Abnormalities of cortical stimulation strength-duration time constant in amyotrophic lateral sclerosis

(cid:1) Strength-duration time constant (SDTC) may now be determined for cortical motor neurones. (cid:1) SDTC is signiﬁcantly reduced in ALS patients and is correlated with ALS functional rating score, and rate of disease progression. (cid:1) An increase in transient Na + conductances may account for the reduction in SDTC.


Introduction
Cortical hyperexcitability has been established as a specific and early feature of amyotrophic laterals sclerosis (ALS), implicated in disease pathogenesis (Kiernan et al., 2011, Menon et al., 2015).Specifically, the evolution of cortical hyperexcitability precedes and correlates with lower motor neuron degeneration (Vucic andKiernan, 2010, Vucic et al., 2008), contributes to development of specific clinical features such as the split-hand phenomenon (Menon et al., 2014b), and seems to underlie an unfavourable disease trajectory, with a faster rate of progression, greater functional disability, and reduced survival (Dharmadasa et al., 2021, Menon et al., 2019, Shibuya et al., 2016).A combination of cortical disinhibition linked to increased facilitation was shown to underlie the development of hyperexcitability in ALS (Menon et al., 2015, Vucic et al., 2023).
Abnormalities of strength-duration properties have been identified in ALS, albeit at a peripheral nerve level (Park et al., 2017).The strength-duration properties of axons describe the relationship between stimulus strength and duration, yielding biomarkers of neuronal excitability, including the strength-duration time constant (SDTC) and rheobase (Kiernan et al., 2020, Krishnan et al., 2009).The SDTC and rheobase reflect function of nodal membrane ion channels and passive membrane properties (Kiernan et al., 2020, Krishnan et al., 2009).In ALS, prolongation of SDTC has been established in ALS and associated with clinical features, such as fasciculations and muscle cramping, as well as neurodegeneration (Geevasinga et al., 2015, Kanai et al., 2006, Kuo et al., 2005, Vucic and Kiernan, 2006, Vucic and Kiernan, 2010).Upregulation of persistent Na + conductance (I NaP ) along with reduction of K + currents have been proposed as mechanisms underlying this increase of SDTC in ALS (Kanai et al., 2006, Kiernan et al., 2020, Kuo et al., 2005, Vucic and Kiernan, 2006).Of specific relevance to disease pathogenesis, modulation of Na + conductances and K + currents result in normalisation of axonal excitability biomarkers, associated with symptomatic improvement in patients (Park et al., 2015, Wainger et al., 2021, Weiss et al., 2016).
At a cortical level, there is a paucity of information regarding abnormalities of strength-duration neuronal properties in ALS patients.Upregulation of persistent Na + conductance and reduced K + currents have been reported in cultured cortical motor neurons of ALS mouse models, resulting in neuronal hyperexcitability and increased neuronal firing frequency (Pieri et al., 2009).Additionally, hyperexcitability of pyramidal cortical neurons located in layer V of the mouse motor cortex has been reported in mouse models (Saba et al., 2016).Suppression of Na + conductances by riluzole was shown to partially normalise cortical neuronal excitability and function in ALS patients (Vucic et al., 2013), similar to mouse models (Bellingham, 2013), underscoring the importance of Na + channel conduction abnormalities in cortical hyperexcitability development (Pradhan and Bellingham, 2021).
Recent advances in transcranial magnetic stimulation (TMS) technologies have enabled the assessment of cortical strengthduration properties in ALS patients.Controllable pulse parameter TMS (cTMS) devices have enabled an estimation of SDTC and rheobase from human motor cortical neurons due to ability to deliver near-rectangular electric field pulses of different durations (D'Ostilio et al., 2016, Peterchev et al., 2013).The SDTC and rheobase can be estimated by measuring resting motor threshold (RMT) or TMS input-output curves (Peterchev et al., 2013).Cortical SDTC estimates generated by RMT methodology reflect properties of cortical neuronal populations, including lower threshold inhibitory and excitatory interneurons that activate pyramidal cells (Aberra et al., 2020).Modelling studies have suggested that cortical SDTC, as estimated by the RMT method, was mediated by transient Na + conductances (I NaT ) of pyramidal neurons located in layers 2/3 and 5, as well as large basket-cells in layer 4 of the primary motor cortex (Menon et al., 2023).Consequently, the present study assessed strength duration properties of cortical neurons in ALS patients utilising the novel cTMS technique.Cortical SDTC and rheobase were assessed and correlated with clinical features, with the specific aim of determining whether alterations in Na + conductances contributed to the development of cortical hyperexcitability, neurodegeneration and disease progression in ALS.

