Anatomy of the Motor System In the CNS, the motor cortex, basal ganglia, cerebellum, and parts of the brainstem, are directly involved in the planning and initiation of movement. In contrast, the precise timing and pattern of movement is generated by MNs located in the spinal cord (Figure 1; Kiehn, 2016). The corticospinal (anterior and lateral) tract is the largest descending tract in humans. The lateral corticospinal tract originates in the primary motor cortex, which lies in the precentral gyrus and sends fibers to muscles in the extremity. This is via contralateral cortical innervation, so that the left motor cortex controls the right extremities and vice versa, to control the voluntary movement of contralateral limbs (Javed and Lui, 2018). MNs outputs are not confined to the peripheral muscles however, but also include excitatory terminals to a group of interneurons, Renshaw cells, and also to other MNs. FIGURE 1 Organization of the human corticospinal tract. MN groups vulnerable and resistant to degeneration in ALS are shown in red and blue, respectively. Glutamate (cortex, spinal cord) and acetylcholine (spinal cord) modulate excitatory input within neurons, whereas GABA and glycine facilitate inhibitory neurotransmission (Ramírez-Jarquín et al., 2014). At the neuromuscular junction (NMJ), only acetylcholine acts at the synapse but interestingly, synaptic transmission between MNs in the spinal cord involves both acetylcholine and glutamate (Bhumbra and Beato, 2018). Renshaw cells are excited through both acetylcholine and glutamate receptors and spinal MNs co-release glutamate to excite Renshaw cells and other MNs, but not to excite muscles (Nishimaru et al., 2005; Bories et al., 2007; Bhumbra and Beato, 2018). Hence, different synaptic transmission systems are present at different postsynaptic targets of MNs (Bhumbra and Beato, 2018). However, MNs are not homogeneous throughout the CNS because they exhibit distinct morphologies and patterns of connectivity, which underlie their different physiological functions. Hence, within a single region, MNs that perform closely related functions can be further subdivided, both anatomically and physiologically. The identities of specific MN subtypes and their target projections are controlled by selective cell-type expression of transcription factors, notably members of the Hox, LIM, Nkx6, and ETS families (Stifani, 2014). This provides the fundamental mechanism for spinal MN diversification and connectivity to specific peripheral muscle targets. Thus, to generate movement, MNs integrate information from sensory structures and transform it into precise temporal and magnitudal activation of muscles. A MN located in the spinal cord innervates up to several hundred fibers within one muscle, which together form the motor unit. Trains of action potentials within the axon cause the release of acetylcholine at the NMJ, which activates nicotinic receptors on the muscle fibers the MN innervates. This initiates a cascade of signaling events in the muscle fiber that leads to its contraction. A motor pool consists of all the individual MNs that innervate a single muscle. A muscle unit (one muscle and its motor pool) is composed of three different types of functional motor units consisting of alpha (α), beta (β), and gamma (γ) MNs, which are classified according to the contractile activity of the muscle fiber innervated. We will now discuss in more detail the anatomy of those structures involved in movement. The Spinal Cord In the spinal cord, MNs are organized into columns (Table 1) based on the location of their target muscle [reviewed in Matise and Sharma (2013) and Stifani (2014)]. Within each column, the MNs innervating each muscle are clustered into motor pools, each containing of 20–300 cells depending on the muscle (Bryan