1 Excitatory and inhibitory neuronal circuits in the spinal cord and their

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Excitatory and inhibitory neuronal circuits in the spinal cord and their role in the control of motor neuron function and degeneration Uri Nimrod Ramírez-Jarquín, and Ricardo Tapia ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00503 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 20, 2018

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ACS Chemical Neuroscience

Excitatory and inhibitory neuronal circuits in the spinal cord and their role in the control of motor neuron function and degeneration

Uri Nimrod Ramírez-Jarquín and Ricardo Tapia* División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510-Ciudad de México, México

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Abstract The complex neuronal networks of the spinal cord coordinate a wide variety of motor functions, including walking, running and voluntary and involuntary movements. This is accomplished by different groups of neurons, called center pattern generators, which control left-right alternation and flexor-extensor patterns. These spinal circuits, located in the ventral horns, are formed by several neuronal types, and the specific function of most of them has been identified by means of studies in vivo and in the isolated spinal cord of mice harboring genetically induced ablation of specific neuronal populations. These studies have shown that the coordinated activity of several interneuron types, mainly GABAergic and glycinergic inhibitory neurons, have a crucial role in the modulation of motor neurons activity that finally excites the corresponding muscles. A pharmacological experimental approach by administering in the spinal cord agonists and antagonists of glutamate, GABA, glycine and acetylcholine receptors to alter their synaptic action has also produced important results, linking the deficits in the synaptic function with the resulting motor alterations. These results have also increased the knowledge of the mechanisms of motor neuron degeneration, which is characteristic of diseases such as amyotrophic lateral sclerosis, and therefore open the possibility of designing new strategies for the prevention and treatment of these diseases. Keywords Excitation-inhibition balance, central pattern generator, neurotransmitters, ALS


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Introduction Neuronal circuits in the ventral horn of the spinal cord are responsible for locomotion and coordinate a wide range of activities, such as walking, running, breathing, and voluntary and involuntary movements. The cytoarchitecture of the neuronal network includes a complex arrangement of motor neurons, interneurons and non-neuronal cells as astrocytes and microglia cells. The physiological activity of this network is responsible for the generation and control of rhythm and pattern of movements, and depends mainly on the interaction of excitatory glutamatergic neurotransmission and inhibitory GABAergic and glycinergic neurotransmission, although acetylcholine, dopamine, serotonin and several modulatory peptides also play important roles (1). The connectivity and function of these neuronal circuits control the rhythm of locomotor movements, including right-left alternation and flexor-extensor equilibrium. The neuronal groups involved in this delicate control of movements are called central patterns generators (CPGs) that can thus be defined as the neuronal networks responsible for the general coordination of locomotion. Modulation of CPGs activity requires a coordinated activity of all the neurons involved, while motor neurons constitute the final efferent pathway to muscles (2, 3). Among the CPG network, interneurons are extremely important, because of the complex connectivity between them and with motor neurons. Whereas glutamate is the main excitatory transmitter in synapses impinging on motor neurons, terminal boutons of interneurons include cholinergic excitatory synapses and, preponderantly, inhibitory GABAergic and glycinergic synapses (1). Computational analysis based on experimental evidence have suggested two functional levels of organization for CPGs function, rhythm generator circuits and pattern formation circuits, which distribute the rhythm to interneuron populations projecting to different motor neuron pools, highlighting the role of inhibitory pathways as modulator of excitatory motor patterns (3-5). Neuronal types involved in locomotor CPGs At least eleven neuronal types of interneurons conform the networks of CPGs, and the knowledge of their projections is relevant to understand the role of these cells in the CPGs activity. They arise from five progenitor cells (p0, p1, p2, p4 and pMN), and mature to different linages, V0D, V0V; V1; V2a, V2b; V3; MN, Hb9, according to their transcription factor (Dbx1/Evx1-, Dbx1/Evx1+, En1, Chx10, Gata2/3, Sim1, Hb9, respectively). Most of these classes of neurons are excitatory, and V2b, V0C/G, V0D, V0V and V1 neurons, as well 3 ACS Paragon Plus Environment