Clinical assessment of participants
Studies were undertaken on 17 ALS patients (mean age 63.7 years; 11 males), diagnosed in accordance with the Gold Coast criteria (Shefner et al., 2020), recruited from the Concord Hospital ALS clinic.None of the patients were taking neuromodulatory medications at the time of the cTMS testing, although the level of caffein was not quantified.It should be stressed that caffeine intake has not been reported to impact on RMT (Ziemann et al., 2015), from which cortical SDTC is calculated (D'Ostilio et al., 2016, Menon et al., 2023).Of relevance, cTMS studies were performed during the same time of day (10:10am to 12:30 pm) in ALS patients and controls, and all participants were well rested.The study was approved by the local hospital ethics committee (Sydney Local Area Health Service and Human Research Ethics Committees).Written informed consent was obtained from every subject prior to participation.
Site of disease onset, disease duration (months) and ALSFRS-R scores were collected in all ALS patients, as was the rate of disease progression in accordance with a previously reported formula (Labra et al., 2016).Muscle strength was determined by the Medical Research Council (MRC) score as follows: (i) upper limbs À shoulder abduction, elbow flexion and extension, wrist extension, finger (first dorsal interosseous) and thumb abduction bilaterally [maximal upper limb MRC score, 60 (normal strength)]; (ii) lower limbs À hip flexion, knee extension, and ankle dorsiflexion bilaterally [maximal lower limb score, 30 (normal strength)].Upper motor neuron (UMN) signs were quantified by using a specific UMN score (Turner et al., 2004).Edinburgh Cognitive and Behavioural ALS Screen (ECAS) was utilised to assess the degree of cognitive dysfunction in ALS (Abrahams et al., 2014).A total ECAS score 105 and ALS-specific subscore < 77 were cutoffs for cognitive dysfunction (Abrahams et al., 2014).

Peripheral nerve assessment
Prior to assessing cortical function, the degree of lower motor neuron dysfunction was assessed.The median nerve was electrically stimulated at the wrist (20 % above threshold) and the compound muscle action potential (CMAP) responses were recorded over the abductor pollicis brevis (APB) muscle.All CMAP responses were recorded using AgCl disc electrodes (10-mm diameter; 3 M, Maplewood, Minnesota) arranged with a belly-tendon montage.The ground electrode was positioned over the dorsum of the hand.Peak-to-peak CMAP amplitude (mV), distal motor onset latency (ms) and minimum F wave latency (ms) were recorded.The split hand index and neurophysiological index were calculated in accordance to previously reported formulae (de Carvalho andSwash, 2000, Menon et al., 2014a).

Assessment of cortical strength duration properties
Cortical strength duration properties were assessed using a commercial cTMS device (ElevateTMS, Rogue Research, Canada) that delivered a TMS pulse of variable duration through a dedicated figure-of-eight coil (70 mm diameter, Rogue Research, Canada) oriented in a position to induce a posterior-anterior directed current in the dominant primary motor cortex.The electric field induced by the cTMS device was varied by pulse width, defined as duration of the first phase of the electric field pulse (D'Ostilio et al., 2016, Peterchev et al., 2013).Pulse widths of 30, 45, 60, 90 and 120 ls were used to estimate SDTC and rheobase, with MÀratio set to 0.1 (predominantly unidirectional monophasic electric field pulse with a main posterior-anterior-directed phase, followed by a longer and smaller second phase in opposite direction) (Menon et al., 2023).
Participants were seated in a comfortable chair, with the 70 mm figure-of-eight coil applied tangentially to the contralateral scalp.The TMS coil handle was oriented at 45°to the sagittal midline, inducing a posterior-anterior initial current in the motor cortex (Menon et al., 2023).Motor evoked potentials (MEPs) were recorded from the dominant abductor pollicis brevis (APB) muscle using AgCl disc electrodes (10-mm diameter; 3 M, Maplewood, Minnesota).The motor hot spot was defined as the area on the contralateral scalp where the largest MEP response was generated by smallest TMS intensity.The coil was moved in 0.5 cm steps to determine the optimal position which was marked and tracked.
The RMT was defined in accordance with the as the lowest TMS intensity required to evoke MEP responses !50 lV in at least 5 out of 10 consecutive MEP trials (Rossini et al., 2015), and was determined for each PW (30, 45, 60, 90, and 120 ls).The PWs were delivered in a pseudo-random sequence to counterbalance any time dependence of RMT estimation, while matching the estimation procedure across subjects (Menon et al., 2023).Visual feedback was provided to ensure that subjects were at rest and facilitated MEP responses were excluded.MEP responses were amplified with a gain of 1000, bandpass filtered 2 Hz -2 kHz, digitized at 5 kHz, and recorded using Signal 7 software (Cambridge Electronic Design, Cambridge, United Kingdom).CMAP responses were amplified with a gain of 1000 and bandpass filtered (3 Hz-5 kHz) using a D440 Isolated amplifier (Digitimer, UK), and sampled at 10 kHz using a 16-bit data acquisition card (National Instruments USB-6341).Data acquisition and stimulus delivery were controlled by QTRACS software (Version 2017, Digitimer, United Kingdom).Skin surface temperature was maintained above 32 °C throughout the tests.

