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.

Intrinsic motoneuron properties in typical human development.

Motoneuron properties and their firing patterns undergo significant changes throughout development and in response to neuromodulators such as serotonin. Here, we examined the age-related development of self-sustained firing and general excitability of tibialis anterior motoneurons in a young development (7-17 years), young adult (18-28 years) and adult (32-53 years) group, as well as in a separate group of participants taking selective serotonin reuptake inhibitors (SSRIs, aged 11-28 years). Self-sustained firing, as measured by ΔF, was larger in the young development (∼5.8 Hz, n = 20) compared to the young adult (∼4.9 Hz, n = 13) and adult (∼4.8 Hz, n = 8) groups, consistent with a developmental decrease in self-sustained firing mediated by persistent inward currents (PIC). ΔF was also larger in participants taking SSRIs (∼6.5 Hz, n = 9) compared to their age-matched controls (∼5.3 Hz, n = 26), consistent with increased levels of spinal serotonin facilitating the motoneuron PIC. Participants in the young development and SSRI groups also had higher firing rates and a steeper acceleration in initial firing rates (secondary ranges), consistent with the PIC producing a steeper acceleration in membrane depolarization at the onset of motoneuron firing. In summary, both the young development and SSRI groups exhibited increased intrinsic motoneuron excitability compared to the adults, which, in the young development group, was also associated with a larger unsteadiness in the dorsiflexion torque profiles. We propose several intrinsic and extrinsic factors that affect both motoneuron PICs and cell discharge which vary during development, with a time course similar to the changes in motoneuron firing behaviour observed in the present study. KEY POINTS: Neurons in the spinal cord that activate muscles in the limbs (motoneurons) undergo increases in excitability shortly after birth to help animals stand and walk. We examined whether the excitability of human ankle flexor motoneurons also continues to change from child to adulthood by recording the activity of the muscle fibres they innervate. Motoneurons in children and adolescents aged 7-17 years (young development group) had higher signatures of excitability that included faster firing rates and more self-sustained activity compared to adults aged ≥18 years. Participants aged 11-28 years of age taking serotonin reuptake inhibitors had the highest measures of motoneuron excitability compared to their age-matched controls. The young development group also had more unstable contractions, which might partly be related to the high excitability of the motoneurons.

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.

General access to cubanes as benzene bioisosteres.

The replacement of benzene rings with sp3-hybridized bioisosteres in drug candidates generally improves pharmacokinetic properties while retaining biological activity1-5. Rigid, strained frameworks such as bicyclo[1.1.1]pentane and cubane are particularly well suited as the ring strain imparts high bond strength and thus metabolic stability on their C-H bonds. Cubane is the ideal bioisostere as it provides the closest geometric match to benzene6,7. At present, however, all cubanes in drug design, like almost all benzene bioisosteres, act solely as substitutes for mono- or para-substituted benzene rings1-7. This is owing to the difficulty of accessing 1,3- and 1,2-disubstituted cubane precursors. The adoption of cubane in drug design has been further hindered by the poor compatibility of cross-coupling reactions with the cubane scaffold, owing to a competing metal-catalysed valence isomerization8-11. Here we report expedient routes to 1,3- and 1,2-disubstituted cubane building blocks using a convenient cyclobutadiene precursor and a photolytic C-H carboxylation reaction, respectively. Moreover, we leverage the slow oxidative addition and rapid reductive elimination of copper to develop C-N, C-C(sp3), C-C(sp2) and C-CF3 cross-coupling protocols12,13. Our research enables facile elaboration of all cubane isomers into drug candidates, thus enabling ideal bioisosteric replacement of ortho-, meta- and para-substituted benzenes.

Post-activation depression from primary afferent depolarization (PAD) produces extensor H-reflex suppression following flexor afferent conditioning.