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as the Ia and the Renshaw cells, are inhibitory (1, 3, 4). Intrasegmentally, these interneurons present a specific location and projections: V0 linage (V0C, V0D, V0C) are commissural interneurons; V0C project ipsilaterally and V0D and V0C show contralateral projections (6); V2a interneurons project ipsilaterally to motor neurons and other interneurons like V0 (7); inhibitory Ia interneurons also present ipsilateral axons that impinge on motor neurons and on control reciprocal inhibitory neurons (8); V3 commissural excitatory neurons project ipsilaterally to other interneurons like Renshaw cells (9); and Renshaw cells are involved in recurrent inhibition by ipsilateral projection to motor neurons (10). A scheme of the connectivity of these neuronal types is shown in Figure 1. The knowledge of the role of the different interneurons, as related to their location, has been greatly increased by recent experiments using genetic manipulation. Selective blockade of V3-neurons activity by expression of tetanus toxin light chain subunit (to decrease neurotransmitter release) or by expression of allastostatin in a transgenic mice model (to open G protein-coupled inward-rectifying potassium channels), resulted in alterations in amplitude and duration of bursts recorded during extensor-flexor and rightleft alternations in the isolated spinal cord. Depleted V3 activity caused also gait disorders in vivo, characterized by an increase of variability in the timing and phasing of stepping movements, as determined by kinematic experiments (9, 11). V0D and V0V neurons represent the most important neurons involved in the control of alternating activation of CPGs for left-right alternation (12). Electrophysiological recordings in the isolated ventral lumbar spinal cord of transgenic mice with total genetic ablation of V0 neuron populations showed incorrect sequential activity of motor neurons pools, causing loss of alternating activity and instead a synchronized activity between leftright and flexor-extensor motor neurons during drug-induced locomotion-like pattern (13). The particular role of each V0-linage associated to the spinal circuits locomotor activity at low or high speed was evaluated by specific genetic ablation of V0D neurons (identify as V0 neurons Pax+). This ablation induced also alterations of drug-induced locomotor-like pattern at low speed, and specific V0V ablation (identified as Pax- neurons) caused changes in the left-right alternation in locomotor electrical activity at high speed. These altered electrophysiological activity recorded in the isolated spinal cord was associated with gait alterations when both neuronal linages were ablated, characterized by the substitution of left-right alternation for a quadrupedal hopping gait at all velocities tested (14). 4 ACS Paragon Plus Environment

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Genetical ablation of V2a neurons by means of the targeted expression of diphtheria toxin A chain under Chx10 gene control (characteristic of V2a neurons) resulted in variability of the burst amplitude recorded in the ventral roots of isolated spinal cord and also in substitution of the normal left-right activity alternation (“trotting”) by synchronous left-right (“galloping”) activity. This altered electrophysiological activity was also associated with gait changes during running of the transgenic mice, in which the “trotting” normal leftright alternation was replaced by “galloping” gait (2, 15). Based on this and other experimental evidence, two configurations have been suggested to explain the V2a-V0V relation: the first model suggests that V2a neurons can be activated by excitatory inputs from ipsilateral flexor centers and excite V0V neurons, which in turn inhibit contralateral flexor centers by activation of inhibitory interneurons like the Renshaw cells through ipsilateral projections (14). The other configuration proposes inputs to the V2a neurons from ipsilateral extensor centers and also direct excitatory inputs from V0V cells to the contralateral flexor motor neuron (16). These possible configurations, that can occur simultaneously in the same segment of the spinal cord, explain the function of the circuits involving V2a and V0V neurons. In the hemisected isolated spinal cord of mice genetically manipulated to express the green fluorescence protein under the Hb9 promoter, in order to identify the commissural Hb9 ventral interneurons, electrophysiological recordings showed that during the onset of electrically- and drug-induced fictive locomotor-like patterns, the firing pattern of Hb9 interneurons switches to a pacemaker mode, displaying bursting activity that contributes to generate the rhythm of the neuronal network activity. Interestingly, motor neurons that also originate from the Hb9 linage do not show a pacemaker activity in these conditions (17, 18). Other recent studies with transgenic mice have addressed the role of the inhibitory interneurons V1 and V2b, which are involved in flexor and extensor alternation during locomotor rhythm. Transgenic mice lacking both interneuron types show incapacity to articulate their limb joints and deficits in limb-driven reflex movements during mechanical stimulation. In vitro extracellular recording of L2 and L5 ventral roots activity in these mice showed a synchronous like-locomotor pattern, suggesting that these interneurons coordinate also the flexor-extensor motor pattern activity (19). The most studied inhibitory interneurons are the Renshaw cells, which originate from p1 progenitors and belong to the V1 interneuron subclass (20). They are located in the ventral regions of layer VII and IX (Renshaw cell area) and their axons bifurcate in 5 ACS Paragon Plus Environment


