**3. What does motor cortex hyperexcitability mean?**

#### **3.1. Is hyperexcitability bad?**

[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

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

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

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

**•** Resting motor threshold increased

**•** Central conduction failure (triple stimulation technique)

**•** CMCT increased

**•** MEP amplitude decreased

in the postsynaptic membrane.

52 Update on Amyotrophic Lateral Sclerosis

glutamatergic neurotransmission through NMDAR.

to antagonists of these receptors [41].

of TMS results in a specific patient.

**•** Resting motor threshold decreased

**•** MEP amplitude increased

**•** SP decreased

**•** SICI reduced and ICF increased

**Hyperexcitability Degeneration**

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

The evidence that motor cortex hyperexcitability plays a role in pathogenesis of ALS is still rather sparse [62]. Hyperexcitability is registered at different stages of the disease, including the pre-symptomatic ones [23, 45]. However, this fact tells nothing about its role in pathogen‐ esis of the disease. It is still unclear when true onset of neurodegeneration occurs; however, the results of studies using transgenic animals demonstrate that the pathological process in ALS may start at the earliest stages of embryogenesis (e.g., at the stage when neural networks are formed) [63]. In this situation, all the objectively detectable alterations may be either primary or secondary (i.e., emerge as one of nonspecific stages of neurodegeneration or via the compensatory mechanism).

The enhancement of excitatory glutamatergic neurotransmission and reduction of inhibitory GABAergic neurotransmission in the neocortex, which underlie hyperexcitability in ALS patients, are believed to damage motor neurons via the excitotoxicity mechanism [4, 7]. The role of excitotoxicity as one of the universal mechanisms causing neuronal death in various nervous system diseases has currently been demonstrated [8, 9, 64]. High sensitivity of motor neurons to excitotoxicity can be related to high expression of glutamate receptors and low expression of calcium-binding proteins [9].

An important argument in favor of the role of hyperexcitability and the underlying molecular processes in ALS pathogenesis is that this phenomenon can be early detected using TMS in asymptomatic SOD1 mutation carriers. Vucic et al. (2008) demonstrated a decrease in SICI in three SOD1 mutation carriers who were asymptomatic at the time the study was performed but developed clinical signs of the disease during a prospective study within 3 years. It is interesting that no neurophysiological signs of motor cortex excitability were revealed in 14 other asymptomatic carriers and they did not present with any clinical manifestations of the disease within the entire study period [45]. Early development of signs of hyperexcitability of neurons of the motor cortex, spinal cord, and even the extramotor areas such as hippocampus (e.g., see [23]) were demonstrated in a large series of experimental studies using a culture of neurons isolated from transgenic animals. Additional evidence is that signs of motor cortex hyperexcitability in ALS patients can be detected before the pyramidal pathways are affected and EMG signs of degeneration of the lower motor neuron emerge. A recent study carried out by Menon et al. (2015) showed that 24 patients with ALS had a statistically significant decrease in motor threshold, duration of cSP and intracortical inhibition, while simultaneously having an increased MER amplitude and intracortical facilitation at onset of the disease [5]. No signs of alterations in central motor conduction time (CMCT) were detected in these patients, thus attesting to the fact that the conduction function of the pyramidal pathway was retained and there were signs of denervation and reinnervation process according to the EMG data. The results of this study demonstrated that motor cortex hyperexcitability precedes degeneration of both the upper and the lower motor neurons and is the earliest neurophysiological signs of neurodegeneration. This supports the hypothesis proposed by Charkot back in 1869 that the upper motor neuron is the first one to be affected in ALS patients ('dying-forward') [65].

The certain effectiveness of riluzole in treating ALS also indirectly confirms the pathogenetic role of hyperexcitability and excitotoxicity. Despite the diversity of its mechanisms of action, riluzole is primarily considered to be a medication reducing excitotoxicity [66]. It has been demonstrated that riluzole can partially normalize SICI and reduce excitability of peripheral nerve axons in ALS patients [67].

The indirect evidence to the pathogenetic role of excitotoxicity was also obtained in studies on using therapeutic repetitive magnetic stimulation in ALS. High-frequency repetitive TMS increases neuronal excitability in the stimulated area (see reviews [68] and [69]). It has been demonstrated in a small study conducted by Di Lazzaro et al. (2004) that high-frequency stimulation may accelerate onset of the disease, while low-frequency stimulation may inhibit it [70]. Hence, high-frequency stimulation may increase excitotoxic degeneration of motor neurons and motor cortex excitability, having an unfavorable effect on the course of the disease.

