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Original Investigation |

Axonal Ion Channel Dysfunction in C9orf72 Familial Amyotrophic Lateral Sclerosis FREE

Nimeshan Geevasinga, MBBS1; Parvathi Menon, MBBS1; James Howells, PhD2; Garth A. Nicholson, PhD3; Matthew C. Kiernan, DSc2; Steve Vucic, PhD1
[+] Author Affiliations
1Westmead Clinical School, University of Sydney, Sydney, Australia
2Brain and Mind Research Institute, University of Sydney, Sydney, Australia
3Department of Neurology, Concord Hospital, Central Clinical School, University of Sydney, Sydney, Australia
JAMA Neurol. 2015;72(1):49-57. doi:10.1001/jamaneurol.2014.2940.
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Published online

Importance  Abnormalities of axonal excitability characterized by upregulation of persistent sodium (Na+) conductances and reduced potassium (K+) currents have been reported in sporadic amyotrophic lateral sclerosis (SALS) phenotypes and linked to the development of clinical features such as fasciculations and neurodegeneration.

Objective  To investigate whether abnormalities of axonal ion channel function, particularly upregulation of persistent Na+ conductances and reduced K+ currents, form the pathophysiological basis of chromosome 9 open reading frame 72 (c9orf72) familial amyotrophic lateral sclerosis (FALS).

Design, Setting, and Participants  This was a prospective study. Clinical and functional assessment, along with motor-nerve excitability studies, were undertaken in 10 clinically affected patients with c9orf72 FALS, 9 asymptomatic c9orf72 mutation carriers, and 21 patients with SALS from 3 hospitals and 2 outpatient clinics.

Main Outcomes and Measures  Axonal excitability variables were measured in patients with c9orf72 ALS and results compared with matched patients with SALS and healthy control participants.

Results  Strength-duration time constant (τSD) was significantly increased in the patients with c9orf72 FALS and those with SALS (mean [SD], c9orf72 FALS: 0.50 [0.02] milliseconds; SALS: 0.52 [0.02] milliseconds; P < .01) when compared with control participants (mean [SD], 0.44 [0.01] milliseconds). In contrast, there were no significant changes of τSD in asymptomatic c9orf72 mutation carriers (P = .42). An accompanying increase in depolarizing threshold electrotonus at 90 to 100 milliseconds (TEd 90-100 milliseconds) was also evident in the c9orf72 FALS (P < .05) and SALS (P < .01) cohorts. Mathematical modeling suggested that an increase in persistent Na+ conductances, along with reduced K+ currents, best explained the changes in axonal excitability. Importantly, these abnormalities in axonal excitability correlated with the motor amplitude (τSD: R = −0.38, P < .05 and TEd 90-100 milliseconds: R = −0.44, P < .01), muscle weakness (TEd 90-100 milliseconds: R = −0.32, P < .05), and the ALS Functional Rating Scale (TEd 90-100 milliseconds: R = −0.34, P < .05).

Conclusions and Relevance  Findings from the present study establish that upregulation of persistent Na+ conductances and reduced K+ currents were evident in both c9orf72 FALS and SALS cohorts, and these changes in axonal excitability were associated with motor neuron degeneration.

Figures in this Article

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and universally fatal neurodegenerative disorder of the motor neurons.1 A genetic etiology has been identified in up to 60% of familial ALS (FALS) and 20% of apparently sporadic ALS (SALS) cohorts, with at least 21 genes and genetic loci implicated in ALS pathogenesis.2 An increased hexanucleotide repeat expansion (GGGGCC) in the first intron of the chromosome 9 open reading frame 72 (c9orf72) gene on chromosome 9p21 was reported to be the most common of the genetic mutations in ALS, underlining approximately 40% of FALS and 20% of SALS cases,3,4 although subsequent studies have established a c9orf72 mutation frequency of 4% to 8%.5

The c9orf72 phenotype may be characterized by ALS and frontotemporal dementia with psychiatric features, although the penetrance and expression of the genotype may vary within and between cohorts.3,4,6,7 The c9orf72-associated ALS phenotype is clinically characterized by coexistence of upper motor neuron (UMN) and lower motor neuron (LMN) signs encompassing multiple body regions, with an earlier age at onset and shorter survival.8 Importantly, LMN signs are characterized by fasciculations and muscle wasting and weakness, clinically indistinguishable from the LMN features in SALS cohorts.

The pathophysiological mechanisms by which c9orf72 gene hexanucleotide expansion leads to the development of neurodegeneration and thereby the clinical features of ALS, particularly fasciculations, remain to be established.3,4 Widespread axonal ion channel dysfunction, including upregulation of persistent sodium (Na+) conductances and reduction of slow and fast potassium (K+) channel conductances, has been extensively documented in SALS, resulting in motor axonal hyperexcitability.918 Importantly, such changes in motor axonal excitability were postulated to underlie motor neuron degeneration and the development of clinical features of ALS, particularly fasciculations.916,19 In addition, motor axonal hyperexcitability, as reflected by upregulation of persistent Na+ conductances, was also reported in FALS cohorts secondary to mutations in the superoxide dismuate–1 (SOD-1) gene and linked to the process of neurodegeneration.

Threshold-tracking techniques may provide unique insights into nodal and internodal axonal membrane properties by sequentially assessing multiple excitability parameters.2022 The strength-duration time constant (τSD), a measure of the rate at which the threshold current for a target potential declines as stimulus duration increases,15,2327 appears to be a biomarker of persistent Na+ conductances.28 This notion is underscored by computer modeling studies of the human motor axon establishing that the τSD reflects the behavior of persistent Na+ conductances.24 In addition, depolarizing threshold electrotonus (TEd) and hyperpolarizing threshold electrotonus (TEh), along with superexcitability and late subexcitability, appear to be robust biomarkers of fast and slow K+ conductances, respectively.28 Consequently, the present study used axonal-excitability techniques to determine whether upregulation of persistent Na+ conductances and reduction of K+ currents was a feature of c9orf72 FALS and whether such changes in axonal excitability were linked to the processes of neurodegeneration, potentially informing the pathophysiological basis of c9orf72 FALS. In addition, the present study also aimed to determine whether axonal ion channel dysfunction was evident in asymptomatic c9orf72 mutation carriers.

