Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder of motor neurons, characterized by upper motor neuron (UMN) and lower motor neuron (LMN) signs . The clinical heterogeneity of the ALS phenotype, however, may result in delayed diagnosis and difficulty in monitoring disease progression and treatment outcomes in a clinical trial setting . Neurophysiological biomarkers provide an objective measure of upper and LMN dysfunction in ALS, enabling an earlier diagnosis and thereby institution of adequate management strategies and recruitment into therapeutic trials. In addition, neurophysiological biomarkers may serve as noninvasive outcome measures in clinical therapeutic trials and provide important insights into disease mechanisms that ultimately lead to uncovering of novel therapeutic targets. In this review, a discussion of upper and LMN neurophysiologic biomarkers is undertaken, focusing on recent advances and their diagnostic and prognostic value in ALS.
NEUROPHYSIOLOGIC BIOMARKERS OF UPPER MOTOR NEURON DYSFUNCTION IN AMYOTROPHIC LATERAL SCLEROSIS
UMN dysfunction appears to be an important pathophysiological and diagnostic biomarker of ALS [1,3,4]. Given the limitations in eliciting UMN signs and relating these to underlying ALS pathophysiology , objective assessment of UMN function would clearly be of pathophysiological and diagnostic utility . Transcranial magnetic stimulation (TMS) technique is a noninvasive and relatively painless neurophysiological tool for assessing UMN function in humans. This technique utilizes a pulsed magnetic field directed at a small cortical area and induces neuronal depolarization in the region to generate an action potential.
When applied in ALS patients, the TMS methodology has resulted in valuable insights into the underlying pathophysiological mechanisms and lead to development of important diagnostic and prognostic biomarkers.
Transcranial magnetic stimulation and amyotrophic lateral sclerosis
Establishing the relationship between upper and LMN dysfunction in ALS appears to be crucial in the understanding of ALS pathophysiology . Cortical dysfunction has been proposed as the primary event in ALS, mediating motor neuron degeneration via a trans-synaptic glutaminergic excitotoxic mechanism, the dying forward hypothesis [1,7,8]. TMS studies have highlighted the importance of UMN dysfunction in ALS pathophysiology , providing critical insights into the underlying pathophysiological mechanisms.
Paired-pulse TMS techniques have provided strong evidence for the importance of cortical hyperexcitability in ALS pathogenesis . Several different paired-pulse paradigms have been developed [10,11], with short interval intracortical inhibition (SICI) and intracortical facilitation (ICF) being most frequently utilized in ALS. In the original ‘constant-stimulus’ paradigm, SICI and ICF were recorded by setting a subthreshold condition stimulus at a predetermined time interval prior to a suprathreshold test stimulus [10–13]. When the interstimulus interval (ISI) was set between 1 and 5 ms, the test response was inhibited, whereas increasing the ISI 7 and 30 ms resulted in facilitation of the test motor evoked potential (MEP) response . A threshold tracking paired-pulse paradigm was developed in order to overcome the limitation of MEP amplitude variability , which limited the constant stimulus method .
A marked reduction or absence of SICI has been reported in sporadic ALS and was accompanied by an increase in ICF, all indicative of cortical hyperexcitability (Fig. 1) [17–21]. Importantly, reduction of SICI was most prominent in the early stages of ALS, preceding the clinical and neurophysiological onset of LMN dysfunction, and correlating with biomarkers of neurodegeneration [19,22,23]. In addition, cortical hyperexcitability appeared to underlie the development of the split-hand phenomenon , pattern of disease spread [25▪] and rate of disease progression in ALS .
Separately, reduction of SICI was also reported in familial ALS patients secondary to mutations in the superoxide dismutase-1 , fused in sarcoma  and c9orf72 genes . The reduction of SICI preceded the clinical onset of familial ALS and correlated with biomarkers of peripheral neurodegeneration [21,29]. Importantly, SICI was within normal limits in asymptomatic mutation carriers [21,28], thereby indicating that factors other than inheritance of the genetic mutation are important in familial ALS pathogenesis. Abnormalities of SICI and ICF have also been documented in atypical ALS phenotypes, with SICI reduction and ICF increases reported in ‘pure LMN’ phenotypes, such as the flail arm and flail leg variants of ALS . In addition, SICI abnormalities were documented in primary lateral sclerosis [31,32], underscoring the importance of cortical dysfunction in ALS.
The notion that cortical hyperexcitability represented compensatory changes in response to LMN degeneration has also been suggested [18,33]. Given that changes in SICI were not evident in ALS mimicking disorders, despite a comparable peripheral disease burden [23,34], argued against this notion. In addition, the partial normalization of SICI with riluzole , an effect lasting 2–3 months and paralleling the clinical effectiveness of riluzole , underscored the importance of cortical hyperexcitability is ALS pathogenesis.
Neuropathological and molecular studies have identified degeneration of inhibitory cortical interneurons  along with disinhibition of the inhibitory interneurons  as potential mechanisms for SICI reduction in ALS. In addition, glutamate-mediated excitotoxicity may also contribute to SICI reduction in ALS [17,39]. As such, preserving the integrity of intracortical inhibitory circuits, and counteracting excitatory cortical circuits, may serve as potential therapeutic options in ALS.
