Widespread innervation of motoneurons by spinal V3 neurons globally amplifies locomotor output in mice.

While considerable progress has been made in understanding the neuronal circuits that underlie the patterning of locomotor behaviours such as walking, less is known about the circuits that amplify motoneuron output to enable adaptable increases in muscle force across different locomotor intensities. Here, we demonstrate that an excitatory propriospinal neuron population (V3 neurons, Sim1 + ) forms a large part of the total excitatory interneuron input to motoneurons (∼20%) across all hindlimb muscles. Additionally, V3 neurons make extensive connections among themselves and with other excitatory premotor neurons (such as V2a neurons). These circuits allow local activation of V3 neurons at just one segment (via optogenetics) to rapidly depolarize and amplify locomotor-related motoneuron output at all lumbar segments in both the in vitro spinal cord and the awake adult mouse. Interestingly, despite similar innervation from V3 neurons to flexor and extensor motoneuron pools, functionally, V3 neurons exhibit a pronounced bias towards activating extensor muscles. Furthermore, the V3 neurons appear essential to extensor activity during locomotion because genetically silencing them leads to slower and weaker mice with a poor ability to increase force with locomotor intensity, without much change in the timing of locomotion. Overall, V3 neurons increase the excitability of motoneurons and premotor neurons, thereby serving as global command neurons that amplify the locomotion intensity.

Locomotor-related propriospinal V3 neurons produce primary afferent depolarization and modulate sensory transmission to motoneurons.

When a muscle is stretched, sensory feedback not only causes reflexes but also leads to a depolarization of sensory afferents throughout the spinal cord (primary afferent depolarization, PAD), readying the whole limb for further disturbances. This sensory-evoked PAD is thought to be mediated by a trisynaptic circuit, where sensory input activates first-order excitatory neurons that activate GABAergic neurons that in turn activate GABAA receptors on afferents to cause PAD, though the identity of these first-order neurons is unclear. Here, we show that these first-order neurons include propriospinal V3 neurons, as they receive extensive sensory input and in turn innervate GABAergic neurons that cause PAD, because optogenetic activation or inhibition of V3 neurons in mice mimics or inhibits sensory-evoked PAD, respectively. Furthermore, persistent inward sodium currents intrinsic to V3 neurons prolong their activity, explaining the prolonged duration of PAD. Also, local optogenetic activation of V3 neurons at one segment causes PAD in other segments, due to the long propriospinal tracts of these neurons, helping to explain the radiating nature of PAD. This in turn facilitates monosynaptic reflex transmission to motoneurons across the spinal cord. In addition, V3 neurons directly innervate proprioceptive afferents (including Ia), causing a glutamate receptor-mediated PAD (glutamate PAD). Finally, increasing the spinal cord excitability with either GABAA receptor blockers or chronic spinal cord injury causes an increase in the glutamate PAD. Overall, we show the V3 neuron has a prominent role in modulating sensory transmission, in addition to its previously described role in locomotion.NEW & NOTEWORTHY Locomotor-related propriospinal neurons depolarize sensory axons throughout the spinal cord by either direct glutamatergic axoaxonic contacts or indirect innervation of GABAergic neurons that themselves form axoaxonic contacts on sensory axons. This depolarization (PAD) increases sensory transmission to motoneurons throughout the spinal cord, readying the sensorimotor system for external disturbances. The glutamate-mediated PAD is particularly adaptable, increasing with either an acute block of GABA receptors or chronic spinal cord injury, suggesting a role in motor recovery.

GABA facilitates spike propagation through branch points of sensory axons in the spinal cord.

Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons directly with optogenetics or indirectly by cutaneous stimulation actually facilitates sensory feedback to motor neurons in rodents and humans. GABAA receptors located at or near nodes of Ranvier of sensory axons cause this facilitation by preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals and so cannot inhibit transmitter release onto motor neurons, unlike GABAB receptors that cause presynaptic inhibition. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other central nervous system axons.

Rehabilitative training improves skilled forelimb motor function after cervical unilateral contusion spinal cord injury in rats.