Studies were undertaken on 10 clinically affected patients with familial c9orf72 ALS as defined by the Awaji criteria (6 men and 4 women; mean age, 63 years; age range, 41-78 years) and 9 asymptomatic c9orf72 mutation carriers (1 man and 8 women; mean age, 45 years; age range, 24-60 years). For comparison, 21 patients with SALS were studied (16 men and 5 women; mean age, 55 years; age range, 32-73 years). All patients with ALS (sporadic and familial) were clinically staged using the ALS Functional Rating Scale–Revised (ALSFRS-R).29 In addition, muscle strength was assessed using the Medical Research Council (MRC) score, with the following muscle groups assessed bilaterally yielding a total MRC score of 90: shoulder abduction; elbow flexion; elbow extension; wrist dorsiflexion; finger abduction; thumb abduction; hip flexion; knee extension; and ankle dorsiflexion.17,30 The degree of UMN dysfunction was assessed by a specific UMN score incorporating the following parameters: brisk jaw jerks (1 point); brisk facial reflex (1 point); pathologically brisk biceps, triceps, supinator, finger, knee, and ankle reflexes (1 point for each, assessed bilaterally); and extensor plantar responses (1 point each and assessed bilaterally). The UMN score ranged from 0 (no UMN dysfunction) to 16 (severe UMN dysfunction).31 Cognitive screening was undertaken only in the patients with c9orf72 FALS and asymptomatic mutation carriers using the Addenbrooke Cognitive Examination–Revised (ACE-R).32 Patients and carriers with diabetes mellitus or chronic renal failure were excluded from the study. None of the patients were taking medications that could affect the results. All participants gave written informed consent to the procedures, which were approved by the South East Sydney Area Health Service Human Research Ethics Committee.

Following clinical staging and phenotyping, the median nerve was stimulated at the wrist and the resultant compound muscle action potential (CMAP) recorded from the abductor pollicis brevis (APB) using surface electrodes. The active recording electrode was placed over the motor point of APB and the reference was placed 4 cm distally at the base of the thumb. Skin temperature was monitored close to the site of stimulation for the duration of each. Prior to excitability studies, CMAP amplitude and onset latency and F-wave latency and frequency were all measured. The neurophysiological index, a marker of peripheral disease burden in ALS, was derived according to a previously reported formula33:

Neurophysiological index = CMAP amplitude (millivolt [mV]) × F-wave frequency/distal motor latency (milliseconds), where F-wave frequency was expressed as the number of F responses recorded in 20 trials.

Axonal Excitability

All patients with ALS underwent axonal excitability studies on the median motor nerve, according to a previously described protocol.21 The median nerve was stimulated at the wrist using 5-mm nonpolarizable Ag-AgCl electrodes (ConMed) with the cathode positioned over the skin crease and anode approximately 10 cm proximally over the lateral forearm. Stimulation was computer controlled and converted to current using an isolated linear bipolar constant current simulator (maximal output ±50 mA; DS5, Digitimer). The CMAP responses were recorded from the APB muscle with the active (G1) electrode positioned over the motor point and the reference (G2) electrode placed at the proximal phalanx 4 cm away.

Test current pulses were applied at 0.5-second intervals and combined with either subthreshold polarizing currents or suprathreshold conditioning stimuli, according to previously described protocols.21 The CMAP amplitude was measured from baseline to negative peak, with the target set to 40% of maximum for all tracking studies. Proportional tracking was used to determine the changes in threshold current required to produce and maintain a target response.34

Electrical stimuli were increased in incremental steps to generate the motor stimulus-response curve. Subsequently, the τSD, a biomarker of persistent Na+ conductance, and rheobase, defined as the threshold current for a stimulus of infinitely long duration, were measured according to the Weiss formula.15,25,34,35

Following the determination of the stimulus strength-duration relationship, threshold electrotonus (TE) was determined using subthreshold polarizing currents of 100-millisecond duration, set to ±40% of controlled threshold current.21,28,34 Test stimuli of 1-millisecond duration were used to produce and maintain a target response of 40% of maximum CMAP amplitude. Three stimulus combinations were tested sequentially: test stimulus alone (measured control threshold current); test stimulus + depolarizing current; and test stimulus + hyperpolarizing current. Threshold was tested at 26 points before, during, and after the 100-millisecond polarizing pulse. The stimulus combinations were repeated until 3 valid estimates were recorded within 15% of target response.21 The following TE changes were recorded with subthreshold depolarizing currents: TEd at 10 to 20 milliseconds; TEd at 40 to 60 milliseconds; and TEd at 90 to 100 milliseconds. In addition, changes in membrane threshold to hyperpolarizing currents at 10 to 20 milliseconds, TEh at 10 to 20 milliseconds and at 90 to 100 milliseconds, and TEh at 90 to 100 milliseconds were also measured.

The current-threshold relationship (I/V), a biomarker of inward and outward rectifying membrane currents,28 was assessed by tracking threshold changes following subthreshold polarizing currents of 200-millisecond duration, which were altered in ramp fashion from +50% (depolarizing) to −100% (hyperpolarizing) of controlled threshold in 10% steps. Conditioning stimuli were alternated with test stimuli until 3 valid threshold estimates were recorded. The following parameters were recorded: (1) resting I/V slope, calculated from polarizing currents between +10% to −10% and (2) hyperpolarizing I/V slope, calculated from polarizing current between −80% and −100%.

Lastly, the recovery cycle of axonal excitability was recorded, according to a well-established protocol.21,36 The following parameters were measured: (1) relative refractory period (in milliseconds), defined as the first intercept at which the recovery curve crosses the x-axis and a biomarker of transient Na+ channel function28; (2) superexcitability (%), defined as the minimum mean threshold change of 3 adjacent points and a biomarker of paranodal fast K+ channel conduction28; and (3) late subexcitability (%), defined as the largest increase in threshold following the superexcitability period and a biomarker of nodal K+ channel conduction.28

Recordings of CMAP responses were amplified and filtered (3 Hz-3 kHz) using a Nicolet-Biomedical EA-2 amplifier (Cardinal Health Viking Select version 11.1.0, Viasys Healthcare Neurocare Group) and sampled at 10 kHz using a 16-bit data-acquisition card (National Instruments PCI-MIO-16E-4). Data acquisition and stimulation delivery were controlled by QTRACS software (TROND-F, version 16/02/2009; Hugh Bostock, PhD, Institute of Neurology, London, England).

