**2. Motor cortex hyperexcitability in ALS**

Although the term 'hyperexcitability' is widely used, it still does not have a commonly accepted definition. According to Bae et al. (2013), it 'means an increased or exaggerated response to a stimulus, which may usually have been expected to evoke a normal response' [7]. We deem that hyperexcitability is discussed in modern TMS studies more broadly: as the ability to respond to stimuli that normally do not evoke any response and, speaking more generally, as the predominance of excitation over inhibition.

Hyperexcitability in ALS patients can be determined using various methods at different levels of the motor system. It can be detected at the level of individual neurons and ion channels in cell cultures in transgenic animals, clinically (presenting as the well-known phenomena, such as fasciculation or cramps) or using the modern neurophysiological techniques such as electromyography (EMG) and TMS [7].

Motor cortex excitability is a complex integral parameter that depends on numerous factors. Hence, the hyperexcitability phenomenon is also a complex and multifactorial process that depends on glutamate synthesis and release, its reuptake and degradation, expression and functional status of several types of glutamate receptors, excitable properties of neuronal membranes, and the status of inhibitory GABAergic neurotransmission. A number of experi‐ mental studies demonstrated that all the aforementioned processes are disturbed in ALS [8, 9]. **Table 1** summarizes the results of some key studies.

A significant advance in understanding the pathophysiological mechanisms of the develop‐ ment of the neurodegenerative process of ALS has been made over the past years after novel neurophysiological and neuroimaging techniques were introduced into the research and clinical practice [2–4]. Transcranial magnetic stimulation (TMS) is a noninvasive brain stimulation method that is used to evaluate the functional status of the upper motor neuron in ALS patients. It has been shown in the recent studies that different TMS parameters are altered in ALS patients [4]. TMS is currently viewed as a valuable research and diagnostic tool

Development of hyperexcitability of the primary motor cortex and the entire motor system is a well-studied phenomenon in ALS. Motor cortex hyperexcitability can be determined using TMS as reduced resting motor threshold and increased motor evoked potential (MEP) amplitude, decreased silent period, reduced effectiveness of short-interval intracortical inhibition (SICI), and increased intracortical facilitation (ICF) (see review [4]). Most authors attribute motor cortex hyperexcitability in ALS patients to enhanced glutamatergic neuro‐ transmission in the neocortex and reduced gamma-aminobutyric acid (GABA)ergic inhibitory neurotransmission, thus suggesting that hyperexcitability provokes degeneration of motor neurons [5–7]. However, no direct evidence has been obtained yet that would unambiguously demonstrate the relationship between motor cortex hyperexcitability in ALS patients and its degeneration. There is an alternative opinion that hyperexcitability can potentially be related to neuroplasticity processes taking place in the motor cortex and compensation for the lost

We would like to discuss the potential relationship of motor cortex hyperexcitability with motor neuron degeneration and neuroplasticity in ALS patients, as well as the possible methods to solve this problem using modern neurophysiological and neuroimaging methods.

Although the term 'hyperexcitability' is widely used, it still does not have a commonly accepted definition. According to Bae et al. (2013), it 'means an increased or exaggerated response to a stimulus, which may usually have been expected to evoke a normal response' [7]. We deem that hyperexcitability is discussed in modern TMS studies more broadly: as the ability to respond to stimuli that normally do not evoke any response and, speaking more

Hyperexcitability in ALS patients can be determined using various methods at different levels of the motor system. It can be detected at the level of individual neurons and ion channels in cell cultures in transgenic animals, clinically (presenting as the well-known phenomena, such as fasciculation or cramps) or using the modern neurophysiological techniques such as

Motor cortex excitability is a complex integral parameter that depends on numerous factors. Hence, the hyperexcitability phenomenon is also a complex and multifactorial process that

**2. Motor cortex hyperexcitability in ALS**

electromyography (EMG) and TMS [7].

generally, as the predominance of excitation over inhibition.

in ALS.

48 Update on Amyotrophic Lateral Sclerosis

function.