Animal models of cervical spinal cord injury (SCI) have frequently utilized partial transection injuries to evaluate plasticity promoting treatments such as rehabilitation training of skilled reaching and grasping tasks. Though highly useful for studying the effects of cutting specific spinal tracts that are important for skilled forelimb motor function, cervical partial-transection SCI-models underappreciate the extensive spread of most human SCIs, thus offering poor predictability for the clinical setting. Conversely, moderate cervical contusion SCI models targeting the spinal tracts important for skilled reaching and grasping can better replicate the increased size of most human SCIs and are often considered more clinically relevant. However, it is unknown whether animals with moderate cervical contusion SCIs that damage key spinal motor tracts can train in skilled reaching and grasping tasks. In this study, we quantify the impact of injury size and distribution on recovery in a skilled motor task called the single pellet reaching, grasping and retrieval (SPRGR) task in rats with cervical unilateral contusion injuries (UCs), and compare to rats with a partial transection SCIs (i.e., dorsolateral quadrant transection; DLQ). We found that UCs damage key tracts important for performing skilled motor tasks, similar to DLQs, but UCs also produce more extensive grey matter damage and more ventral white matter damage than DLQs. We also compared forelimb functionality at 1, 3, and 5 weeks of rehabilitative motor training between trained and untrained rats and found a more severe drop in SPRGR performance than in DLQ SCIs. Nevertheless, despite more severe injuries and initially low SPRGR performance, rehabilitative training for contusion animals resulted in significant improvements in SPRGR performance and proportionally more recovery than DLQ rats. Our findings show that rehabilitative motor training can facilitate considerable amounts of motor recovery despite extensive spinal cord damage, especially grey matter damage, thus supporting the use of contusion or compression SCI models and showing that ventral grey and white matter damage are not necessarily detrimental to recovery after training.

Branching points of primary afferent fibers are vital for the modulation of fiber excitability by epidural DC polarization and by GABA in the rat spinal cord.

The aim of the study was to examine whether the sustained increases in the excitability of afferent fibers traversing the dorsal columns evoked by their polarization depend on the branching points of these fibers. To this end, the effects of epidural polarization were compared in four spinal regions in deeply anesthetized rats; two with the densest collateralization of muscle afferent fibers (above motor nuclei and Clarke's column) and two where the collateralization is more sparse (rostral and caudal to motor nuclei, respectively. The degree of collateralization in different segments was reconstructed in retrogradely labeled afferent fibers in the rat. Nerve volleys evoked in peripheral nerves by electrical stimulation of the dorsal columns within these regions were used as a measure of the excitability of the stimulated fibers. Potent increases in the excitability were evoked by polarization above motor nuclei and Clarke's column, both during constant direct current (DC) polarization (1 µA for 1 min) and for at least 30 min following DC polarization. Smaller excitability increases occurred during the polarization within other regions and were thereafter either absent or rapidly declined after its termination. The postpolarization increases in excitability were counteracted by the GABAA receptor antagonist bicuculline and the α5GABAA extrasynaptic receptor antagonist L655708 and enhanced by the GABAA receptor agonist muscimol and by ionophoretically applied GABA. As extrasynaptic α5GABAA receptors have been found close to Na channels within branching points, these results are consistent with the involvement of branching points in the induction of the sustained postpolarization increases in fiber excitability.NEW & NOTEWORTHY Polarization of sensory fibers traversing dorsal columns of the spinal cord may considerably increase the excitability of these fibers. We show that this involves the effects of current at branching points of afferent fibers and depends on extrasynaptic effects of GABA. These results contribute to our understanding of the mechanism underlying plasticity of activation of nerve fibers and may be used to increase the effectiveness of epidural stimulation in humans and recovery of spinal functions.

5-HT1D receptors inhibit the monosynaptic stretch reflex by modulating C-fiber activity.