Mathematical Modeling of Axonal Excitability Changes

To model the axonal excitability changes evident in patients with c9orf72 FALS and those with SALS, mathematical simulations were undertaken using an established model of the human motor axon.13,3740 Nodal transient and persistent Na+ currents were modeled using data from voltage-clamp and latent addition studies in human axons.24,41 This model incorporates slow and fast K+ currents at both the node and internode; the hyperpolarization-activated current Ih at the internode; and leak and pump currents at both the node and internode.42 The discrepancy between the model and the group data was calculated as the weighted sum of the squares of the error terms: (xm − x̄),2 where xm is the model threshold and x̄ is the mean threshold for the group data. The weights were 0.5 for the τSD and 1 for each of the other threshold measurement types (ie, TE, current-threshold relationship, and the recovery cycle). The model was first adjusted to fit the normal control data using an interactive least-squares procedure, which minimized the discrepancy between the model and data.42

Statistical Analysis

Axonal excitability studies were compared with 34 age-matched control individuals (13 men and 21 women; mean [SD] age, 51.1 [2.2] years), with the t test used for assessing differences between means among patients, carriers, and control participants. All of the axonal excitability data were normally distributed as assessed by the Shapiro-Wilk test. Pearson correlation coefficients were used to examine the relationship between parameters. Because measures of excitability may vary with age and temperature, parameters were compensated for age, temperature, and sex, if required before statistical analysis, using the relations established previously in control individuals.43 The ACE-R score was nonparametrically distributed and a Mann-Whitney U test was used to compare differences between c9orf72 FALS and asymptomatic mutation carriers. P < .05 was considered statistically significant, and the probability values were corrected for multiple comparisons. Results were expressed as mean ± standard error of the mean (SEM) or median (interquartile range).

Clinical Phenotype

The clinical features for the 10 clinically affected patients with FALS, 9 asymptomatic c9orf72 mutation carriers, and 21 patients with SALS are summarized in the Table. At the time of assessment, the patients with c9orf72 FALS and SALS presented with the classic ALS phenotype. Bulbar-onset disease was evident in 40% of patients with c9orf72 FALS and 29% of patients with SALS, while limb-onset disease was reported in 60% of patients with c9orf72 FALS and 71% of those with SALS. At the time of assessment, the mean (SEM) age at onset was unexpectedly higher in the c9orf72 FALS cohort (c9orf72 FALS: 63 [3.8] years and SALS: 55 [2.5] years; P = .08), while the mean (SEM) disease duration from symptom onset was comparable between the patients with FALS and those with SALS (c9orf72 FALS: 14.3 [3.9] months and SALS: 12.5 [2] months; P = .35).

Table Graphic Jump LocationTable.  Clinical Details for the Patients With C9orf72 FALS and SALS and AMCs

The mean (SEM) ALSFRS-R score in the c9orf72 FALS cohort was 39 (3.2) and was comparable with that in the SALS cohort (41.6 [1.4]; P = .22), indicating a similar degree of functional impairment. In addition, the mean (SEM) degree of muscle weakness was similar between the groups as reflected by the upper limb (c9orf72 FALS: 54 [2.2] and SALS: 53.8 [1.8]; P = .25; maximal score of 60 when normal strength), lower limb (c9orf72 FALS: 28 [0.7] and SALS: 28 [0.8]; P = .20; maximal score 30 when muscle strength normal), and total MRC scores (c9orf72 FALS: 82 [2.3] and SALS: 82 [1.5]; P = .48; maximal score 90 when muscle strength normal). In addition, there was a comparable degree of UMN dysfunction in both the patients with c9orf72 FALS and those with SALS (mean [SEM] UMN score, c9orf72 FALS: 10.7 [1.8] and SALS: 10 [0.6]). While frontotemporal dementia was not evident in the current c9orf72 ALS cohort, there was a mild reduction of the ACE-R score, suggesting the presence of subtle cognitive dysfunction in patients with c9orf72 FALS (c9orf72 carriers: ACE-R median, 86 [interquartile range, 76-92] and asymptomatic mutation carriers: ACE-R median, 97 [interquartile range, 95-98]; P < .05).

Neurological examination findings were normal in all of the asymptomatic c9orf72 mutation carriers. Specifically, there was no clinical evidence of UMN or LMN dysfunction at the time of assessment. To date, all of the asymptomatic mutation carriers have remained well.

Neurophysiology

The mean (SEM) CMAP amplitudes were significantly reduced in the patients with c9orf72 FALS and SALS when compared with normal control participants (c9orf72 FALS: 3.4 [0.3] mV; SALS: 5.6 [0.8] mV; and control participants: 8.8 [0.4] mV; F = 17.9; P < .01). Importantly, there were no significant differences in CMAP amplitudes between the c9orf72 FALS and SALS cohorts (P = .11). In addition, the neurophysiological index was also significantly reduced in the patients with c9orf72 FALS and those with SALS when compared with control participants (mean [SEM], c9orf72 FALS: 1.1 [0.2]; SALS: 1.2 [0.2]; and control participants: 2.5 [0.1]; P < .01). While these findings indicate a significant degree of LMN dysfunction in the c9orf72 FALS and SALS cohorts, there was a trend for the CMAP amplitude to be lower in the patients with c9orf72 FALS, despite a comparable clinical disease burden. This discordant finding could be potentially explained by a greater sensitivity of neurophysiological techniques in detecting LMN dysfunction.44 Of further relevance, clinical and electrical evidence of fasciculations were evident in all the patients with c9orf72 FALS and SALS.

In contrast, there was no significant differences in the mean (SEM) CMAP amplitude (c9orf72 mutation carriers: 7.0 [0.6] mV and control participants: 8.8 [0.4] mV) and neurophysiological index (asymptomatic c9orf72 mutation carriers: 2.5 [0.3] and control participants: 2.5 [0.1]) between the c9orf72 mutation carriers and normal control participants, reaffirming the structural integrity of the peripheral nerves at the time of assessment in asymptomatic control participants.