et al., 1972; McHanwell and Biscoe, 1981). α-MNs located in the spinal cord are archetypal MNs that innervate extrafusal muscle fibers, thus creating force to move the skeleton (Table 2). In contrast, γ-MNs innervate intrafusal fibers, which modulate the sensitivity of muscle spindles to stretch (Table 2) (Hunt and Kuffler, 1951; Kuffler et al., 1951; Kanning et al., 2010). β-MNs are not as well characterized as α-MNs but they innervate both intrafusal and extrafusal muscle fibers (Bessou et al., 1965). Both α and γ-MNs have large dendritic trees but γ-MNs have fewer large dendrites than α-MNs (7–11) and they also branch less (Westbury, 1982). The somas of γ-MNs are smaller than those of α-MNs and they also possess thinner axons, which reflects their slower conduction velocity (<55 m/s in γ-MN vs. ∼70–90 m/s in α-MNs in cats) (Table 2) (Westbury, 1982). γ-MNs receive only indirect sensory inputs. Therefore, γ-MNs do not directly participate in spinal reflexes (Eccles et al., 1960; Stifani, 2014), but they contribute to the modulation of muscle contraction instead. Table 1 Segmental organization of spinal cord columns. Pools of MNs that innervate muscles of similar embryonic origin are stereotypically localized within the ventral spinal cord, known as motor columns. The medial motor column (brown) is present in the whole spinal cord and it comprises MNs that innervate the long muscles of the back and the body wall musculature. The spinal accessory column (purple) and the phrenic column (red) are found along the five first cervical segments (C1 to C5) and between C3 and C5, respectively. The preganglionic column (yellow) extends from the first thoracic segment (T1) to the second lumbar segment (L2), and between sacral segments 2 and 4 (S2 to S4) where the Onuf’s nuclei (∗) are found. MNs in the preganglionic column innervate neurons of the sympathetic ganglia. The hypaxial motor column (blue) is restricted to the thoracic spinal cord (T1 to T12). The lateral motor column (green), connected to the limbs, comprises the cervical and thoracic spinal cord (from C5 to T1) and the lumbar spinal cord (L1 to L5). Table 2 Comparison of α- and γ-spinal motor neurons. Spinal α-MN Spinal γ-MN Target muscle fiber Extrafusal1 Intrafusal1 Soma size Larger2,3,4,5 Smaller2,3,4,5 Axon diameter Larger2 Thinner2 Dendrite branching More2 Less2 Motor unit size (innervation ratio) Larger6 Smaller6 Membrane input resistance Larger7 Smaller7 Firing Subtype-dependent8 Subtype-dependent8 Axon conduction velocity Faster2,7,9 Slower2,7,9 Afterhyperpolarization duration Subtype-dependent7,9 Variable7,9 Spinal reflex Yes10 No10 Affected in ALS Yes11,12 Less11,12 Affected in aging Yes13,14 No13,14 Markers Osteopontin15RBFOX3/NeuN16Hb9::GFP5NKAα117 (adult) Err316Weak NeuN5,16NKAα317 (adult)ESRRG16GFRα15HTR1D18 (early marker)WNT7A19 (late embryonic stage) ESRRG, estrogen-related receptor gamma; GFRα1, GDNF family receptor alpha 1; HTR1D, serotonin receptor 1D; NAKα1/3, Na+/K+-ATPases 1/3; RBFOX3, RNA binding protein fox-1 homolog 3. 1(Kuffler et al., 1951), 2(Burke et al., 1977), 3(Westbury, 1982), 4(Friese et al., 2009), 5(Shneider et al., 2009), 6(Adal and Barker, 1965), 7(Kemm and Westbury, 1978), 8(Murphy and Martin, 1993), 9(Gustafsson and Lipski, 1979), 10(Eccles et al., 1960), 11(Mohajeri et al., 1998), 12(Lalancette-Hebert et al., 2016), 13(Swash and Fox, 1972), 14(Hashizume et al., 1988), 15(Misawa et al., 2012), 16(Friese et al., 2009), 17(Edwards et al., 2013), 18(Enjin et al., 2012), 19(Ashrafi et al., 2012). A distinct group of MNs in the sacral spinal cord termed ‘Onuf’s’ neurons, innervate the striated muscles of the external urethra, external anal sphincter via the pudental nerve, and the ischiocavernosus and bulbocavernosus muscles in males (Sato et al., 1978; Nagashima et al., 1979; Kuzuhara