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ascending and descending branches (10). Renshaw neurons regulate the motor output by recurrent inhibitory circuits to the motor neurons, exerting feedback modulation of their excitatory action, both directly and through inhibition of Ia inhibitory neurons that impinge on the reciprocal motor neuron involved in flexor and extensor alternation activity. Recurrent inhibition by the Renshaw cells exerts a longer inhibitory synaptic action than the more phasic inhibition induced by Ia interneurons, probably because the latter is a pure glycinergic neurotransmission whereas Renshaw cells release both GABA and glycine and GABAergic transmission follows a slower time course (21, 22). In the isolated spinal cord of the perinatal transgenic mice in which the excitatory neurotransmission has been suppressed by genetical elimination of the vesicular glutamate transporter 2 (VgluT2), the flexor-extensor alternation persists, due probably to the activity of inhibitory Ia interneurons, thus defining a minimal inhibitory network needed to produce flexor-extensor like-locomotor pattern (23). Excitatory and inhibitory modulation of motor neuron excitability and locomotion Besides the electrophysiological recordings of neuronal activity in the isolated spinal cord of transgenic mice and the consequent motor alterations reviewed in the previous section, the role of excitatory and inhibitory neurotransmissions in the control of motor neuron excitability has been studied by means of

pharmacologic strategies in vivo.

Hyperexcitation of glutamatergic pathways has been induced in rats by the intrathecal infusion of excitatory amino acids or glutamate receptor agonists, using miniosmotic pumps, which caused motor behavioral changes such as palsy of the tail, hind-limb paralysis and urinary incontinency (24, 25). By means of the acute administration of drugs by microdialysis or by chronic infusion with miniosmotic pumps directly in the tissue of the rat lumbar spinal cord, we have shown that AMPA (α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) induces muscle hyperexcitability, progressive motor deficits leading to complete paralysis of the hindlimbs and intense degeneration of motor neurons in the infused region (26-28). This neuronal loss is due to calcium entry through the calcium-permeable AMPA receptors (29), and both the motor alterations and the motor neuron loss are prevented by the administration of trophic factors such as vascular endothelial growth factor (28, 30, 31) or of some energy substrates, mainly pyruvate (32, 33). These experiments clearly show that overexcitation of spinal motor neurons leads to their death by excitotoxicity, mediated probably by calcium-induced structural and functional mitochondrial energy deficits, and have allowed also to establish a correlation 6 ACS Paragon Plus Environment

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between the number of motor neurons affected in the infused segments of the spinal cord and the consequent motor alterations (34). Recently, some of our results in the rat were replicated in the mouse, where the denervation of neuromuscular junctions in the corresponding muscles was reported (35). Mainly because of the finding of decreased GLT1 transporter in the spinal cord of some amyotrophic lateral sclerosis (ALS) patients (36, 37), the effect of glutamate transport inhibitors infused in spinal cord tissue or intrathecally has been studied. We have shown that in the rat neither the acute (26) nor the chronic (38) infusion in the lumbar spinal cord of unspecific glutamate transport inhibitors like pyrrolidine dicarboxylate caused any motor neuron degeneration or motor behavior alteration, even when the extracellular concentration of glutamate, measured by microdialysis and HPLC, was greatly increased (26, 38). In a recent report, using a conditional mouse model for deleting the astroglial transporters GLT1 and GLAST in the spinal cord, it was shown that this depletion induced progressive motor neuron loss, severe paralysis and shortened lifespan, and that these alterations are prevented with parampanel, an AMPA receptor antagonist (39). A different approach designed by our group consisted in stimulating the release of neurotransmitters