The monosynaptic stretch reflex (MSR) plays an important role in feedback control of movement and posture but can also lead to unstable oscillations associated with tremor and clonus, especially when increased with spinal cord injury (SCI). To control the MSR and clonus after SCI, we examined how serotonin regulates the MSR in the sacrocaudal spinal cord of rats with and without a chronic spinal transection. In chronic spinal rats, numerous 5-HT receptor agonists, including zolmitriptan, methylergonovine, and 5-HT, inhibited the MSR with a potency highly correlated to their binding affinity to 5-HT1D receptors and not other 5-HT receptors. Selective 5-HT1D receptor antagonists blocked this agonist-induced inhibition, although antagonists alone had no action, indicating a lack of endogenous or constitutive receptor activity. In normal uninjured rats, the MSR was likewise inhibited by 5-HT, but at much higher doses, indicating a supersensitivity after SCI. This supersensitivity resulted from the loss of the serotonin transporter SERT with spinal transection, because normal and injured rats were equally sensitive to 5-HT after SERT was blocked or to agonists not transported by SERT (zolmitriptan). Immunolabeling revealed that the 5-HT1D receptor was confined to superficial lamina of the dorsal horn, colocalized with CGRP-positive C-fibers, and eliminated by dorsal rhizotomy. 5-HT1D receptor labeling was not found on large proprioceptive afferents or α-motoneurons of the MSR. Thus serotonergic inhibition of the MSR acts indirectly by modulating C-fiber activity, opening up new possibilities for modulating reflex function and clonus via pain-related pathways. NEW & NOTEWORTHY Brain stem-derived serotonin potently inhibits afferent transmission in the monosynaptic stretch reflex. We show that serotonin produces this inhibition exclusively via 5-HT1D receptors, and yet these receptors are paradoxically mostly confined to C-fibers. This suggests that serotonin acts by gating of C-fiber activity, which in turn modulates afferent transmission to motoneurons. We also show that the classic supersensitivity to 5-HT after spinal cord injury results from a loss of SERT, and not 5-HT1D receptor plasticity.

Locomotor-related V3 interneurons initiate and coordinate muscles spasms after spinal cord injury.

Spinal cord injury leads to a devastating loss of motor function and yet is accompanied by a paradoxical emergence of muscle spasms, which often involve complex muscle activation patterns across multiple joints, reciprocal muscle timing, and rhythmic clonus. We investigated the hypothesis that spasms are a manifestation of partially recovered function in spinal central pattern-generating (CPG) circuits that normally coordinate complex postural and locomotor functions. We focused on the commissural propriospinal V3 neurons that coordinate interlimb movements during locomotion and examined mice with a chronic spinal transection. When the V3 neurons were optogenetically activated with a light pulse, a complex coordinated pattern of motoneuron activity was evoked with reciprocal, crossed, and intersegmental activity. In these same mice, brief sensory stimulation evoked spasms with a complex pattern of activity very similar to that evoked by light, and the timing of these spasms was readily reset by activation of V3 neurons. Given that V3 neurons receive abundant sensory input, these results suggest that sensory activation of V3 neurons is alone sufficient to generate spasms. Indeed, when we silenced V3 neurons optogenetically, sensory evoked spasms were inhibited. Also, inhibiting general CPG activity by blocking N-methyl-d-aspartate (NMDA) receptors inhibited V3 evoked activity and associated spasms, whereas NMDA application did the opposite. Furthermore, overwhelming the V3 neurons with repeated optogenetic stimulation inhibited subsequent sensory evoked spasms, both in vivo and in vitro. Taken together, these results demonstrate that spasms are generated in part by sensory activation of V3 neurons and associated CPG circuits. NEW & NOTEWORTHY We investigated whether locomotor-related excitatory interneurons (V3) play a role in coordinating muscle spasm activity after spinal cord injury (SCI). Unexpectedly, we found that these neurons not only coordinate reciprocal motor activity but are critical for initiating spasms, as well. More generally, these results suggest that V3 neurons are important in initiating and coordinating motor output after SCI and thus provide a promising target for restoring residual motor function.

Extrasynaptic α5GABAA receptors on proprioceptive afferents produce a tonic depolarization that modulates sodium channel function in the rat spinal cord.