Single pulse transcranial magnetic stimulation
Single pulse TMS biomarkers have provided corroborating evidence for the importance of cortical hyperexcitability in ALS . Abnormalities of resting motor threshold (RMT), which reflects corticomotoneuronal membrane excitability [40–42], have been well documented in ALS. Longitudinal studies in sporadic ALS patients have established an initial reduction of RMT, indicative of cortical hyperexcitability, followed by a progressive increase and eventual cortical inexcitability in later stages of ALS [18,43]. RMT is particularly reduced in ALS patients exhibiting profuse fasciculations, increased deep-tendon reflexes and a relatively preserved muscle bulk , supporting the notion that cortical hyperexcitability is an early and important pathophysiological mechanism in ALS.
Significant increases in the MEP amplitude, which reflects the density of corticomotoneuronal projections onto motor neurons , have also been documented ALS, including familial and atypical ALS phenotypes . Increases in MEP amplitude have been reported in early stages of ALS and correlate with neurophysiological biomarkers of axonal degeneration . As for SICI, the increase in MEP amplitude is not evident in ALS mimicking disorders , reaffirming the importance of cortical hyperexcitability in ALS pathogenesis [20,46].
Of further relevance, a significant reduction in contralateral and ipsilateral cortical silent period (CSP) duration has been established as an early biomarker of ALS [18–21,46–48]. The reduction of CSP, a biomarker of long-latency cortical inhibitory interneuronal function acting via GABAB receptors , appears to be specific for ALS [20,46,50]. Although the precise mechanisms underlying CSP reduction in ALS remain to be fully elucidated, degeneration of long-latency cortical inhibitory interneurons, acting via GABAB receptor system, seems a possible explanation. Separately, given that ipsilateral CSP is mediated by transcallosal glutamatergic fibers projecting onto inhibitory interneurons in the contralateral motor cortex , degeneration of these transcallosal fibers or their target inhibitory interneurons may account for ipsilateral CSP abnormalities. In addition, abnormalities of central motor conduction time have been documented in ALS , and related to degeneration of UMNs [52–54].
TMS has evolved to be an important diagnostic tool in ALS, providing objective assessment of UMN dysfunction. In the absence of a pathognomic test, the diagnosis of ALS relies on identification of upper and LMN signs with evidence of disease progression [3,4]. Identification of UMN signs in ALS remains clinically challenging , thereby limiting the sensitivity of currently utilized diagnostic criteria [2,4,55,56]. The threshold tracking TMS has identified cortical hyperexcitability as a sensitive and specific diagnostic biomarker of ALS at an early stage in the disease process , with SICI reduction reliably differentiating ALS from mimicking disorders. Furthermore, identification of subclinical UMN dysfunction has enabled a more definite diagnosis at earlier stage in the disease process in atypical ALS phenotypes [30,32]. Incorporation of the TMS technique into ALS diagnostic criteria may hasten ALS diagnosis, thereby enabling earlier recruitment into clinical trials, where neuroprotective therapies may be more effective.
BIOMARKERS OF LOWER MOTOR NEURON DYSFUNCTION IN AMYOTROPHIC LATERAL SCLEROSIS
Compound motor action potential and neurophysiological index
The compound motor action potential (CMAP) is obtained by providing a supramaximal stimulus to a motor nerve and recording the resulting muscle response. Although the CMAP itself has not generally been considered a biomarker in ALS, most LMN neurophysiologic biomarkers depend on it. The CMAP amplitude decreases with disease progression, although early in the disease course, it may be preserved because of collateral reinnervation. Nevertheless, there are considerable data, obtained from studies described below, suggesting that CMAP amplitude reduces with disease progression.
The neurophysiological index is obtained by dividing the CMAP amplitude with the distal motor latency and multiplying the product by F-wave persistence . The basic concept underlying the neurophysiological index is that with disease progression, CMAP amplitude declines, distal motor latency is prolonged and F wave persistence reduces, resulting in neurophysiological index reduction. Although few longitudinal studies in ALS have pursued this concept, a recent study has shown that the neurophysiological index is correlated with disease progression in ALS . Unfortunately, there have been no longer term studies mirroring clinical trial designs and test-retest repeatability remains to be determined.
Motor unit number estimation
Motor unit number estimation (MUNE) is a compelling biomarker of LMN dysfunction attempting to approximate the number of motor neurons innervating a target muscle . The basic principle of MUNE techniques is to divide the maximal CMAP amplitude by the average surface-recorded motor unit potential (SMUP). A number of MUNE techniques have been reported and differ in the manner by which SMUPs are derived. The original MUNE technique utilized the incremental stimulation method (Fig. 2), whereby the stimulus intensity at one point in the nerve was gradually increased from subthreshold until 10 increments in the motor response were recorded . The average SMUP amplitude was calculated by dividing the response amplitude with the number of steps. Subsequently, multiple point and adapted multiple-point stimulation techniques were developed in order to overcome the problem of ‘alternation’ that affects the incremental stimulation method which resulted in artificially lower MUNE counts [60–62]. In the multipoint stimulation techniques, the nerve is stimulated at different points along its length with low-intensity pulses to obtain a series of low-threshold motor unit potentials with the amplitude of each being measured . A number of additional approaches incorporating F-wave responses  and spike-triggered averaging  have also been reported.