Strength-Duration Time Constant and Rheobase

The τSD is a biomarker of persistent Na+ conductances at the node of Ranvier.15,2427,45 The mean (SEM) τSD was significantly increased in the patients with c9orf72 FALS (0.5 [0.02] milliseconds; P < .05) and SALS (0.52 [0.02] milliseconds; P < .01) when compared with control participants (Figure 1A and B). In contrast, there were no significant differences in the τSD between asymptomatic c9orf72 mutation carriers and control participants (asymptomatic c9orf72 mutation carriers: 0.45 [0.04] and control participants: 0.44 [0.01]; P = .42; Figure 1A and B).

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Figure 1.
Strength-Duration Time Constant in Amyotrophic Lateral Sclerosis (ALS)

The strength-duration time constant reflects nodal persistent Na+ channel conductances. A, The strength-duration time constant was significantly increased in patients with c9orf72 familial ALS (FALS, triangles) and sporadic ALS (SALS, circles) when compared with asymptomatic c9orf72 mutation carriers (AMCs, diamonds) and control participants (squares). B, Mean strength-duration time constant was significantly increased in patients with FALS and SALS. C, Rheobase was significantly reduced in patients with FALS and SALS. The error bars indicate standard errors of the mean.aP < .05.bP < .01.cP < .001.

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Of further relevance, rheobase was significantly reduced in both patients with c9orf72 FALS (mean [SEM], 1.61 [0.5] mA; P < .001; Figure 1A and C) and SALS (mean [SEM], 1.35 [0.10] mA; control participants: 2.27 [0.23] mA; P < .001; Figure 1A and C). In contrast, rheobase was slightly increased in the asymptomatic c9orf72 mutation carriers (mean [SEM], 2.64 [0.72] mA; Figure 1A and C) but again this increase was not significant.

Threshold Electrotonus

Threshold electrotonus provides insight into nodal and internodal membrane conductances. Previously, 2 types of abnormalities of TE have been described in SALS, namely the type I abnormality, in which there is a greater change in response to a subthreshold depolarizing pulse, and the type II abnormality, in which there is a sudden decrease in membrane excitability marked by an abrupt increase in threshold.13 In the present study, the type I abnormality was evident in 40% of the patients with c9orf72 FALS and 29% of those with SALS, while the type II abnormality was not evident. Furthermore, neither the type I nor type II abnormalities were evident in the asymptomatic c9orf72 mutation carriers.

Group data analysis disclosed the presence of a significant difference in TE between patients with FALS and SALS, asymptomatic c9orf72 mutation carriers, and control participants (Figure 2A). Specifically, mean (SEM) TEd (90-100 milliseconds) was significantly increased in both the c9orf72 FALS (51.0% [1.9%]; P < .05) and SALS (50.3% [1.4%]; P < .01) cohorts when compared with control participants (45.6% [0.6%]) (Figure 2B). Although there was a greater threshold change to hyperpolarizing subthreshold conditioning pulses in the patients with c9orf72 FALS and SALS (c9orf72 FALS: −128.7% [8.7%]; SALS: −122.5% [0.6%]; and control participants: −117.5% [3.2%]; P = .13; Figure 2), this difference was not significant.

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Figure 2.
Threshold Electrotonus (TE) Measures in Amyotrophic Lateral Sclerosis (ALS)

Threshold electrotonus reflects changes in membrane excitability in response to long-duration polarizing currents. Threshold reduction with depolarization is represented in an upward direction and hyperpolarization in a downward direction. A, Depolarizing TE (TEd) was significantly increased in c9orf72 familial ALS (FALS, triangles) and sporadic ALS (SALS, circles) when compared with asymptomatic c9orf72 mutation carriers (AMCs, diamonds) and control participants (squares). In contrast, hyperpolarizing TE was not significant (NS) between groups. B, Mean TEd at 90 to 100 milliseconds was significantly increased in c9orf72 FALS and SALS. The error bars indicate standard errors of the mean.aP < .05.bP < .01.

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Current-Threshold Relationship

The I/V relationship estimates the inward and outward rectifying properties of nodal and internodal axonal segments, with the hyperpolarizing I/V gradient reflecting conductances via inward rectifying currents.34,46 The hyperpolarizing I/V gradient was significantly increased in patients with c9orf72 FALS (mean [SEM], 0.42 [0.02]; P < .05) and SALS (mean [SEM], 0.41 [0.02]; P < .05) when compared with asymptomatic mutation carriers (mean [SEM], 0.34 [0.03]) and control participants (mean [SEM], 0.36 [0.01]).

Recovery Cycle of Excitability

The mean (SEM) relative refractory period duration was not significantly different when compared with control participants (c9orf72 FALS: 3.7 [0.2] milliseconds, P = .06; SALS: 3.8 [0.3] milliseconds, P = .08; asymptomatic c9orf72 mutation carriers: 3.2 [0.1] milliseconds, P = .36; and control participants: 3.3 [0.1] milliseconds). In addition, there were no significant differences in superexcitability and late subexcitability between groups.

Correlation With Clinical Parameters and Disease Duration

Combining measures of axonal excitability, clinical assessment, and disease severity, it was evident that τSD (R = −0.38; P < .05; Figure 3A) and TEd (90-100 milliseconds) (R = −0.44; P < .01; Figure 3B) in patients with c9orf72 FALS and SALS correlated with the CMAP amplitude, thereby suggesting that the increase in the τSD and TEd (90-100 milliseconds) were potentially linked to axonal degeneration. In addition, the TEd (90-100 milliseconds) was significantly correlated with the ALSFRS-R (R = −0.34; P < .05) and MRC upper limb scores (R = −0.32; P < .05). Taken together, these findings suggest that abnormalities of axonal excitability, particularly increased the τSD and TEd (90-100 milliseconds), are late features of c9orf72 FALS and linked to the process of neurodegeneration.