Activation of GABAA receptors on sensory axons produces a primary afferent depolarization (PAD) that modulates sensory transmission in the spinal cord. While axoaxonic synaptic contacts of GABAergic interneurons onto afferent terminals have been extensively studied, less is known about the function of extrasynaptic GABA receptors on afferents. Thus, we examined extrasynaptic α5GABAA receptors on low-threshold proprioceptive (group Ia) and cutaneous afferents. Afferents were impaled with intracellular electrodes and filled with neurobiotin in the sacrocaudal spinal cord of rats. Confocal microscopy was used to reconstruct the afferents and locate immunolabelled α5GABAA receptors. In all afferents α5GABAA receptors were found throughout the extensive central axon arbors. They were most densely located at branch points near sodium channel nodes, including in the dorsal horn. Unexpectedly, proprioceptive afferent terminals on motoneurons had a relative lack of α5GABAA receptors. When recording intracellularly from these afferents, blocking α5GABAA receptors (with L655708, gabazine, or bicuculline) hyperpolarized the afferents, as did blocking neuronal activity with tetrodotoxin, indicating a tonic GABA tone and tonic PAD. This tonic PAD was increased by repeatedly stimulating the dorsal root at low rates and remained elevated for many seconds after the stimulation. It is puzzling that tonic PAD arises from α5GABAA receptors located far from the afferent terminal where they can have relatively little effect on terminal presynaptic inhibition. However, consistent with the nodal location of α5GABAA receptors, we find tonic PAD helps produce sodium spikes that propagate antidromically out the dorsal roots, and we suggest that it may well be involved in assisting spike transmission in general. NEW & NOTEWORTHY GABAergic neurons are well known to form synaptic contacts on proprioceptive afferent terminals innervating motoneurons and to cause presynaptic inhibition. However, the particular GABA receptors involved are unknown. Here, we examined the distribution of extrasynaptic α5GABAA receptors on proprioceptive Ia afferents. Unexpectedly, these receptors were found preferentially near nodal sodium channels throughout the afferent and were largely absent from afferent terminals. These receptors produced a tonic afferent depolarization that modulated sodium spikes, consistent with their location.

Eliciting inflammation enables successful rehabilitative training in chronic spinal cord injury.

Rehabilitative training is one of the most successful therapies to promote motor recovery after spinal cord injury, especially when applied early after injury. Polytrauma and management of other medical complications in the acute post-injury setting often preclude or complicate early rehabilitation. Therefore, interventions that reopen a window of opportunity for effective motor training after chronic injury would have significant therapeutic value. Here, we tested whether this could be achieved in rats with chronic (8 weeks) dorsolateral quadrant sections of the cervical spinal cord (C4) by inducing mild neuroinflammation. We found that systemic injection of a low dose of lipopolysaccharide improved the efficacy of rehabilitative training on forelimb function, as assessed using a single pellet reaching and grasping task. This enhanced recovery was found to be dependent on the training intensity, where a high-intensity paradigm induced the biggest improvements. Importantly, in contrast to training alone, the combination of systemic lipopolysaccharide and high-intensity training restored original function (reparative plasticity) rather than enhancing new motor strategies (compensatory plasticity). Accordingly, electrophysiological and tract-tracing studies demonstrated a recovery in the cortical drive to the affected forelimb muscles and a restructuration of the corticospinal innervation of the cervical spinal cord. Thus, we propose that techniques that can elicit mild neuroinflammation may be used to enhance the efficacy of rehabilitative training after chronic spinal cord injury.

Cyclosporine-immunosuppression does not affect survival of transplanted skin-derived precursor Schwann cells in the injured rat spinal cord.

A major goal of Schwann cell (SC) transplantation for spinal cord injury (SCI) is to fill the injury site to create a bridge for regenerating axons. However, transplantation of peripheral nerve SCs requires an invasive biopsy, which may result in nerve damage and donor site morbidity. SCs derived from multipotent stem cells found in skin dermis (SKP-SCs) are a promising alternative. Regardless of source, loss of grafted SCs post-grafting is an issue in studies of regeneration, with survival rates ranging from ∼1 to 20% after ≥6 weeks in rodent models of SCI. Immune rejection has been implicated in these low survival rates. Therefore, our aim was to explore the role of the immune response on grafted SKP-SC survival in Fischer rats with a spinal hemisection injury. We compared SKP-SC survival 6 weeks post-transplantation in: (I) cyclosporine-immunosuppressed rats (n=8), (II) immunocompetent rats (n=9), and (III) rats of a different sub-strain than the SKP-SC donor rats (n=7). SKP-SC survival was similar in all groups, suggesting immune rejection was not a main factor in SKP-SC loss observed in this study. SKP-SCs were consistently found on laminin expressed at the injury site, indicating detachment-mediated apoptosis (i.e., anoikis) might play a major role in grafted cell loss.