by 4-aminopyridine,

a potassium

channel blocker

that

when

administered in vivo in the hippocampus increases the release of glutamate and GABA and produces neurodegeneration that is prevented by blockade of glutamate receptors (40, 41). Using the miniosmotic pumps procedure mentioned above (28), we have recently shown that the intraspinal administration of 4-aminopyridine induces temporary muscle fasciculation and motor deficits but no motor neuron degeneration. These effects were prevented by antagonists of both AMPA and NMDA (N-methyl-D-aspartate) receptors, suggesting that the 4-aminopyrydine-induced motor neuron hyperexcitation is due to the overactivation of excitatory glutamate receptors consequent to the exacerbated release of glutamate (42). The role of spinal inhibitory circuits in the regulation of motor neuron excitability has been also explored using the previously described pharmacological approach in vivo. Acute and chronic infusion of the GABAA receptor blocker bicuculline induces temporary muscular hyperexcitability and motor deficits, as well permanent phalange flaccidity, changes associated to moderate motor neuron loss; in contrast, blockade of glycine receptors with strychnine did not cause any significant effect. The excitotoxic effect of bicuculline was notably potentiated by 4-aminopyridine and by a low dose of AMPA, since 7 ACS Paragon Plus Environment


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motor neuron loss was more intense and total paralysis occurred (42, 43). These results suggest a close functional link between excitatory glutamatergic transmission and the inhibitory circuits mediated by GABA in the regulation of motor neuron excitability, and this GABAergic modulatory role seems to be intrasegmental, because the chronic blockade of glycinergic neurotransmission, which is mainly intersegmental (44, 45) did not induce any sign of hyperexcitability or motor neuron degeneration. This is in line with the description of the spinal circuits described in the previous section, where ipsilateral flexor-extensor alternations are governed by GABAergic neurons which directly affect the motor neuron activity within each spinal segment. Table 1 summarizes the findings of these pharmacological approach, in relation to the neuronal types described in the previous section of this review. Structural alterations of spinal neuronal circuits and motor neuron degeneration Synaptic

dysfunction

and

presynaptic

alterations

have

been

described

in

neurodegenerative diseases, including ALS, mainly in experimental models (46). Axonal transport deficits and low axonal mitochondrial motility were observed in primary cultured hippocampal neurons from mutant SOD1 (superoxide dismutase type 1) mice (the most used transgenic model of familial ALS, fALS), as well as protein accumulation by mislocation of the mutant enzyme in synaptic boutons (47). Mislocalization of proteins has also been associated to apoptosis in mouse cortical neuronal cultures expressing mutant SOD1 (48). In the same transgenic model, boutons afferent to lumbar spinal motor neurons innervating the medial gastrocnemius muscle showed a significant decrease in the number of presynaptic terminals immunolabeled with synaptophysin, at the time of onset of the motor deficiency symptoms (49). Alterations of neuromuscular synapses at type II muscle fibers (fast fatigable fibers) but not type I (slow fibers) have been also found in SOD1 mutant mice since presymptomatic stages (50). In our pharmacological model of motor neuron degeneration induced by chronic AMPA receptor overactivation we have observed an increase of presynaptic boutons afferent to lumbar motor neurons, identified by synaptophysin and by markers of GABAergic and glycinergic boutons, at early degenerative stages, whereas the number of these boutons decreased at late degenerative stages, when paralysis has been established (Ramírez-Jarquín and Tapia, in preparation). Transgenic fALS mice have been also used to study the alterations and role of the cholinergic presynaptic C-boutons afferent to motor neurons. Recently, morphometric 8 ACS Paragon Plus Environment