**Table 1.** Potential mechanisms of development of motor neuron hyperexcitability in ALS patients.

Today, TMS is the key and actually the only method for clinical investigation of motor cortex excitability. The use of the entire range of TMS parameters allows one to perform a relatively differentiated study of various factors contributing to motor cortex excitation [26]. TMS can be employed for assessing the functional status of the corticospinal tract due to the ability to excite motor cortex neurons by induced electrical current followed by propagation of excitation to alpha motor neurons of spinal cord. This causes contraction of muscle fibers within a certain motor unit, which can be recorded by cutaneous electrodes as a MEP [27]. TMS assesses the functional status of neuronal contours of the motor cortex [28].

Motor threshold is the key parameter used to assess motor cortex excitability. The motor threshold represents the density of corticospinal projections and can be regarded as a bio‐ marker of neuronal membrane excitability [26]. Decreased motor threshold is considered to be one of the key signs of motor cortex hyperexcitability.

According to most studies, the motor threshold in ALS patients is increased, probably being indicative of degeneration of cortical motor neurons [29–35]. Meanwhile, a paradoxical decrease in the motor threshold when examining patients at the onset of the disease was shown in some studies [36–39]. The motor threshold is likely to decrease at the onset of ALS, probably until clinical signs appear, and subsequently increases as motor neurons die. A statistically significant direct correlation between the motor threshold and disease duration was demon‐ strated in some studies [29, 40].

Physiologically, the motor threshold is primarily determined by rapid AMPA receptor (AMPAR)-mediated glutamatergic neurotransmission in the neocortex and excitability of motor neuron membranes that depends on voltage-gated sodium channels [41]. In ALS, AMPA receptor-mediated glutamatergic neurotransmission increases and properties of sodium channels change (presenting as increased conductance) [23, 24, 42, 43]. Alteration in functional properties of potential-gated sodium channels has been revealed: more rapid recovery after inactivation, increased permeability to sodium, and increased density of ion channels [21]. Pieri et al. demonstrated a decrease in the action potential threshold, an increase in pulse frequency, and an increase in persistent sodium current in cortical motor neurons isolated in G93A mutant mice [22].

MEP amplitude is determined by the number of reduced motor units and the number of activated alpha motor neurons in the spinal cord. Increased stimulation intensity enhances MEP amplitude due to superimposition of late I-waves and I1-wave [28, 44]. Like the motor threshold, MEP amplitude is determined by the density of corticospinal projections. Mean‐ while, MEP amplitude to a greater extent represents the function of neurons with lower excitability or those located farther from the stimulation site [4]. GABAergic drugs reduce MEP amplitude, which results from the scheme of generation of late I-waves modulated by inserted inhibitory GABAergic interneurons [41]. Noteworthy, the motor threshold and MEP ampli‐ tude are modulated by drugs belonging to different pharmacological classes, thus emphasiz‐ ing the difference between the mechanisms of their formation.

Identically to the motor threshold, MEP amplitude in ALS patients changes in opposite directions depending on stage of the disease. MEP amplitude decreases in most cases, being accompanied by increased motor threshold and representing a decrease in motor neuron number and reduction of density of corticospinal projections [4]. On the contrary, some patients with the reduced motor threshold may have increased MEP amplitude and the ratio between MEP amplitude and M-response amplitude [45, 46]. The increased slope ratio of the amplitude vs intensity curve is additional evidence to motor cortex hyperexcitability in ALS patients as it demonstrates a more pronounced amplitude increment with increasing stimu‐ lation intensity compared to the norm. The increase in MEP amplitude and the slope ratio of the amplitude vs intensity curve in ALS patients is probably related to both enhanced glutamatergic and reduced inhibitory GABAergic neurotransmission in the neocortex [4].

The *cortical silent period* (cSP) represents inhibition of voluntary muscular activity during a certain period after a magnetic stimulus was applied [47]. It has been demonstrated that the first one-third of cSP is mostly controlled by inhibitory mechanisms at the spinal cord level, while the remaining two thirds are of cortical origin and are related to inhibitory neurotrans‐ mission through GABA(B) receptors [27].