### **3.2. Is hyperexcitability good?**

the pre-symptomatic ones [23, 45]. However, this fact tells nothing about its role in pathogen‐ esis of the disease. It is still unclear when true onset of neurodegeneration occurs; however, the results of studies using transgenic animals demonstrate that the pathological process in ALS may start at the earliest stages of embryogenesis (e.g., at the stage when neural networks are formed) [63]. In this situation, all the objectively detectable alterations may be either primary or secondary (i.e., emerge as one of nonspecific stages of neurodegeneration or via

The enhancement of excitatory glutamatergic neurotransmission and reduction of inhibitory GABAergic neurotransmission in the neocortex, which underlie hyperexcitability in ALS patients, are believed to damage motor neurons via the excitotoxicity mechanism [4, 7]. The role of excitotoxicity as one of the universal mechanisms causing neuronal death in various nervous system diseases has currently been demonstrated [8, 9, 64]. High sensitivity of motor neurons to excitotoxicity can be related to high expression of glutamate receptors and low

An important argument in favor of the role of hyperexcitability and the underlying molecular processes in ALS pathogenesis is that this phenomenon can be early detected using TMS in asymptomatic SOD1 mutation carriers. Vucic et al. (2008) demonstrated a decrease in SICI in three SOD1 mutation carriers who were asymptomatic at the time the study was performed but developed clinical signs of the disease during a prospective study within 3 years. It is interesting that no neurophysiological signs of motor cortex excitability were revealed in 14 other asymptomatic carriers and they did not present with any clinical manifestations of the disease within the entire study period [45]. Early development of signs of hyperexcitability of neurons of the motor cortex, spinal cord, and even the extramotor areas such as hippocampus (e.g., see [23]) were demonstrated in a large series of experimental studies using a culture of neurons isolated from transgenic animals. Additional evidence is that signs of motor cortex hyperexcitability in ALS patients can be detected before the pyramidal pathways are affected and EMG signs of degeneration of the lower motor neuron emerge. A recent study carried out by Menon et al. (2015) showed that 24 patients with ALS had a statistically significant decrease in motor threshold, duration of cSP and intracortical inhibition, while simultaneously having an increased MER amplitude and intracortical facilitation at onset of the disease [5]. No signs of alterations in central motor conduction time (CMCT) were detected in these patients, thus attesting to the fact that the conduction function of the pyramidal pathway was retained and there were signs of denervation and reinnervation process according to the EMG data. The results of this study demonstrated that motor cortex hyperexcitability precedes degeneration of both the upper and the lower motor neurons and is the earliest neurophysiological signs of neurodegeneration. This supports the hypothesis proposed by Charkot back in 1869 that the upper motor neuron is the first one to be affected in ALS patients ('dying-forward') [65].

The certain effectiveness of riluzole in treating ALS also indirectly confirms the pathogenetic role of hyperexcitability and excitotoxicity. Despite the diversity of its mechanisms of action, riluzole is primarily considered to be a medication reducing excitotoxicity [66]. It has been demonstrated that riluzole can partially normalize SICI and reduce excitability of peripheral

the compensatory mechanism).

54 Update on Amyotrophic Lateral Sclerosis

expression of calcium-binding proteins [9].

nerve axons in ALS patients [67].

Excitotoxicity and hyperexcitability have been for a long time regarded only as damaging phenomena facilitating neuronal death. However, findings obtained in a number of studies suggest that hyperexcitability can also have a compensatory effect, at least at early stages. Furthermore, molecular alterations that accompany excitotoxicity are similar to the processes taking place when neuroplasticity is ensured. We present the results of some studies confirm‐ ing the validity of this alternative view of the hyperexcitability problem in patients with ALS and other neurodegenerative diseases.

#### *3.2.1. Motor cortex excitability and neuroplasticity*

The term 'neuroplasticity' means brain's ability to alter structurally and functionally in response to internal and external factors. This ability is currently attributed both to strength‐ ening or weakening of the existing neural connections and to the formation of new connections or destruction of the old ones [71]. Starting with the first description of cellular mechanisms of neuroplasticity, this universal property of neural tissue is inseparably associated with alteration in neuronal excitability. Long-term potentiation was shown to be predominantly of postsynaptic origin and is mostly connected to the glutamatergic system [72–74].