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Figure 3.
Correlation of Axonal Excitability Parameters With Motor Amplitudes

The strength-duration time constant (A) and depolarizing threshold electrotonus (TEd) at 90 to 100 milliseconds (B) significantly correlated with the compound muscle action potential (CMAP) amplitude, thereby suggesting that strength-duration time constant and TEd at 90 to 100 milliseconds were linked with the process of axonal degeneration. FALS indicates familial amyotrophic lateral sclerosis; SALS, sporadic ALS.

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Mathematical Modeling of Abnormal Excitability Properties

To assist in interpreting the changes observed in axonal excitability, a mathematical model of the human motor axon was adjusted to provide a close match to the control group (Figures 4 and 5). The model was then used to explore which membrane parameter changes could reproduce the changes seen in the combined FALS and SALS case recordings. The axonal excitability data in c9orf72 FALS and SALS were best modeled by a 30% reduction in the nodal slow K+ conductance along with an increase in persistent Na+ conductances and increase in Ih current, reducing the overall discrepancy by 81.7%. The modeled changes resulted in a net depolarization of resting membrane potential of 0.8 mV.

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Figure 4.
Mathematical Modeling of Axonal Excitability Parameters in Amyotrophic Lateral Sclerosis

Comparison of group data (control participants, open circles; combined c9orf72 and sporadic ALS, filled circles) and mathematical model (open triangles, normal model; c9orf72 and sporadic ALS model, filled triangles). A, Threshold electrotonus group data. B, Modeled threshold electrotonus. C, Current-threshold relationship. D, Modeled current-threshold relationship.

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Figure 5.
Mathematical Modeling of Strength-Duration Time Constant in Amyotrophic Lateral Sclerosis (ALS)

Comparisons of recorded and modeled strength-duration time constant data. The strength-duration time constant was significantly increased in patients with c9orf72 familial ALS (FALS) and sporadic ALS (SALS). Modeled strength-duration time constant for normal and combined SALS and FALS data sets.

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The present study established a pattern of axonal excitability abnormalities in c9orf72 FALS that resembled changes observed in patients with SALS. Specifically, the τSD was significantly longer and TEd at 90 to 100 milliseconds significantly greater in the c9orf72 FALS and SALS cohorts. Mathematical modeling suggested that the changes in axonal excitability were best explained by a reduction of slow nodal K+ currents along with an increase in persistent Na+ conductances, inward rectification, and Na+ pump activity. Importantly, the abnormalities of axonal excitability correlated with functional (ALSFRS-R and MRC upper limb scores) and neurophysiological (CMAP amplitude) biomarkers of peripheral disease burden. In contrast, axonal excitability was normal in asymptomatic c9orf72 mutation carriers. Taken together, the findings from the present series have identified a potential role for a reduction of slow nodal K+ currents and an upregulation of persistent Na+ conductances in the pathophysiology of c9orf72-associated FALS.

The τSD, or chronaxie, is a measure of the rate at which the threshold current declines as stimulus duration increases.24 The τSD is a biomarker of persistent Na+ conductances,10,12,15,23,2528,34,45,47 a notion supported by computer modeling of the human motor axon.24 Prolongation of the τSD has previously been established as a feature of SALS, attributed to upregulation of persistent Na+ conductances.9,10,12,15 Importantly, upregulation of persistent Na+ conductances has been linked to the development of axonal degeneration in ALS,9,10,19 including the split-hand pattern of degeneration,18 as well as motor axonal hyperexcitability and generation of fasciculations.9,10,12,15,17,18 In addition, prolongation of the τSD was also reported in SOD-1 FALS and correlated with biomarkers of peripheral disease burden,48 thereby suggesting that upregulation of persistent Na+ conductances contributed to the development of clinical features and neurodegeneration in SOD-1 FALS, a finding in keeping with transgenic SOD-1 mouse model studies.49

The present study establishes that prolongation of the τSD, and thereby upregulation of persistent Na+ conductances, was a pathophysiological feature of c9orf72 FALS, a notion supported by mathematical modeling of axonal excitability. The correlation between prolongation of the τSD and biomarkers of peripheral disease burden (including the CMAP amplitude) suggests that persistent Na+ conductances may contribute to motor neuron degeneration in c9orf72 FALS. Importantly, findings from the present study imply the existence of a common pathogenic pathway in ALS, irrespective of the underlying genotype.

In addition to prolongation of the τSD, there were significant changes in TEd. Specifically, the type I abnormality of TE, in which there was a greater change in response to a subthreshold depolarizing pulse, was evident in the c9of72 FALS cohort but not asymptomatic mutation carriers. In addition, the mean TEd (90-100 milliseconds) was significantly increased in c9orf72 FALS and SALS, correlating with peripheral biomarkers of peripheral disease burden, thereby suggesting that reduction of slow K+ currents contributed to the development of axonal hyperexcitability and clinical features in c9orf72 FALS. Importantly, mathematical modeling indicated that reduction of slow K+ currents in a large part accounted for the changes observed in axonal excitability, suggesting an important role for slow K+ channels in c9orf72 pathophysiology. This notion is further underscored by recent patch-clamp studies reporting that retigabine, a K+ channel activator, reduces axonal hyperexcitability and improves motor neuron survival in pluripotent stem cells derived from patients with c9orf72 FALS, thereby implying a therapeutic potential for retigabine.49 Of further relevance, a similar degree of reduction in slow K+ currents in the patients with c9orf72 FALS and SALS underscores the existence of common pathogenic pathways.

It could also be argued that the observed reduction in slow K+ conductances contributed to the increase in the τSD through depolarization of the resting membrane potential. Such a hypothesis cannot be discounted given that modeling studies suggested that reduction in slow K+ plays a bigger role in reducing the discrepancy in the measure of the τSD than the increase in fraction of persistent Na+ channels. Given that axonal excitability parameters, such as superexcitability and TE, which are reduced with resting membrane potential depolarization,20 remained unchanged argues against the notion that membrane depolarization solely accounted for the increase in the τSD.

In contrast to previous studies in patients with SALS,9,10 paranodal fast K+ channel function appears to be preserved in patients with c9orf72 FALS and the current SALS cohort. Specifically, superexcitability and TEd (10-20 milliseconds), both biomarkers of paranodal fast K+ channels,28 were not significantly altered in the c9orf72 FALS and SALS cohorts. Importantly, paranodal fast K+ channel dysfunction appears to evolve with disease progression and seems most pronounced immediately prior to motor axonal loss.50 Consequently, the discordant findings between the current and previous ALS cohorts may relate to differences in the extent and stage of motor axonal loss, reflecting the membrane properties of surviving motor axons at the time of assessment.