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studies using different parameters such as area, volume and size, have found that the Cboutons number is unchanged at presymptomatic ages, but their size increases during days 8-30 although these alterations were observed only in male mice (51). In another work in fALS mice the number and area of C-boutons was found increased at presymptomatic ages but decreased at symptomatic stages (52). The presymptomatic changes observed have been proposed as a compensatory neuroprotective mechanism by means of increasing motor neuron excitability. This has been suggested because reduction of motor neuron excitability by chronic administration of CNQX (6-cyano-7nitroquinoxaline-2,3-dione), an AMPA receptor antagonist, produces further enlargement of C-boutons, and because antagonists of metabotropic cholinergic receptors delay denervation and the decline of muscular force; these effects are mediated by mTOR, thus suggesting that C-bouton volume increase facilitates motor neuron excitability through mTOR signaling and thus represent an endogenous protective mechanism during early stages of the disease in mutant SOD1 mice (53). As amply discussed in a recent review (54), there is a great variability and conflict in the results obtained in the numerous studies on the role of C-boutons in neuron excitability and neuroprotection, due to several factors like differences in the parameters considered, experimental design and variances in data processing. Therefore, the role of these cholinergic boutons as a factor in the pathophysiology of ALS or as a compensatory protective mechanism, is still unclear. Conclusion Spinal motor circuits show high complexity, conformed by a wide spectrum of interneurons, non-neuronal cells and motor neurons, and the cytoarchitecture and activity of these neuronal networks are crucial for the maintenance of synaptic connections. The advances in the knowledge of the neuronal circuitry in the ventral spinal cord reviewed in this article reveal also a great complexity in the function of the different types of neurons involved in the control of motor excitability. This was perhaps to be expected when considering the numerous and distinct possible muscular movements required for walking, running and hoping, which necessitates greatly coordinated flexor-extensor and right-left alternations. The identification and role of particular neurons, through the experimental deletion of specific neuronal types and its consequences, in terms of both the electrical activity of the neurons and the motor alterations, has permitted a better understanding of these mechanisms. Since each neuronal type is characterized by an excitatory or inhibitory action on its neighbor interneurons and finally on the motor neurons that 9 ACS Paragon Plus Environment


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innervate the muscles, the identification of the neurotransmitters used by each neuronal type is very relevant to understand the excitation-inhibition balance inside the spinal cord necessary for the correct coordination of movements. The pharmacological approach in vivo, that allows the study of the motor consequences of the disruption of the excitatory and inhibitory pathways has been also very useful in this respect. This excitatory-inhibitory balance is extremely delicate and its perturbations clearly induce deleterious functional alterations and neuronal death. Motor neuron degeneration in ALS has been associated with glutamate-induced excitotoxicity, but growing evidence suggest that motor hyperexcitability and motor neuron degeneration can be also the consequence of inhibitory failure in which alterations of GABAergic, glycinergic and cholinergic neurotransmission are involved. Thereby, procedures designed to prevent inhibitory failure represent an opportunity to explore new strategies for the treatment of motor neuron degeneration in ALS. AUTHOR INFORMATION Corresponding Author *(RT). División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México, México. Phone: +52 55 56225642. E-mail: [email protected]

(UNRJ). División de Neurociencias, Instituto de Fisiología Celular and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México. E-mail: [email protected]

Author contribution The two authors jointly planned the manuscript and wrote the review. Conflict interest The authors declare that not have any conflict of interest Acknowledgments This work, and most of the work of the authors’ laboratory cited, were supported by Consejo Nacional de Ciencia y Tecnología (México) (CONACyT, project 240817) and 10 ACS Paragon Plus Environment

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Dirección General de Asuntos del Personal Académico (DGAPA, UNAM, project IN204516).

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Figure legend Figure 1. Schematic representation of the bilateral ventral intrasegmental spinal circuits (CPGs) involved in locomotor activity, showing the connections of the neuronal types that have been identified. Ipsilateral inhibition mediated by Renshaw (RC) and Ia neurons control, respectively, the recurrent and the reciprocal inhibition involved in the control of ipsilateral flexor (fMN) and extensor (eMN) motor neurons function. When V2a neurons activate V0V, these neurons excite the contralateral fMNs directly and inhibit eMN activity by exciting the Renshaw cells (RC). V0D neurons act similarly to V0V but at different pattern speed. V0C neurons inhibit ipsilateral control motor neuron excitability by means of RC and Ia activation. Although Hb9 connections are still unclear, they are important during the onset of pattern activity. See the text for details.


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1 Excitatory and inhibitory neuronal circuits in the spinal cord and their

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