A series of studies have shown the alteration in motor cortex excitability and reorganization of the cortical representation of muscles after motor learning and acquisition of new skills [75, 76]. The TMS data confirmed that there is a relationship between neuroplastic alterations and an increase in motor cortex excitability [77–79]. Thus, Tyč and Boyadjian (2011) performed TMS mapping of cortical representation of the deltoid and brachioradialis muscles in healthy volunteers before and after a 6-week training (playing darts three to four times a week). The boundaries of cortical representation of the muscles under study were expanded after training, which was accompanied by an increase in the slope ratio of the amplitude-intensity curve, being indicative of the increase in motor cortex excitability [80]. According to Perez et al. (2004), a 32-minute training causes statistically significant increase in the slope ratio of the amplitudeintensity curve and a decrease in intracortical inhibition [81]. Increased motor cortex excita‐ bility presenting as increased MER amplitude and reduced motor threshold is observed in healthy volunteers not only after actual training but also after they had imagined the move‐ ments [82]. Professional sportsmen and musicians were found to have increased motor cortex excitability and increased ability of the motor cortex to undergo plastic alterations [83, 84].

To sum up, the aforementioned facts indicate that motor cortex excitability and glutamatergic neurotransmission increase during neuroplastic alterations.

#### *3.2.2. Hyperexcitability and neuroplasticity in pathology*

The most forcible evidence for the possible relationship between hyperexcitability and neuroplasticity was observed in patients with Alzheimer's disease (AD). Neurodegeneration in this disease is not limited to the structures involved in the cognitive function and affects other brain regions as well, including the primary motor cortex. At the late stages of the disease, AD patients often have motor disorders, including spasticity and pathologic plantar responses, attesting to involvement of the upper motor neuron [85]. Furthermore, lesions of the motor cortex, predominantly giant pyramidal cells of Betz, were detected in AD patients in patho‐ morphological studies [86]. In this connection, investigation of the structural and functional status of the motor cortex in AD patients is of interest as a model of clinically asymptomatic neurodegeneration of the motor cortex.

In AD patients, identically to those with ALS, motor cortex hyperexcitability can be recorded using TMS. It has been demonstrated in many studies that the motor threshold decreases in AD patients. Decreased intracortical inhibition and short cSP duration were also detected in some studies (although not all of them) (see reviews [87, 88]). There is controversy in data on disrupted inhibitory neurotransmission in AD patients; however, decreased SICI in AD is most probably not associated with dysfunction of the GABAergic system [89]. The enhanced glutamatergic neurotransmission is currently the predominant conception of the development of motor cortex hyperexcitability in AD patients.

It is interesting to mention that motor cortex hyperexcitability in AD patients is predominantly attributed to neuroplasticity processes. Hyperexcitability is believed to develop via the compensatory mechanism as a response to disruption of associative connections [90].

Motor cortex hyperexcitability in AD patients is accompanied by reorganization of the cortical representation of muscles according to TMS mapping presenting as displacement of the center of gravity of the cortical representation in the frontomedial direction with respect to localiza‐ tion of hot spots in patients without motor disorders [90]. Ferreri et al. (2011) believe that this may attest to plastic brain alterations aimed at maintaining normal motor activity as the neuron number decreases progressively [91]. fMRI studies in patients at early stages of AD, identically to those with ALS, showed areas with increased activation, which is also regarded as a result of compensatory changes [92]. In their recent study, Guerra et al. (2015) demonstrated the relationship between motor cortex hyperexcitability and neuroplasticity. Examination of seven patients with vascular dementia and nine AD patients showed a statistically significant decrease in motor threshold compared to healthy volunteers of comparable age. Parameters related to motor cortex excitability (the area of cortical representation of muscles and the area of active cortical points) showed a statistically significant correlation with neuroplastic reorganization of the motor cortex assessed based on the distance between the center of gravity of the maps and hot spot localization. The authors drew a conclusion that motor cortex hyperexcitability may promote neuroplasticity [93].