Determination of cortical strength-duration properties
Estimation of cortical SDTC and rheobase were performed according to previously reported methodology (D'Ostilio et al., 2016, Peterchev et al., 2013).As previously discussed, an MEP response is generated when depolarisation strength (or factor) reaches threshold (rheobase).Consequently, the relationship between RMT and PW can be modelled by a previously reported equation representing the strength-duration curve: representing the modelled RMT and r SDTC; PW ð Þ the depolarisation factor (Peterchev et al., 2013).Empirical RMT PW ð Þ values were fitted to the parametric model RMT 0 PW ð Þby minimizing the sum of five pulse widths (30, 40, 60, 90, 120 ls) for calculation of SDTC.
The electric field pulse waveform was recorded for each pulse width at MÀratio of 0.1 using a digital oscilloscope (DSO2C15, Hantek, Poland) with waveforms digitised and stored for offline calculation of SDTC and rheobase.Estimation of SDTC and rheobase was performed by purpose-built software in MATLAB (version R2020b, The Mathworks, Inc., Natick, MA, USA).

Statistical analysis
SDTC and rheobase values were compared to previously reported 17 healthy controls (7 males, 10 females; mean age of 51 years, range 29-79 years) (Menon et al., 2023).While the controls were younger than ALS patients, there were no correlations between SDTC (P = 0.461) or rheobase (P = 0.118) with age.Data was initially assessed for normality by using the Schapiro-Wilk test.Two-way mixed model analysis of variance (ANOVA) was used to evaluate for differences in the RMT strength-duration curves between ALS patients and controls across the five pulse widths (30, 45, 60, 90, 120 ls).Greenhouse-Geisser correction was applied to correct for violations of sphericity when required.Students t test were used to assess for differences in SDTC and rheobase between groups.Pearson's correlation coefficient or Spearman's p was used for analysing associations between SDTC and clinical parameters.Statistical analysis was performed using SPSS statistics software (Version 28).Data is expressed as mean ± standard deviation or median (interquartile range).P value < 0.05 were deemed to be significant.

Data sharing statement
The data will be shared for reasonable research requests by emailing the corresponding author.

Clinical characteristics
Limb onset disease was evident in 71 % patients, characterised by concurrent upper and lower motor neuron signs (Table 1).Bulbar-onset disease, characterised by dysarthria, dysphagia and tongue wasting with fasciculations and absence of limb involvement in first 6 months from symptoms onset, was observed in 29 % patients.The mean age at assessment was 64 ± 3 years, with mean disease duration from symptom onset being 16.8 ± 3.7 mon ths.Mean ALSFRS-R score was 41 ± 0.7, indicating a moderate level of dysfunction.A moderate rate of disease progression was 0was evident in the current ALS cohort, as was a moderate degree of upper and lower limb weakness (Table 1).Twelve percent of patients were classified as Kings clinical stage 1, 71 % stage 2, and 17 % as stage 3.No patients were classified as stage 4. At the time of testing 19 % of ALS patients were taking riluzole.
The total ECAS score was reduced in 64.2 % and ALS-specific subscore was reduced in 50 % of ALS patients (Strong et al., 2017).The mean total ECAS and ALS subscores were reduced (Table 1), indicative of mild cognitive dysfunction.There were no cases of frontotemporal dementia.

Peripheral nerve studies
The degree of lower motor neuron dysfunction was initially assessed in ALS patients.The mean peak-to-peak CMAP amplitude (ALS 4.6 ± 0.7 mV; Control 13.2 ± 1.3 mV, t 26.2 = -6.084,P < 0.001) and neurophysiological index (ALS 1.0 ± 0.2 Control 3.5 ± 0.3; t 28.8 = -5.793,P < 0.001) were significantly reduced in ALS patients.A reduction of the split hand index (<7.4)was evident in 75 % of ALS patients with mean SI of 5.4 ± 0.7.Taken together, these findings indicate a significant degree of LMN dysfunction in ALS patients, with preferential wasting of the thenar group of muscles.