Studies utilizing various MUNE techniques in ALS patients and healthy controls have reported good intrarater and interrater reliability [61,65–67]. In addition, progressive linear decline in MUNE counts has been reported in ALS , suggesting utility as a potential biomarker of disease progression in a clinical trial setting . An advantage of MUNE over CMAP is that it is theoretically capable of capturing disease progression very early in the disease course, at a time when CMAP size remains stable because of ongoing reinnervation .
Motor unit number index
Motor unit number index (MUNIX) is a relatively new method of assessing of motor unit counts based on patients performing a voluntary contraction at various intensity levels and the surface interference patterns being subsequently captured and decomposed to obtain a normalized motor unit size [69,70]. This value is then divided into the maximal CMAP value to obtain the MUNIX. The MUNIX technique has a distinct advantage in requiring little less training and practice. The technique cannot be reliably performed in patients with severe limitations in voluntary muscle activation and in animal models.
Recently, the utility of the MUNIX technique as a biomarker of LMN dysfunction in ALS has been established [71,72▪], suggesting a utility of the MUNIX technique in clinical trials. In addition, the MUNIX technique exhibited a comparable sensitivity to the multipoint stimulation method at detecting LMN loss , underscoring the potential of the more easily applied MUNIX technique for monitoring ALS disease progression.
Electrical impedance myography
Electrical impedance myography (EIM) utilizes a small, high-frequency electrical current applied across two electrodes positioned over a muscle with the resulting surface voltages measured between a second pair of electrodes, from which the resistive and capacitive properties of the tissue are obtained (Fig. 3). This technique does not rely on the inherent electrical activity of the tissue, but rather on how the tissue impacts the applied current. Thus, the technique is sensitive structural and compositional change in the muscle that accompanies ALS, including myofiber atrophy and the presence of increasing fat within the muscle. Animal and human studies have shown that EIM is very sensitive to disease progression and is highly reliable [74–77]. These studies have also shown that EIM values correlate with more standard approaches including handheld dynamometry and MUNE. Major additional advantages to the technique include EIM's being easy to perform, its requiring minimal training and its capability of being applied to proximal, truncal and even bulbar muscles [78,79]. A recent multicenter study [80▪▪] suggests that the use of EIM could provide more than a five-fold reduction in sample size requirements for ALS clinical therapeutic trials over standard outcome measures such as the ALS functional rating scale-revised (ALSFRS-R). EIM technology is now also being incorporated into standard EMG needles such that it may also be able to serve as a diagnostic biomarker alongside standard EMG . Finally, relatively inexpensive EIM devices  may make it possible for patients or caregivers to obtain data on themselves at home on a more frequent basis than is possible in standard clinical trials. This is being investigated as part of a study entitled ‘ALS testing through home based outcome measures’ that is currently ongoing .
Axonal excitability techniques utilize a combination of subthreshold and suprathreshold condition stimuli, preceding a test stimulus, to provide information about nodal and intermodal axonal ion channel function [84,85]. Upregulation of persistent Na+ conductances along with reduced slow and fast K+ currents has been reported in sporadic and familial forms of ALS [29,86–89]. Importantly, the changes in axonal excitability have been linked to development of muscle cramps, fasciculations and motor neuron degeneration [90,91,92▪], and have been associated with an adverse prognosis in ALS . Of relevance, the neurophysiological effectiveness of the Na+ channel blocker flecainide, which reduced the rate of neurophysiological index reduction in ALS patients, was associated with stabilization of axonal excitability , suggesting a potential monitoring role for axonal excitability in future clinical ALS trials.
The present review highlights the importance of neurophysiological biomarkers in assessing upper and LMN dysfunction in ALS. In addition to exhibiting diagnostic utility, the neurophysiological biomarkers may aid in the prognosis and monitoring of the effects of therapeutic agents in a clinical trial setting. Importantly, however, while the utility of the neurophysiological biomarkers has been suggested, future studies should evaluate their potential in a multicenter setting in larger ALS cohorts that exhibit heterogeneity of the clinical phenotype.
Financial support and sponsorship
Funding support from the National Health and Medical Research Council of Australia (Project grant numbers APP1024915) and the National Institutes of Health (Grant K24 NS060951) is gratefully acknowledged.
Conflicts of interest
S.B.R. has equity in, and serves as a consultant and scientific advisor to, Myolex, Inc. a company that designs impedance devices for clinical and research use; he is also a member of the company's Board of Directors. The company also has an option to license patented impedance technology of which S.B.R. is named as an inventor. S.V. has no conflicts to report.
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