An increase in the hyperpolarizing I/V gradient was also evident in the c9orf72 FALS and SALS cohorts and is best explained by an increase in inward rectifying currents (Ih). The increase in Ih most likely represents an adaptive response to the increased demands on surviving motor axons, perhaps secondary to axonal hyperexcitability due to reduction in slow K+ currents and upregulation of persistent Na+ conductances.

In contrast to abnormalities of axonal excitability evident in the patients with c9orf72 FALS and SALS, there were no significant changes in asymptomatic c9orf72 mutation carriers. This finding is in keeping with previous studies in SOD-1 asymptomatic mutation carriers,48 implying that factors other than inheritance of the genetic mutation may be required to trigger the disease process.

The mechanisms by which axonal ion channel dysfunction leads to neurodegeneration and adverse survival in ALS51 remain to be elucidated, although it has been postulated that an influx of Na+ ions results in reverse operation of the Na+/calcium (Ca2+)-exchanger, intra-axonal accumulation of Ca2+, and ultimately activation of Ca2+-dependent enzyme pathways leading to motor neuron degeneration.5256 In addition, reduction of slow K+ currents and upregulation of persistent Na+ conductances would increase the depolarizing drive, thereby leading to axonal hyperexcitability and the development of cramps and fasciculations, both prominent symptoms in ALS.1,5762

Corresponding Author: Steve Vucic, PhD, Department of Neurology, Westmead Hospital, Cnr Hawkesbury and Darcy Roads, Westmead, NSW 2145, Australia (s.vucic@neura.edu.au).

Accepted for Publication: August 22, 2014.

Published Online: November 10, 2014. doi:10.1001/jamaneurol.2014.2940.

Author Contributions: Dr Vucic had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Nicholson, Kiernan, Vucic.

Acquisition, analysis, or interpretation of data: Geevasinga, Menon, Howells, Kiernan, Vucic.

Drafting of the manuscript: Geevasinga, Menon, Howells, Kiernan, Vucic.

Critical revision of the manuscript for important intellectual content: Nicholson, Kiernan, Vucic.

Statistical analysis: Geevasinga, Menon, Vucic.

Obtained funding: Kiernan, Vucic.

Administrative, technical, or material support: Geevasinga, Menon, Howells, Vucic.

Study supervision: Nicholson, Kiernan, Vucic.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was supported in part by research grants from the Motor Neuron Disease Research Institute of Australia (Dr Geevasinga) and the National Health and Medical Research Council of Australia (project grants 510233, 1024915, and 1055778; Drs Kiernan and Vucic). This work was also supported by funding to Forefront, a collaborative research group dedicated to the study of frontotemporal dementia and motor neuron disease, from the National Health and Medical Research Council of Australia (program grant 1037746, Dr Kiernan) and Pfizer Neuroscience (grant WS2343874, Dr Geevasinga).