Assessment of cortical strength duration properties in ALS
Cortical strength-duration curves recorded in ALS patients and healthy controls with MÀratio set to 0.1 and are depicted in Fig. 1.Two-way mixed model ANOVA disclosed a significant difference in the strength-duration curves (F (2.1) = 3.853, P = 0.023), whereby the RMT values were reduced in ALS patients when compared to controls.

Discussion
The present study established abnormalities of cortical strength-duration properties in ALS by determining motor thresholds across different TMS pulse widths.Specifically, cortical SDTC was significantly reduced in ALS and was more prominent in cognitively impaired patients.The reduction in SDTC appeared to be an early feature of ALS, correlating with a faster rate of disease progression, greater functional disability, more prominent muscle weakness and lower motor neuron degeneration as reflected by CMAP amplitude and the neurophysiological index.Given that modelling studies have demonstrated that increases in I NaT may reduce SDTC (Menon et al., 2023), the present findings suggest that an increase of cortical I NaT could contribute to disease pathogenesis in ALS, by increasing neuronal excitability.

Pathophysiological mechanisms underlying SDTC abnormalities in ALS
Modelling approaches suggested that TMS maximally depolarizes intracortical axonal terminations in the superficial gyral crown and lip regions of the primary motor cortex (M1), with layer 5 pyramidal cells exhibiting the lowest thresholds, followed by layer 4 inhibitory basket cells and layer 2/3 pyramidal neurons (Aberra et al., 2020, Menon et al., 2023).Cortical SDTC appears to be dependent on the density and distribution of voltage-gated Na + channels located in the intracortical axonal terminal branches of neurons located in layers 2-to-5 of M1, such that an increase in Na + conductances, but not persistent Na + , fast or slow K + currents, were associated with smaller SDTC values (Menon et al., 2023).
In the present study, a significant reduction of cortical SDTC was evident in ALS patients and associated with a more aggressive phenotype, as characterised by a faster rate of disease progression, greater functional disability, and more prominent muscle weak-ness.Additionally, the reduction of cortical SDTC correlated with neurophysiological biomarkers of lower motor neuron degeneration (CMAP amplitude and neurophysiological index), suggesting that increased I NaT contributed to motor neuron degeneration and thereby ALS pathogenesis.The correlation between cortical SDTC and CMAP amplitude could also imply a contribution of Na + channel dysfunction at the level of the spinal motor neuron level.
The reduction of cortical SDTC is in part mediated by an increase in transient Na + conductances, resulting in more frequent action potential generation and thereby higher firing frequencies of cortical neurons.This higher neuronal firing frequency could result in hyperexcitability of descending corticomotoneuronal tracts and ultimately neurodegeneration via an glutamatergic excitotoxic mechanism (Eisen et al., 1992).Increased expression of the Na V 1.6 isoform has been reported in the primary motor cortex of the SOD-1 mouse model and varies with disease progression (Saba et al., 2016, Saba et al., 2019).Increased expression of Na + channels was associated with upregulation of cortical I NaP , but not transient Na + conductances (Pieri et al., 2009, Pradhan and Bellingham, 2021, Saba et al., 2016, Saba et al., 2019).The discordant findings may relate to pathophysiological differences between ALS patients and mouse models (Picher-Martel et al., 2016), with cortical I NaT being dysfunctional in ALS patients.It should be acknowledged that kinetics and spatial distribution of transient and persistent Na + conductances in cortical axons are poorly characterized, and consequently a contribution from I NaP cannot be discounted.It should also be acknowledged that the correlation between cortical SDTC and CMAP amplitude suggests that upregulation of transient Na + channel conductance at the spinal motor neuronal level could also contribute to abnormalities of cortical strength duration properties evident in the current ALS cohort.The preservation of a negative correlation between SDTC and rheobase, a finding also evident in healthy controls, may suggest that reduction of SDTC is mediated by functional changes (i.e increase in transient Na + conductance) with a minimal contribution of neuronal degeneration.
Reduction of SDTC was more prominent in cognitively impaired ALS patients.Specifically, cortical SDTC reduction was greater in ALS patients exhibiting patients reduced total ECAS and ALSspecific scores.These findings are in keeping with a previous study reporting a greater degree of cortical hyperexcitability in cogni- tively impaired ALS patients (Higashihara et al., 2021).The mechanisms underlying SDTC reduction may relate to evolution of TDP-43 pathology.The ECAS score is a valid predictor of regional distribution of TDP-43 pathology (Gregory et al., 2020), with reduction of the ALS-specific components being associated with TDP-43 deposition in the fronto-temporal cortical regions (Gregory et al., 2020).SDTC reduction could be mediated by deposition of TDP-43 and thereby dysfunction of pyramidal and interneuronal populations.Alternatively, degeneration or dysfunction of projecting fibers from extra-motor cortical regions to the primary motor cortex could also contribute to aberrant modulation of pyramidal and interneuronal populations, resulting in SDTC reduction.The latter notion is supported by neuroimaging studies demonstrating an association between abnormalities of functional connectivity and cortical hyperexcitability in ALS (Geevasinga et al., 2017).
Resting motor threshold is also a biomarker of voltage gated Na + conductances (Vucic et al., 2023, Ziemann et al., 2015).Reduction in RMTs has been reported in early stages of ALS, preceding the onset of muscle wasting and associated with hyper-reflexia (Mills and Nithi, 1997).Taken together, the reduction in RMT suggests that increased cortical neuronal transient Na + conductances occur in early stages of ALS and contribute to hyperexcitability.The reduction in RMT is not a universal finding, with studies reporting either normal (Menon et al., 2017, Mills and Nithi, 1997, Zanette et al., 2002), or increased RMT (Berardelli et al., 1991, Eisen et al., 1990, Urban et al., 2001).A potential explanation for these discordant findings may relate to clinical and pathological heterogeneity of ALS, with increased transient Na + conductances evident in early stages of disease, progressing to loss of transient Na + conductances due to either neurodegeneration or Na + channel dysfunction.This notion is supported by findings of a significant correlation between SDTC and disease duration in the present study.Longitudinal studies of cortical SDTC changes would be required to determine whether a loss of I NaT is evident with disease progression.