As opposed of the cortical motor zones, most AD patients at the dementia stage have decreased connectivity and activation of the hippocampus, medial temporal, and prefronal cortex when performing cognitive tasks involving short-term memory [94]. Meanwhile, activity of these areas is increased in AD patients at the moderate cognitive impairment stage and in asymp‐ tomatic carriers of mutations in the presenilin-1 gene [95–97]. This is believed to result from turning on of the compensatory mechanisms. It should be mentioned that transgenic animals at the presymptomatic stage and stage of initial manifestations (which approximately corre‐ sponds to the moderate cognitive impairment stage in humans) showed an increased expres‐ sion of a number of genes promoting synaptic plasticity [98]. In particular, transgenic mice showed enhanced expression of the AMPAR subunit genes at the early stages of the patho‐ logical process [99]. Furthermore, experimental studies revealed an increase in synaptic plasticity and hyperexcitability in hippocampal neurons, which preceded β-amyloid deposi‐ tion [100, 101]. Schneider et al. (2001) reported that hippocampal neurons in transgenic mice with presenilin mutation are characterized not only by hyperexcitability and reduced thresh‐ old to excitotoxic damage but also by facilitation of long-term synaptic potentiation [102]. In their pathomorphological study, Bell et al. (2007) showed that patients with moderate cognitive impairments had increased density of presynaptic boutons in glutamatergic synapses, while the density of presynaptic boutons in AD patients was reduced [103]. On the contrary, expression of the genes involved in synaptic plasticity and energy exchange was shown to decrease at the late stages of the disease [98, 104]. These data give grounds for assuming that the increase in neuronal excitability in AD patients at early stages of the disease may occur via the compensatory mechanism.

#### *3.2.3. Compensatory hyperexcitability in ALS: evidence from fundamental research*

To sum up, the aforementioned facts indicate that motor cortex excitability and glutamatergic

The most forcible evidence for the possible relationship between hyperexcitability and neuroplasticity was observed in patients with Alzheimer's disease (AD). Neurodegeneration in this disease is not limited to the structures involved in the cognitive function and affects other brain regions as well, including the primary motor cortex. At the late stages of the disease, AD patients often have motor disorders, including spasticity and pathologic plantar responses, attesting to involvement of the upper motor neuron [85]. Furthermore, lesions of the motor cortex, predominantly giant pyramidal cells of Betz, were detected in AD patients in patho‐ morphological studies [86]. In this connection, investigation of the structural and functional status of the motor cortex in AD patients is of interest as a model of clinically asymptomatic

In AD patients, identically to those with ALS, motor cortex hyperexcitability can be recorded using TMS. It has been demonstrated in many studies that the motor threshold decreases in AD patients. Decreased intracortical inhibition and short cSP duration were also detected in some studies (although not all of them) (see reviews [87, 88]). There is controversy in data on disrupted inhibitory neurotransmission in AD patients; however, decreased SICI in AD is most probably not associated with dysfunction of the GABAergic system [89]. The enhanced glutamatergic neurotransmission is currently the predominant conception of the development

It is interesting to mention that motor cortex hyperexcitability in AD patients is predominantly attributed to neuroplasticity processes. Hyperexcitability is believed to develop via the

Motor cortex hyperexcitability in AD patients is accompanied by reorganization of the cortical representation of muscles according to TMS mapping presenting as displacement of the center of gravity of the cortical representation in the frontomedial direction with respect to localiza‐ tion of hot spots in patients without motor disorders [90]. Ferreri et al. (2011) believe that this may attest to plastic brain alterations aimed at maintaining normal motor activity as the neuron number decreases progressively [91]. fMRI studies in patients at early stages of AD, identically to those with ALS, showed areas with increased activation, which is also regarded as a result of compensatory changes [92]. In their recent study, Guerra et al. (2015) demonstrated the relationship between motor cortex hyperexcitability and neuroplasticity. Examination of seven patients with vascular dementia and nine AD patients showed a statistically significant decrease in motor threshold compared to healthy volunteers of comparable age. Parameters related to motor cortex excitability (the area of cortical representation of muscles and the area of active cortical points) showed a statistically significant correlation with neuroplastic reorganization of the motor cortex assessed based on the distance between the center of gravity of the maps and hot spot localization. The authors drew a conclusion that motor cortex

compensatory mechanism as a response to disruption of associative connections [90].

neurotransmission increase during neuroplastic alterations.