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Kiernan  MC, Vucic  S, Cheah  BC,  et al.  Amyotrophic lateral sclerosis. Lancet. 2011;377(9769):942-955.
PubMed   |  Link to Article
Andersen  PM, Al-Chalabi  A.  Clinical genetics of amyotrophic lateral sclerosis: what do we really know? Nat Rev Neurol. 2011;7(11):603-615.
PubMed   |  Link to Article
Renton  AE, Majounie  E, Waite  A,  et al; ITALSGEN Consortium.  A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72(2):257-268.
PubMed   |  Link to Article
DeJesus-Hernandez  M, Mackenzie  IR, Boeve  BF,  et al.  Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72(2):245-256.
PubMed   |  Link to Article
Majounie  E, Renton  AE, Mok  K,  et al; Chromosome 9-ALS/FTD Consortium; French research network on FTLD/FTLD/ALS; ITALSGEN Consortium.  Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. 2012;11(4):323-330.
PubMed   |  Link to Article
Snowden  JS, Harris  J, Richardson  A,  et al.  Frontotemporal dementia with amyotrophic lateral sclerosis: a clinical comparison of patients with and without repeat expansions in C9orf72. Amyotroph Lateral Scler Frontotemporal Degener. 2013;14(3):172-176.
PubMed   |  Link to Article
Williams  KL, Fifita  JA, Vucic  S,  et al.  Pathophysiological insights into ALS with C9ORF72 expansions. J Neurol Neurosurg Psychiatry. 2013;84(8):931-935.
PubMed   |  Link to Article
van Rheenen  W, van Blitterswijk  M, Huisman  MH,  et al.  Hexanucleotide repeat expansions in C9ORF72 in the spectrum of motor neuron diseases. Neurology. 2012;79(9):878-882.
PubMed   |  Link to Article
Vucic  S, Kiernan  MC.  Axonal excitability properties in amyotrophic lateral sclerosis. Clin Neurophysiol. 2006;117(7):1458-1466.
PubMed   |  Link to Article
Kanai  K, Kuwabara  S, Misawa  S,  et al.  Altered axonal excitability properties in amyotrophic lateral sclerosis: impaired potassium channel function related to disease stage. Brain. 2006;129(pt 4):953-962.
PubMed   |  Link to Article
Nakata  M, Kuwabara  S, Kanai  K,  et al.  Distal excitability changes in motor axons in amyotrophic lateral sclerosis. Clin Neurophysiol. 2006;117(7):1444-1448.
PubMed   |  Link to Article
Tamura  N, Kuwabara  S, Misawa  S,  et al.  Increased nodal persistent Na+ currents in human neuropathy and motor neuron disease estimated by latent addition. Clin Neurophysiol. 2006;117(11):2451-2458.
PubMed   |  Link to Article
Bostock  H, Sharief  MK, Reid  G, Murray  NM.  Axonal ion channel dysfunction in amyotrophic lateral sclerosis. Brain. 1995;118(pt 1):217-225.
PubMed   |  Link to Article
Horn  S, Quasthoff  S, Grafe  P, Bostock  H, Renner  R, Schrank  B.  Abnormal axonal inward rectification in diabetic neuropathy. Muscle Nerve. 1996;19(10):1268-1275.
PubMed   |  Link to Article
Mogyoros  I, Kiernan  MC, Burke  D, Bostock  H.  Strength-duration properties of sensory and motor axons in amyotrophic lateral sclerosis. Brain. 1998;121(pt 5):851-859.
PubMed   |  Link to Article
Kiernan  M, Burke  D. Threshold electrotonus and the assessment of nerve excitability in amyotrophic lateral sclerosis. In: Eisen  A, ed. Clinical Neurophysiology of Motor Neuron Diseases. Amsterdam, the Netherlands: Elsevier; 2004:359-366.
Menon  P, Kiernan  MC, Vucic  S.  ALS pathophysiology: insights from the split-hand phenomenon. Clin Neurophysiol. 2014;125(1):186-193.
PubMed   |  Link to Article
Shibuya  K, Misawa  S, Nasu  S,  et al.  Split hand syndrome in amyotrophic lateral sclerosis: different excitability changes in the thenar and hypothenar motor axons. J Neurol Neurosurg Psychiatry. 2013;84(9):969-972.
PubMed   |  Link to Article
Kanai  K, Kuwabara  S, Arai  K, Sung  JY, Ogawara  K, Hattori  T.  Muscle cramp in Machado-Joseph disease: altered motor axonal excitability properties and mexiletine treatment. Brain. 2003;126(pt 4):965-973.
PubMed   |  Link to Article
Kiernan  MC, Bostock  H.  Effects of membrane polarization and ischaemia on the excitability properties of human motor axons. Brain. 2000;123(pt 12):2542-2551.
PubMed   |  Link to Article
Kiernan  MC, Burke  D, Andersen  KV, Bostock  H.  Multiple measures of axonal excitability: a new approach in clinical testing. Muscle Nerve. 2000;23(3):399-409.
PubMed   |  Link to Article
Krishnan  AV, Lin  CS, Park  SB, Kiernan  MC.  Axonal ion channels from bench to bedside: a translational neuroscience perspective. Prog Neurobiol. 2009;89(3):288-313.
PubMed   |  Link to Article
Bostock  H.  The strength-duration relationship for excitation of myelinated nerve: computed dependence on membrane parameters. J Physiol. 1983;341:59-74.
PubMed   |  Link to Article
Bostock  H, Rothwell  JC.  Latent addition in motor and sensory fibres of human peripheral nerve. J Physiol. 1997;498(pt 1):277-294.
PubMed   |  Link to Article
Mogyoros  I, Kiernan  MC, Burke  D.  Strength-duration properties of human peripheral nerve. Brain. 1996;119(pt 2):439-447.
PubMed   |  Link to Article
Mogyoros  I, Kiernan  MC, Burke  D.  Strength-duration properties of sensory and motor axons in carpal tunnel syndrome. Muscle Nerve. 1997;20(4):508-510.
PubMed   |  Link to Article
Mogyoros  I, Lin  CS, Kuwabara  S, Cappelen-Smith  C, Burke  D.  Strength-duration properties and their voltage dependence as measures of a threshold conductance at the node of Ranvier of single motor axons. Muscle Nerve. 2000;23(11):1719-1726.
PubMed   |  Link to Article
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Kiernan  MC, Isbister  GK, Lin  CS, Burke  D, Bostock  H.  Acute tetrodotoxin-induced neurotoxicity after ingestion of puffer fish. Ann Neurol. 2005;57(3):339-348.
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Figures

Place holder to copy figure label and caption
Figure 1.
Strength-Duration Time Constant in Amyotrophic Lateral Sclerosis (ALS)

The strength-duration time constant reflects nodal persistent Na+ channel conductances. A, The strength-duration time constant was significantly increased in patients with c9orf72 familial ALS (FALS, triangles) and sporadic ALS (SALS, circles) when compared with asymptomatic c9orf72 mutation carriers (AMCs, diamonds) and control participants (squares). B, Mean strength-duration time constant was significantly increased in patients with FALS and SALS. C, Rheobase was significantly reduced in patients with FALS and SALS. The error bars indicate standard errors of the mean.aP < .05.bP < .01.cP < .001.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Threshold Electrotonus (TE) Measures in Amyotrophic Lateral Sclerosis (ALS)

Threshold electrotonus reflects changes in membrane excitability in response to long-duration polarizing currents. Threshold reduction with depolarization is represented in an upward direction and hyperpolarization in a downward direction. A, Depolarizing TE (TEd) was significantly increased in c9orf72 familial ALS (FALS, triangles) and sporadic ALS (SALS, circles) when compared with asymptomatic c9orf72 mutation carriers (AMCs, diamonds) and control participants (squares). In contrast, hyperpolarizing TE was not significant (NS) between groups. B, Mean TEd at 90 to 100 milliseconds was significantly increased in c9orf72 FALS and SALS. The error bars indicate standard errors of the mean.aP < .05.bP < .01.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.
Correlation of Axonal Excitability Parameters With Motor Amplitudes

The strength-duration time constant (A) and depolarizing threshold electrotonus (TEd) at 90 to 100 milliseconds (B) significantly correlated with the compound muscle action potential (CMAP) amplitude, thereby suggesting that strength-duration time constant and TEd at 90 to 100 milliseconds were linked with the process of axonal degeneration. FALS indicates familial amyotrophic lateral sclerosis; SALS, sporadic ALS.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.
Mathematical Modeling of Axonal Excitability Parameters in Amyotrophic Lateral Sclerosis

Comparison of group data (control participants, open circles; combined c9orf72 and sporadic ALS, filled circles) and mathematical model (open triangles, normal model; c9orf72 and sporadic ALS model, filled triangles). A, Threshold electrotonus group data. B, Modeled threshold electrotonus. C, Current-threshold relationship. D, Modeled current-threshold relationship.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.
Mathematical Modeling of Strength-Duration Time Constant in Amyotrophic Lateral Sclerosis (ALS)

Comparisons of recorded and modeled strength-duration time constant data. The strength-duration time constant was significantly increased in patients with c9orf72 familial ALS (FALS) and sporadic ALS (SALS). Modeled strength-duration time constant for normal and combined SALS and FALS data sets.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable.  Clinical Details for the Patients With C9orf72 FALS and SALS and AMCs