Conclusion
In conclusion, the present study established a reduction of cortical SDTC in ALS, which was associated with a more aggressive phenotype, cognitive impairment, and lower motor neuronal dysfunction.Increased I NaT is likely to mediate the reduction of cortical SDTC in ALS, thereby contributing to hyperexcitability.Direct and indirect modulation of I NaT in ALS patients was shown to normalizes cortical hyperexcitability ALS (Vucic et al., 2013, Wainger et al., 2021).Consequently, modulation of transient Na + channels could be therapeutically beneficial in ALS, with measurement of cortical SDTC potentially serving as an objective marker of target engagement.contributions were supported by the National Institutes of Health (U.S.A.) under Award Numbers R01MH128422, R01NS117405 and RF1MH124943.The funding sources did not have a role in study design, and the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding sources.

Fig. 1 .
Fig. 1.Cortical strength duration curves.Cortical strength-duration curves for amyotrophic lateral sclerosis (ALS) patients and controls recorded at 5 pulsewidths.The strength-duration curve for ALS patients was significantly reduced when compared to controls.

Fig. 2 .
Fig. 2. Cortical strength duration time constant.Cortical strength-duration time constant (SDTC) was significantly reduced in amyotrophic lateral sclerosis (ALS) patients when compared to controls.

Fig. 3 .
Fig. 3. Cortical strength duration time constant is reduced with cognitive impairment.(A) Cortical strength-duration time constant (SDTC) was significantly reduced in amyotrophic lateral sclerosis (ALS) patients exhibiting an abnormal Total Edinburgh Cognitive and Behavioural ALS Screen (ECAS) Score (h1 0 5) compared to controls.In ALS patients with a normal Total ECAS Score (!105) the SDTC was comparable to controls.(B) The SDTC was significantly reduced in ALS patients with an abnormal ECAS ALS-Specific Score compared to controls.Individual participant SDTC values are depicted by the black dot markers.

Fig. 4 .
Fig. 4. Correlation between strength duration time constant and clinical measures.Cortical strength duration time constant was significantly correlated with (A) disease duration, (B) ALS functional rating score-revised (ALSFRS-R), and (C) rate of disease progression.

Table 1
Demographic and clinical features of ALS patients:Upper limb (UL) or lower limb (LL) onset disease was most frequently reported by ALS patients.The ALS functional rating score-revised (ALSFRS-R), Medical Research Council (MRC) sum scores, and upper motor neuron (UMN) score were recorded in all ALS patients.Cognitive function was assessed by using the total Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and ALS-specific subscores.A total ECAS score < 105 and ALS-specific subscore < 77 are regarded as abnormal.In three ALS patients, cognitive assessment was not undertaken (ND) due to poor English language.All data are expressed as mean (standard error of mean, SEM) or median (interquartile range, IQR).