*3.2.2. Hyperexcitability and neuroplasticity in pathology*

56 Update on Amyotrophic Lateral Sclerosis

neurodegeneration of the motor cortex.

of motor cortex hyperexcitability in AD patients.

hyperexcitability may promote neuroplasticity [93].

Several recently published studies using transgenic animals expressing mSOD1 have com‐ promised the conception of the pathological role of excitability of motor neurons in ALS. Saxena et al. (2013) demonstrated that hyperexcitability can be a defense mechanism prevent‐ ing degeneration of motor neurons. Thus, reduced excitability of motor neurons decreased accumulation of mutant SOD1, whereas increased excitability was accompanied by the greater number of intracellular aggregates and quicker death of motor neurons. It was also demon‐ strated that mutant protein accumulation increases after AMPAR blockade and decreases when AMPA is introduced. Stronger excitatory stimulation may contribute to the decrease in severity of endoplasmic stress and accumulation of abnormal proteins, thus exhibiting protective properties. Interestingly, mutant SOD1 accumulation was first detected in the least excitable fast fatiguing (FF) motor neurons of the spinal cord [105]. Leroy et al. (2014) studied the excitability of various populations of spinal cord motor neurons isolated from G93A mutant mice [106]. In this study, the electrophysiological signs of hyperexcitability were revealed only in degeneration-resistant motor neurons. The authors believe that their findings indicate that hyperexcitability does not cause degeneration; instead, it can be a defense mechanism [106].

#### *3.2.4. Neuroplasticity in ALS: evidence from fMRI studies*

Nowadays, fMRI is the key method for studying neuroplasticity in ALS patients. Numerous studies have revealed the changes in activation patterns of various brain regions in ALS both in rest and using various paradigms [2]. In this publication, the changes in activation patterns in ALS patients performing motor tasks are of special interest.

In the study performed by Konrad et al. (2002), ALS patients and healthy volunteers underwent fMRI as they performed a motor paradigm (bending fingers) [107]. Significant changes in the patterns of cortex activation were observed: a forward displacement of the activation cluster, into the supplementary motor area, and an increase in its volume. Activation volume in the inferior frontal gyrus (Brodmann area 6) in the contralateral hemisphere and parietal lobes increased bilaterally. The authors have put forward a hypothesis that these changes represent the structural and functional rearrangement of the motor system induced by degeneration of the upper and lower motor neuron [107]. Lule et al. (2007) reported that an increase in activation of the primary motor and premotor cortex in ALS patients is revealed not only when performing the movement but also when imaging it [108]. Stanton et al. (2007) detected that activation in the sensorimotor cortex (Brodmann areas 1, 2, and 4), the inferior parietal lobule, and the superior temporal gyrus increased when the ALS patients performed a motor task, while activation in the dorsolateral prefrontal cortex decreased [109]. It is noteworthy that these changes were observed when comparing ALS patients not only to healthy volunteers but also to patients with peripheral nerve disorders [109]. This confirms that involvement of the upper motor neuron plays a crucial role in development of these changes and does not give grounds for considering them as just a response to the development of muscle fatigue.

Thus, an analysis of the results of fMRI with the motor paradigm in ALS patients demonstrates that the activation areas expand when a motor task is being performed. Interestingly, the changes in activation during fMRI can have a prognostic value. Poujois et al. (2013) reported that activation in the somatosensory and parietal cortex increases in ALS patients performing a simple motor task compared to the control group. The dynamic follow-up for 1 year has shown that activity of the contralateral parietal lobe has a statistically significant negative correlation with the rate of disease progression (*p* = 0.001) [110].

These findings give grounds for suggesting that expansion of activation areas as an ALS patient performs a motor task has the compensatory mechanism and is probably aimed at maintaining the motor function in response to progressive degeneration of cortical motor neurons. Meanwhile, certain researchers believe that the activation areas can increase due to degener‐ ation of inhibitory interneurons [2]. The relationship between alterations in activation patterns in ALS patients according to fMRI data and changes in motor cortex excitability according to TMS data has not been studied yet. It should be mentioned that the positive prognostic significance of neuroplastic alterations demonstrated by Poujois et al. (2013) is in contrast with the views about the negative role of hyperexcitability in this disease.