Motor threshold is the key parameter used to assess motor cortex excitability. The motor threshold represents the density of corticospinal projections and can be regarded as a bio‐ marker of neuronal membrane excitability [26]. Decreased motor threshold is considered to

According to most studies, the motor threshold in ALS patients is increased, probably being indicative of degeneration of cortical motor neurons [29–35]. Meanwhile, a paradoxical decrease in the motor threshold when examining patients at the onset of the disease was shown in some studies [36–39]. The motor threshold is likely to decrease at the onset of ALS, probably until clinical signs appear, and subsequently increases as motor neurons die. A statistically significant direct correlation between the motor threshold and disease duration was demon‐

Physiologically, the motor threshold is primarily determined by rapid AMPA receptor (AMPAR)-mediated glutamatergic neurotransmission in the neocortex and excitability of motor neuron membranes that depends on voltage-gated sodium channels [41]. In ALS, AMPA receptor-mediated glutamatergic neurotransmission increases and properties of sodium channels change (presenting as increased conductance) [23, 24, 42, 43]. Alteration in functional properties of potential-gated sodium channels has been revealed: more rapid recovery after inactivation, increased permeability to sodium, and increased density of ion channels [21]. Pieri et al. demonstrated a decrease in the action potential threshold, an increase in pulse frequency, and an increase in persistent sodium current in cortical motor neurons isolated in

MEP amplitude is determined by the number of reduced motor units and the number of activated alpha motor neurons in the spinal cord. Increased stimulation intensity enhances MEP amplitude due to superimposition of late I-waves and I1-wave [28, 44]. Like the motor threshold, MEP amplitude is determined by the density of corticospinal projections. Mean‐ while, MEP amplitude to a greater extent represents the function of neurons with lower excitability or those located farther from the stimulation site [4]. GABAergic drugs reduce MEP amplitude, which results from the scheme of generation of late I-waves modulated by inserted inhibitory GABAergic interneurons [41]. Noteworthy, the motor threshold and MEP ampli‐ tude are modulated by drugs belonging to different pharmacological classes, thus emphasiz‐

Identically to the motor threshold, MEP amplitude in ALS patients changes in opposite directions depending on stage of the disease. MEP amplitude decreases in most cases, being accompanied by increased motor threshold and representing a decrease in motor neuron number and reduction of density of corticospinal projections [4]. On the contrary, some patients with the reduced motor threshold may have increased MEP amplitude and the ratio between MEP amplitude and M-response amplitude [45, 46]. The increased slope ratio of the amplitude vs intensity curve is additional evidence to motor cortex hyperexcitability in ALS patients as it demonstrates a more pronounced amplitude increment with increasing stimu‐ lation intensity compared to the norm. The increase in MEP amplitude and the slope ratio of the amplitude vs intensity curve in ALS patients is probably related to both enhanced glutamatergic and reduced inhibitory GABAergic neurotransmission in the neocortex [4].

be one of the key signs of motor cortex hyperexcitability.

ing the difference between the mechanisms of their formation.

strated in some studies [29, 40].

50 Update on Amyotrophic Lateral Sclerosis

G93A mutant mice [22].

It was demonstrated in most studies that patients with sporadic and familial ALS had either decreased cSP or no cSP at all, which is regarded as significant evidence attesting to intracort‐ ical inhibitory dysfunction in ALS patients [48–50].

*Paired pulse stimulation* is used to assess intracortical inhibition and excitation processes. The method involves sequential generation of two pulses: the conditioning (S1) and testing (S2) pulses. The physiological effects of paired pulse stimulation are determined by the intensity of S1 and the interval between S1 and S2 pulses. Application of the sub-threshold S1 pulse 1– 6 ms before the supra-threshold S2 pulse reduces MEP amplitude compared to isolated application of a supra-threshold stimulus [51]. This phenomenon is known as SICI. Contrari‐ wise, an increase of the interstimulus interval to 8–20 ms rises the amplitude of MEP to the testing pulse (ICF). Several protocols have been proposed where the conditioning pulse has the supra- and sub-threshold intensity. MEP amplitude increases at the long interstimulus interval (long-interval intracortical inhibition, LICI) and decreases at the short interval (shortinterval intracortical facilitation, SICF) [27].

The neurophysiological mechanisms of formation of these phenomena remain insufficiently studied; however, it has been demonstrated rather convincingly that they have the predomi‐ nantly intracortical origin. Thus, SICI and LICI protocols cause suppression of amplitude and the number of late descending waves [51, 52]. Intracortical inhibition processes are assumed to be caused by activation of neocortical inhibitory interneurons under the action of the conditioning pulse; SICI being mediated by inhibition through GABA(A) receptors and LICI, through GABA(B) receptors. The ICF phenomenon is presumably caused by activation of glutamatergic neurotransmission through NMDA glutamate receptors [26].