Pericytes impair capillary blood flow and motor function after chronic spinal cord injury.

Blood vessels in the central nervous system (CNS) are controlled by neuronal activity. For example, widespread vessel constriction (vessel tone) is induced by brainstem neurons that release the monoamines serotonin and noradrenaline, and local vessel dilation is induced by glutamatergic neuron activity. Here we examined how vessel tone adapts to the loss of neuron-derived monoamines after spinal cord injury (SCI) in rats. We find that, months after the imposition of SCI, the spinal cord below the site of injury is in a chronic state of hypoxia owing to paradoxical excess activity of monoamine receptors (5-HT1) on pericytes, despite the absence of monoamines. This monoamine-receptor activity causes pericytes to locally constrict capillaries, which reduces blood flow to ischemic levels. Receptor activation in the absence of monoamines results from the production of trace amines (such as tryptamine) by pericytes that ectopically express the enzyme aromatic L-amino acid decarboxylase (AADC), which synthesizes trace amines directly from dietary amino acids (such as tryptophan). Inhibition of monoamine receptors or of AADC, or even an increase in inhaled oxygen, produces substantial relief from hypoxia and improves motoneuron and locomotor function after SCI.

Dynamic motor compensations with permanent, focal loss of forelimb force after cervical spinal cord injury.

Incomplete cervical lesion is the most common type of human spinal cord injury (SCI) and causes permanent paresis of arm muscles, a phenomenon still incompletely understood in physiopathological and neuroanatomical terms. We performed spinal cord hemisection in adult rats at the caudal part of the segment C6, just rostral to the bulk of triceps brachii motoneurons, and analyzed the forces and kinematics of locomotion up to 4 months postlesion to determine the nature of motor function loss and recovery. A dramatic (50%), immediate and permanent loss of extensor force occurred in the forelimb but not in the hind limb of the injured side, accompanied by elbow and wrist kinematic impairments and early adaptations of whole-body movements that initially compensated the balance but changed continuously over the follow-up period to allow effective locomotion. Overuse of both contralateral legs and ipsilateral hind leg was evidenced since 5 days postlesion. Ipsilateral foreleg deficits resulted mainly from interruption of axons that innervate the spinal cord segments caudal to the lesion, because chronic loss (about 35%) of synapses was detected at C7 while only 14% of triceps braquii motoneurons died, as assessed by synaptophysin immunohistochemistry and retrograde neural tracing, respectively. We also found a large pool of propriospinal neurons projecting from C2-C5 to C7 in normal rats, with topographical features similar to the propriospinal premotoneuronal system of cats and primates. Thus, concurrent axotomy at C6 of brain descending axons and cervical propriospinal axons likely hampered spontaneous recovery of the focal neurological impairments.

Compartmentalization in the triceps brachii motoneuron nucleus and its relation to muscle architecture.

Each fascicle of the triceps brachii (TB) can be independently recruited during movement execution. To investigate the anatomical basis of this selective control and to gain insight into its functional role in adult rats, we carried out a triple retrograde tracing of the motoneurons (MNs) innervating each TB head, and performed muscle ATPase histochemistry and histology to correlate the size of the MN pools with the number and type of muscle fibers innervated. No double-labeled MNs were found, demonstrating that each TB head is innervated by a completely independent MN subnucleus. Absolute cell counts determined that the long fascicle had the largest MN subnucleus, followed by the medial and the lateral fascicles. MNs of the three fascicles intermingled extensively in the rostral part of the spinal motor column, while the caudal part of the column comprised mostly MNs innervating the long fascicle. Muscle histology and average innervation ratios estimated from absolute MN counts showed that the medial head was predominantly formed by small type I fibers and motor units (69 fibers/MN). In contrast, the lateral fascicle comprised a great quantity of large type IIb fibers and motor units (179 fibers/MN), whereas the long head consisted of a more balanced mixture of fiber types and motor units (99 fibers/MN). Taking into account the mechanical and physiological heterogeneity of the TB, our findings suggest that each fascicle may be considered an independent muscle with specific functional roles.