et al., 1980; Roppolo et al., 1985). These MNs are histologically similar to limb α-MNs (Mannen et al., 1977) and they are located anteromedial to the anterolateral nucleus and extend between the distal part of the S1 segment and the proximal part of S3. α-motor units can be subdivided according to their contractile properties, into fast-twitch (F) and slow-twitch (S) fatigue-resistant types (Table 3) (Burke et al., 1973). In addition, fast-twitch α-motor units can be further categorized into fast-twitch fatigable [FF] and fast-twitch fatigue-resistant [FR] types, based on the length of time they sustain contraction. The basis of this classification is the duration of the twitch contraction time (Burke et al., 1973). F- and S-MNs also exhibit different afterhyperpolarization duration (AHP) properties. AHP is the phenomenon by which the membrane potential undershoots the resting potential following an action potential. S-MNs have a longer AHP than F-MNs, indicating that S-MNs have a longer “waiting period” before they can be stimulated by an action potential. Thus, they cannot fire at the same frequency as F-MNs (Eccles et al., 1957), so the larger FF-MNs take longer to reach an activation threshold. Similarly, other electrical properties differ between S- and F-MNs (Table 3), including their input resistance (a measure of resistance over the plasma membrane) and rheobase (a measure of the current needed to generate an action potential). S-MNs have a higher input resistance than F-MNs, underlying Hennenman’s size principle which postulates that S-motor units are the first to be recruited during movement, followed by FR and then FF units (Henneman, 1957; Mendell, 2005). Hence, a slow movement generating a small force will only recruit S-MNs, whereas a quick and strong movement will also recruit F-MNs, as well as S-MNs. Table 3 Comparison of fast (FF, fast-fatigable; FR, fast-resistant) and slow (S) spinal α-motor neurons. Spinal α-MN F S Target muscle fiber IIb (FF), IIa (FR)1 I1 Soma size Similar2,3,4,5,6 Similar2,3,4,5,6 Axon diameter Larger7,8 Thinner7,8 Dendrite branching More4,9 Less4,9 Motor unit size (innervation ratio) Larger1,10 Smaller1,10 Membrane input resistance Smaller11,12,13 Larger11,12,13 Firing Phasic14,15 Tonic14,15 Axon conduction velocity Faster1,13 Slower1,13 Afterhyperpolarization duration Shorter14 Longer14 Recruitment Late15 Early15 Affected in ALS Early16,17,18 Late16,17,18 Affected in aging Early19,20,21 Late19,20,21 Markers CHODL22 CALCA22 SV2a23 SK324 ESRRB22 (adult) CALCA, calcitonin-related polypeptide alpha; CHODL, chondrolectin; SV2A, synaptic vesicle glycoprotein 2a; SK3, postsynaptic Ca2+-activated K+ 3; ESRRB, estrogen-related receptor beta. 1(Burke et al., 1973), 2(Kernell and Zwaagstra, 1981), 3(Burke et al., 1982), 4(Cullheim et al., 1987), 5(Vinsant et al., 2013), 6(Hadzipasic et al., 2014), 7(Burke et al., 1977), 8(Dukkipati et al., 2018), 9(Ulfhake and Kellerth, 1981), 10(Burke and Tsairis, 1973), 11(Bakels and Kernell, 1993), 12(Gardiner, 1993), 13(Zengel et al., 1985), 14(Eccles et al., 1957), 15(Zajac and Faden, 1985), 16(Frey et al., 2000), 17(Hegedus et al., 2007), 18(Pun et al., 2006), 19(Hashizume et al., 1988), 20(Kadhiresan et al., 1996), 21(Kanda and Hashizume, 1989), 22(Enjin et al., 2010), 23(Chakkalakal et al., 2010), 24(Deardorff et al., 2013). In addition, at least eleven types of interneurons are involved in the control of movement, as part of central pattern generators in the spinal cord. Interneurons arise from five progenitor cells and, according to the expression of distinct transcription factors, they mature into different lineages. This includes excitatory V2a, V3, MN and Hb9 neurons and inhibitory V0C/G,V0D, V0V, V1, V2b, Ia and Renshaw cells (belonging to the V1 interneuron subclass), which display