Suppression of the extensor H-reflex by flexor afferent conditioning is thought to be produced by a long-lasting inhibition of extensor Ia afferent terminals via GABAA receptor-activated primary afferent depolarization (PAD). Given the recent finding that PAD does not produce presynaptic inhibition of Ia afferent terminals, we examined in 28 participants if H-reflex suppression is instead mediated by post-activation depression of the extensor Ia afferents triggered by PAD-evoked spikes and/or by a long-lasting inhibition of the extensor motoneurons. A brief conditioning vibration of the flexor tendon suppressed both the extensor soleus H-reflex and the tonic discharge of soleus motor units out to 150 ms following the vibration, suggesting that part of the H-reflex suppression during this period was mediated by postsynaptic inhibition of the extensor motoneurons. When activating the flexor afferents electrically to produce conditioning, the soleus H-reflex was also suppressed but only when a short-latency reflex was evoked in the soleus muscle by the conditioning input itself. In mice, a similar short-latency reflex was evoked when optogenetic or afferent activation of GABAergic (GAD2+ ) neurons produced a large enough PAD to evoke orthodromic spikes in the test Ia afferents, causing post-activation depression of subsequent monosynaptic EPSPs. The long duration of this post-activation depression and related H-reflex suppression (seconds) was similar to rate-dependent depression that is also due to post-activation depression. We conclude that extensor H-reflex inhibition by brief flexor afferent conditioning is produced by both post-activation depression of extensor Ia afferents and long-lasting inhibition of extensor motoneurons, rather than from PAD inhibiting Ia afferent terminals. KEY POINTS: Suppression of extensor H-reflexes by flexor afferent conditioning was thought to be mediated by GABAA receptor-mediated primary afferent depolarization (PAD) shunting action potentials in the Ia afferent terminal. In line with recent findings that PAD has a facilitatory role in Ia afferent conduction, we show here that when large enough, PAD can evoke orthodromic spikes that travel to the Ia afferent terminal to evoke EPSPs in the motoneuron. These PAD-evoked spikes also produce post-activation depression of Ia afferent terminals and may mediate the short- and long-lasting suppression of extensor H-reflexes in response to flexor afferent conditioning. Our findings highlight that we must re-examine how changes in the activation of GABAergic interneurons and PAD following nervous system injury or disease affects the regulation of Ia afferent transmission to spinal neurons and ultimately motor dysfunction in these disorders.

Facilitation of sensory transmission to motoneurons during cortical or sensory-evoked primary afferent depolarization (PAD) in humans.

Sensory and corticospinal tract (CST) pathways activate spinal GABAergic interneurons that have axoaxonic connections onto proprioceptive (Ia) afferents that cause long-lasting depolarizations (termed primary afferent depolarization, PAD). In rodents, sensory-evoked PAD is produced by GABAA receptors at nodes of Ranvier in Ia afferents, rather than at presynaptic terminals, and facilitates spike propagation to motoneurons by preventing branch-point failures, rather than causing presynaptic inhibition. We examined in 40 human participants whether putative activation of Ia-PAD by sensory or CST pathways can also facilitate Ia afferent activation of motoneurons via the H-reflex. H-reflexes in several leg muscles were facilitated by prior conditioning from low-threshold proprioceptive, cutaneous or CST pathways, with a similar long-lasting time course (∼200 ms) to phasic PAD measured in rodent Ia afferents. Long trains of cutaneous or proprioceptive afferent conditioning produced longer-lasting facilitation of the H-reflex for up to 2 min, consistent with tonic PAD in rodent Ia afferents mediated by nodal α5-GABAA receptors for similar stimulation trains. Facilitation of H-reflexes by this conditioning was likely not mediated by direct facilitation of the motoneurons because isolated stimulation of sensory or CST pathways did not alone facilitate the tonic firing rate of motor units. Furthermore, cutaneous conditioning increased the firing probability of single motor units (motoneurons) during the H-reflex without increasing their firing rate at this time, indicating that the underlying excitatory postsynaptic potential was more probable, but not larger. These results are consistent with sensory and CST pathways activating nodal GABAA receptors that reduce intermittent failure of action potentials propagating into Ia afferent branches. KEY POINTS: Controlled execution of posture and movement requires continually adjusted feedback from peripheral sensory pathways, especially those that carry proprioceptive information about body position, movement and effort. It was previously thought that the flow of proprioceptive feedback from Ia afferents was only reduced by GABAergic neurons in the spinal cord that sent axoaxonic projections to the terminal endings of sensory axons (termed GABAaxo neurons). Based on new findings in rodents, we provide complementary evidence in humans to suggest that sensory and corticospinal pathways known to activate GABAaxo neurons that project to dorsal parts of the Ia afferent also increase the flow of proprioceptive feedback to motoneurons in the spinal cord. These findings support a new role for spinal GABAaxo neurons in facilitating afferent feedback to the spinal cord during voluntary or reflexive movements.

