conductances develops in presymptomatic mice expressing the G93A SOD-1 mutation.36

Prolongation of τ_SD has also been linked to the process of axonal regeneration and sprouting.11 14 Increased expression of persistent nodal Na⁺ channels and alteration in Na⁺ channel gating properties have been reported with axonal regeneration40 and appear to underlie the increase in persistent Na⁺ channel conductances and thereby prolongation of τ_SD. The finding that τ_SD correlated with the CMAP amplitude and NI supports the notion that in FALS patients, the increase in persistent Na⁺ channel conductances may be associated with the process of axonal degeneration.

The strength–duration time constant may also be affected by passive membrane properties at the node of Ranvier, with τ_SD classically increased with membrane depolarisation.27 Membrane depolarisation seems an unlikely explanation for the prolonged τ_SD in the present study, since other indices of membrane excitability did not suggest such a change. Specifically, superexcitability,41 the most sensitive parameter of membrane potential and typically reduced by depolarisation, remained unchanged in FALS patients, as did threshold electrotonus, which is normally ‘fanned in’ with depolarisation.41 In addition, axonal demyelination may be associated with prolongation of the τ_SD.42 As ALS is not recognised as a demyelinating disorder,1 this could not explain the prolonged τ_SD evident in the present study.

In contrast to prolongation of τ_SD, there were no significant changes in other axonal excitability parameters in FALS patients. Notably, the greater changes in threshold to both depolarising and hyperpolarising subthreshold conditioning pulses as well as increased superexcitability evident in SALS patients were not evident in FALS patients. Further, the type I abnormality of threshold electronus was not evident in FALS patients. Taken together, these findings suggest that paranodal fast and slow K⁺ channel conduction remained intact in FALS.

What are the pathophysiological mechanisms underlying neurodegeneration in FALS?

The finding that τ_SD correlated with SICI, a measure of cortical excitability, may provide indirect support for the dying forward process in FALS, which proposes that corticomotoneurons drive anterior horn cell loss.16 Specifically, given that upregulation of persistent Na⁺ currents has been previously linked to the processes of axonal degeneration,14 this correlation may suggest that cortical hyperexcitability is associated with motor neuron loss in FALS. Of further clinical relevance, riluzole, the only disease-modifying treatment for ALS, blocks persistent Na⁺ channels43 and reduces expression of persistent Na⁺ currents in mutant SOD-1 (G93A) motor neurons.36 Alternatively, the correlation between τ_SD and SICI could represent a compensatory upregulation of the upper motor neuron system attempting to overcome the lower motor neuron dysfunction.

In addition, the present study may also provide insight into the mechanisms underlying the clinical features of FALS, particularly cramps and fasciculations. Fasciculations, prominent features of ALS, reflect ectopic activity in motor axons.4 Upregulation of persistent Na⁺ conductances would increase depolarising drive, thereby resulting in axonal hyperexcitability and fasciculations.

In conclusion, the present study has documented upregulation of persistent Na⁺ conductances in FALS. This upregulation of persistent Na⁺ conductances correlated with peripheral markers of disease burden and measures of cortical excitability, thereby suggesting that the increase in persistent Na⁺ conductances is linked to the process of axonal degeneration and cortical hyperexcitability. Findings from the present series may provide support for a trial of novel neuroprotective strategies directed at persistent Na⁺ conductances in ALS patients.