specific locations and projections within the spinal cord (Ramírez-Jarquín et al., 2014). The Brainstem Cranial nerve nuclei are populations of neurons in the brainstem that are associated with one or more cranial nerves. They provide afferent and efferent (sensory, motor, and autonomic) innervation to the structures of the head and neck (Sonne and Lopez-Ojeda, 2018). The more posterior and lateral nuclei tend to be sensory, and the more anterior nuclei are usually motor nuclei. Trigeminal MNs innervate the muscles of mastication, whereas facial MNs supply the superficial muscles of the face, and ambiguous MNs supply the muscles of the soft palate, pharynx, and larynx. The oculomotor (III), trochlear (IV) and abducens (VI) nuclei are somatic efferents innervating the extraocular muscles within the orbit. The oculomotor nucleus contains MNs that innervate four of the six extraocular muscles (superior, medial and inferior recti, inferior oblique), plus the levator palpebrae superioris muscle. These muscles display a unique composition of six fiber types, distinct from other skeletal muscles that possess marked fatigue resistance (Table 4). Oculomotor units are amongst the smallest of the motor units, in contrast to skeletal muscle motor units that have higher maximum MN discharge rates. Furthermore, α-MNs in oculomotor units have higher resting membrane potentials (∼61 mV) than spinal cord α-MNs (∼70 mV), and they also discharge at higher frequencies (∼100 Hz during steady state and ∼600 Hz during saccadic eye movements, compared to ∼100 Hz for spinal cord α-MNs) (Table 4) (Robinson, 1970; Fuchs et al., 1988; Torres-Torrelo et al., 2012). Oculomotor neurons are almost continually active at high frequencies when maintaining eye position (Fuchs et al., 1988; De La Cruz et al., 1989), and this level of activity places high metabolic demand on these cells (Robinson, 1970; Porter and Baker, 1996; Brockington et al., 2013). Table 4 Comparison of α-spinal motor neurons and oculomotor neurons. Spinal α-MN Oculomotor neuron Target muscle fiber Single fiber type1 Multiple fiber types1 Soma size Larger2,3 Smaller2,3 Dendrite branching Larger2 Smaller2 Motor unit size (innervation ratio) Larger4,5,6,7 Smaller4,5,6,7 Resting potential Smaller8,9,10 Higher8,9,10 Discharge frequency 100 Hz8,9,10 100–600 Hz8,9,10 Affected in ALS Yes11,12,13,14 No11,12,13,14 Affected in aging Yes15,16 No15,16 1(Zhou et al., 2011), 2(Durand, 1989), 3(Shoenfeld et al., 2014), 4(Burke et al., 1971), 5(Burke and Tsairis, 1973), 6(Enoka, 1995), 7(Guéritaud et al., 1985), 8(Robinson, 1970), 9(Fuchs et al., 1988), 10(Torres-Torrelo et al., 2012), 11(Nimchinsky et al., 2000), 12(Hedlund et al., 2010), 13(Valdez et al., 2012), 14(Comley et al., 2016), 15(Hashizume et al., 1988), 16(Swash and Fox, 1972). The Cortical Motor System The motor cortex is the region of the cerebral cortex responsible for mediating voluntary movements. In rodents, the primary cortex (M1) is large and comprises almost all of the frontal cortex (Gioanni and Lamarche, 1985; Neafsey et al., 1986; Brecht et al., 2004; Yu et al., 2008; Hira et al., 2013; Paxinos, 2014), whereas in primates, the frontal cortex is compartmentalized into specialized premotor subfields and M1 is relatively small in comparison (Ferrier, 1875; Leyton and Sherrington, 1917; Asanuma and Rosén, 1972; Dickey et al., 2013; Riehle et al., 2013; Young et al., 2013; Ebbesen and Brecht, 2017). M1 plays a central role in controlling movement. This involves specialized UMNs located in layer V of this region (Broadman area 4), the giant Betz cells or corticospinal MNs. These MNs are the cortical components of the MN circuit that initiates and modulates precise voluntary movement, through long-range projections to the spinal cord. Approximately ∼30–50% of corticospinal