References

Kiernan  MC, Vucic  S, Cheah  BC,  et al.  Amyotrophic lateral sclerosis. Lancet. 2011;377(9769):942-955.
PubMed   |  Link to Article
Andersen  PM, Al-Chalabi  A.  Clinical genetics of amyotrophic lateral sclerosis: what do we really know? Nat Rev Neurol. 2011;7(11):603-615.
PubMed   |  Link to Article
Renton  AE, Majounie  E, Waite  A,  et al; ITALSGEN Consortium.  A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72(2):257-268.
PubMed   |  Link to Article
DeJesus-Hernandez  M, Mackenzie  IR, Boeve  BF,  et al.  Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72(2):245-256.
PubMed   |  Link to Article
Majounie  E, Renton  AE, Mok  K,  et al; Chromosome 9-ALS/FTD Consortium; French research network on FTLD/FTLD/ALS; ITALSGEN Consortium.  Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. 2012;11(4):323-330.
PubMed   |  Link to Article
Snowden  JS, Harris  J, Richardson  A,  et al.  Frontotemporal dementia with amyotrophic lateral sclerosis: a clinical comparison of patients with and without repeat expansions in C9orf72. Amyotroph Lateral Scler Frontotemporal Degener. 2013;14(3):172-176.
PubMed   |  Link to Article
Williams  KL, Fifita  JA, Vucic  S,  et al.  Pathophysiological insights into ALS with C9ORF72 expansions. J Neurol Neurosurg Psychiatry. 2013;84(8):931-935.
PubMed   |  Link to Article
van Rheenen  W, van Blitterswijk  M, Huisman  MH,  et al.  Hexanucleotide repeat expansions in C9ORF72 in the spectrum of motor neuron diseases. Neurology. 2012;79(9):878-882.
PubMed   |  Link to Article
Vucic  S, Kiernan  MC.  Axonal excitability properties in amyotrophic lateral sclerosis. Clin Neurophysiol. 2006;117(7):1458-1466.
PubMed   |  Link to Article
Kanai  K, Kuwabara  S, Misawa  S,  et al.  Altered axonal excitability properties in amyotrophic lateral sclerosis: impaired potassium channel function related to disease stage. Brain. 2006;129(pt 4):953-962.
PubMed   |  Link to Article
Nakata  M, Kuwabara  S, Kanai  K,  et al.  Distal excitability changes in motor axons in amyotrophic lateral sclerosis. Clin Neurophysiol. 2006;117(7):1444-1448.
PubMed   |  Link to Article
Tamura  N, Kuwabara  S, Misawa  S,  et al.  Increased nodal persistent Na+ currents in human neuropathy and motor neuron disease estimated by latent addition. Clin Neurophysiol. 2006;117(11):2451-2458.
PubMed   |  Link to Article
Bostock  H, Sharief  MK, Reid  G, Murray  NM.  Axonal ion channel dysfunction in amyotrophic lateral sclerosis. Brain. 1995;118(pt 1):217-225.
PubMed   |  Link to Article
Horn  S, Quasthoff  S, Grafe  P, Bostock  H, Renner  R, Schrank  B.  Abnormal axonal inward rectification in diabetic neuropathy. Muscle Nerve. 1996;19(10):1268-1275.
PubMed   |  Link to Article
Mogyoros  I, Kiernan  MC, Burke  D, Bostock  H.  Strength-duration properties of sensory and motor axons in amyotrophic lateral sclerosis. Brain. 1998;121(pt 5):851-859.
PubMed   |  Link to Article
Kiernan  M, Burke  D. Threshold electrotonus and the assessment of nerve excitability in amyotrophic lateral sclerosis. In: Eisen  A, ed. Clinical Neurophysiology of Motor Neuron Diseases. Amsterdam, the Netherlands: Elsevier; 2004:359-366.
Menon  P, Kiernan  MC, Vucic  S.  ALS pathophysiology: insights from the split-hand phenomenon. Clin Neurophysiol. 2014;125(1):186-193.
PubMed   |  Link to Article
Shibuya  K, Misawa  S, Nasu  S,  et al.  Split hand syndrome in amyotrophic lateral sclerosis: different excitability changes in the thenar and hypothenar motor axons. J Neurol Neurosurg Psychiatry. 2013;84(9):969-972.
PubMed   |  Link to Article
Kanai  K, Kuwabara  S, Arai  K, Sung  JY, Ogawara  K, Hattori  T.  Muscle cramp in Machado-Joseph disease: altered motor axonal excitability properties and mexiletine treatment. Brain. 2003;126(pt 4):965-973.
PubMed   |  Link to Article
Kiernan  MC, Bostock  H.  Effects of membrane polarization and ischaemia on the excitability properties of human motor axons. Brain. 2000;123(pt 12):2542-2551.
PubMed   |  Link to Article
Kiernan  MC, Burke  D, Andersen  KV, Bostock  H.  Multiple measures of axonal excitability: a new approach in clinical testing. Muscle Nerve. 2000;23(3):399-409.
PubMed   |  Link to Article
Krishnan  AV, Lin  CS, Park  SB, Kiernan  MC.  Axonal ion channels from bench to bedside: a translational neuroscience perspective. Prog Neurobiol. 2009;89(3):288-313.
PubMed   |  Link to Article
Bostock  H.  The strength-duration relationship for excitation of myelinated nerve: computed dependence on membrane parameters. J Physiol. 1983;341:59-74.
PubMed   |  Link to Article
Bostock  H, Rothwell  JC.  Latent addition in motor and sensory fibres of human peripheral nerve. J Physiol. 1997;498(pt 1):277-294.
PubMed   |  Link to Article
Mogyoros  I, Kiernan  MC, Burke  D.  Strength-duration properties of human peripheral nerve. Brain. 1996;119(pt 2):439-447.
PubMed   |  Link to Article
Mogyoros  I, Kiernan  MC, Burke  D.  Strength-duration properties of sensory and motor axons in carpal tunnel syndrome. Muscle Nerve. 1997;20(4):508-510.
PubMed   |  Link to Article
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