Disruption of intracortical inhibition and facilitation under paired-pulse stimulation is currently believed to be the most convincing evidence that motor cortex hyperexcitability develops in ALS patients and has been detected in numerous studies [6, 53, 54, 55]. It should be mentioned that a decrease in efficiency of SICI is revealed at the earliest stages of the disease, including asymptomatic SOD1 mutation carriers, and correlates with axonal degeneration of peripheral motor neurons [45]. Assessment of the disruptions of intracortical inhibition using the new threshold tracking technique is considered to be a potential diagnostic tool in ALS demonstrating high sensitivity and specificity [56, 57].

The role of inhibitory interneurons dysfunction in pathogenesis of ALS has been actively studied over the past years [58–60]. Degeneration of inhibitory interneurons both in the spinal cord and in the motor cortex was demonstrated in several pathomorphological studies carried out in the 1990s. Nihei et al. (1993) reported a reduced amount of parvalbumin-positive interneurons in the motor cortex in ALS patients [61]. In addition, Petri et al. (2003) demon‐ strated that the level of GABA(A) receptor alpha-1 subunit mRNA decreases in ALS patients [20]. Dysfunction of inhibitory neurotransmission in the neocortex has also been confirmed in neuroimaging studies presenting as a decrease in GABA amount according to magnetic resonance spectroscopy [18] and decreased 11C-flumazenil binding as shown by PET [19].

The TMS data additionally confirm that inhibitory neurotransmission in the neocortex is disrupted in ALS patients. It should be mentioned that TMS has been used to show the disturbance of inhibition in the motor cortex both mediated by GABA(A) (decreased SICI) and by GABA(B) receptors (decreased LICI and sCP), being indicative of degeneration of inter‐ neurons or GABA metabolism disruption rather than dysfunction of receptors of this mediator in the postsynaptic membrane.

Other factors that cause SICI disruption in ALS patients are being discussed. Thus, riluzole was reported to induce partial recovery of SICI in ALS patients, which gives grounds for suggesting that enhancement of glutamatergic neurotransmission plays a role in disturbance of intracortical inhibition in ALS patients. NMDA receptor antagonists, memantine and amantadine, were shown to increase SICI and decrease ICF in several pharmaco-TMS studies [41]. These data suggest that decreased intracortical inhibition in ALS patients can be related not only to reduction of inhibitory GABAergic neurotransmission but also to enhancement of glutamatergic neurotransmission through NMDAR.

Increased intracortical facilitation is another evidence for involvement of NMDAR in patho‐ genesis of ALS. This phenomenon is currently predominantly attributed to glutamatergic neurotransmission through NMDAR, which is confirmed, in particular, by its reduction due to antagonists of these receptors [41].

Hence, a large body of data attesting to the development of motor cortex hyperexcitability in ALS patients can currently be obtained by TMS. Signs attesting to degeneration of the upper motor neuron are detected simultaneously (**Table 2**). Importantly, some TMS parameters (MEP threshold and amplitude) can change in opposite directions and attest to motor cortex hyperexcitability at onset of the disease and degeneration as the disease progresses. Mean‐ while, such signs of hyperexcitability as decreased cSP and efficiency of SICI are detected even in patients with pronounced degeneration of motor neurons. This phenomenon in ALS patients was referred to as 'dying but overactive' [7]; it can cause difficulties for interpretation of TMS results in a specific patient.


**Table 2.** Motor cortex hyperexcitability and degeneration in ALS: TMS findings.

**Table 3** summarizes the TMS data on the potential mechanisms responsible for the signs of motor cortex hyperexcitability in ALS patients. It should be mentioned that the variety of TMS procedures makes it possible not only to reveal the dysfunction of individual mediator systems but also to perform differentiated evaluation of neurotransmission through different types of glutamate and GABA receptors in ALS patients.


**Table 3.** Potential mechanisms of formation of TMS signs of motor cortex hyperexcitability in ALS.

Based on TMS findings, it is rather promising and reasonable to suggest that motor cortex hyperexcitability and the underlying molecular events (e.g., excitotoxicity) cause degeneration of motor neurons in ALS patients. We attempt to discuss the specific potential mechanisms underlying motor cortex hyperexcitability in ALS and present the evidence for its pathogenetic relationship with degeneration of motor neuron.