projections originate from M1 MNs and they begin modulating their firing rate several hundred ms before movement of the limb is initiated (Georgopoulos et al., 1982; Porter and Lemon, 1993). In most mammals, the axons of cortical MNs terminate at spinal interneurons, but they also make direct connections to MNs (Lemon, 2008; Rathelot and Strick, 2009). This constitutes the final efferent pathway to the muscle to generate or suppress movement (Ramírez-Jarquín and Tapia, 2018). Motor Neurons Selectively Degenerate in ALS Patients Lesions to motor structures in humans and experimental animals lead to impairments in normal movement. In ALS, as MNs degenerate, the ability to control movement of the muscles is progressively lost. Specific MNs in the brain, brainstem and spinal cord are selectively targeted, and pathology appears first in these restricted MN populations. In fact, the name “Amyotrophic Lateral Sclerosis” reflects the strikingly selective degeneration of MNs in ALS. It is derived from a combination of three words; “Lateral” refers to the lateral spinal cord, given that corticospinal MNs are particularly vulnerable to degeneration; “Amyotrophic” is from the Greek “amyotrophia,” meaning lacking muscle nourishment; and “Sclerosis” (fibrosis) refers to gliosis of the crossed corticospinal tract in the dorsolateral quadrant of the spinal cord (Charcot, 1874; Frey et al., 2000; Pun et al., 2006). In the brain, UMNs in the primary cortex are also amongst the first to degenerate in ALS, and similarly, in the brainstem, the hypoglossal MNs that innervate the muscles of the tongue involved in swallowing and breathing, are also targeted early in disease course. In the brainstem, ALS can also affect trigeminal MNs, the facial MNs and ambiguous MNs. However, other MN subgroups within this region are relatively resistant to degeneration, including MNs of the oculomotor (III), trochlear (IV) and abducens (VI) nuclei, innervating the extraocular muscles (Mannen et al., 1977; Schrøder and Reske-Nielsen, 1984). Hence, eye movements remain relatively preserved throughout disease course (Kanning et al., 2010) and as a consequence, eye tracking devices are often used to aid communication in the later stages of ALS (Caligari et al., 2013). Whilst it has been reported that oculomotor neurons may be affected at disease end stage, this was recently attributed to dysfunction of the dorsolateral prefrontal cortex, the frontal eye field and the supplementary eye field, confirming the relative resistance of pure oculomotor functions in ALS (Shaunak et al., 1995; Proudfoot et al., 2015). Widespread loss of GABAergic interneurons has also been described in ALS, in both the cortex (Stephens et al., 2001; Maekawa et al., 2004) and the spinal cord (Stephens et al., 2006; Hossaini et al., 2011). MRI studies of ALS patients has revealed that very specific neuronal networks are vulnerable to degeneration in ALS (Bede et al., 2016). However, whilst TDP-43 pathology is the signature pathological hallmark of almost all ALS cases, it can arise in areas of the CNS that are not particularly vulnerable to degeneration (Geser et al., 2008). Significant TDP-43 pathology is present in the substantia nigra and basal ganglia, which are not affected in ALS, as well as in the motor gyrus, midbrain and spinal cord. Curiously, pathological forms of TDP-43 are also detectable in the occipital lobe, amygdala, orbital gyrus and hippocampus (Geser et al., 2008). Hence, whilst major degeneration of corticobulbar, LMN, pyramidal and frontotemporal networks underlie the widespread clinical symptoms of ALS, it remains unclear how other circuits, such as the visual, sensory, autonomic and auditory systems, remain relatively protected in ALS. These unaffected networks, however, have not been well studied in ALS patients.