GABA Increases Sensory Transmission In Monkeys.

Sensory input flow is central to voluntary movements. For almost a century, GABA was believed to modulate this flow by inhibiting sensory axons in the spinal cord to sculpt neural inputs into skilled motor output. Instead, here we show that GABA can also facilitate sensory transmission in monkeys and consequently increase spinal and cortical neural responses to sensory inputs challenging our understanding of generation and perception of movement.

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.

Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds.

Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure-activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles.

Self-directed rehabilitation training intensity thresholds for efficient recovery of skilled forelimb function in rats with cervical spinal cord injury.

Task specific rehabilitation training is commonly used to treat motor dysfunction after neurological injures such as spinal cord injury (SCI), yet the use of task specific training in preclinical animal studies of SCI is not common. This is due in part to the difficulty in training animals to perform specific motor tasks, but also due to the lack of knowledge about optimal rehabilitation training parameters to maximize recovery. The single pellet reaching, grasping and retrieval (SPRGR) task (a.k.a. single pellet reaching task or Whishaw task) is a skilled forelimb motor task used to provide rehabilitation training and test motor recovery in rodents with cervical SCI. However, the relationships between the amount, duration, intensity, and timing of training remain poorly understood. In this study, using automated robots that allow rats with cervical SCI ad libitum access to self-directed SPRGR rehabilitation training, we show clear relationships between the total amount of rehabilitation training, the intensity of training (i.e., number of attempts/h), and performance in the task. Specifically, we found that rats naturally segregate into High and Low performance groups based on training strategy and performance in the task. Analysis of the different training strategies showed that more training (i.e., increased number of attempts in the SPRGR task throughout rehabilitation training) at higher intensities (i.e., number of attempts per hour) increased performance in the task, and that improved performance in the SPRGR task was linked to differences in corticospinal tract axon collateral densities in the injured spinal cords. Importantly, however, our data also indicate that rehabilitation training becomes progressively less efficient (i.e., less recovery for each attempt) as both the amount and intensity of rehabilitation training increases. Finally, we found that Low performing animals could increase their training intensity and transition to High performing animals in chronic SCI. These results highlight the rehabilitation training strategies that are most effective to regain skilled forelimb motor function after SCI, which will facilitate pre-clinical rehabilitation studies using animal models and could be beneficial in the development of more efficient clinical rehabilitation training strategies.

Selective Synthesis of 1-Dialkylamino-2-alkylbicyclo-[1.1.1]pentanes.

Compared to the abundance of methodologies accessing 1-substituted and 1,3-disubstuted bicyclo[1.1.1] pentanes (BCPs), the synthetic accessibility of BCPs with substitutions at the methylene position has been limited. Herein, we report a selective synthesis of 1-dialkylamino-2-alkylbicyclo[1.1.1]pentanes from prefunctionalized starting materials. We also achieved further functionalizations of these 1,2-disubstituted BCPs and developed a synthesis of these compounds with minimum necessity of chromatographic purifications starting from 1,3-dichloroacetone. Finally, we also detailed the synthesis of a 1,2,3-trisubstituted BCP analogue.

Estimation of self-sustained activity produced by persistent inward currents using firing rate profiles of multiple motor units in humans.

Persistent inward calcium and sodium currents (IP) activated during motoneuron recruitment help synaptic inputs maintain self-sustained firing until derecruitment. Here, we estimate the contribution of the IP to self-sustained firing in human motoneurons of varying recruitment threshold by measuring the difference in synaptic input needed to maintain minimal firing once the IP is fully activated compared with the larger synaptic input required to initiate firing before full IP activation. Synaptic input to ≈20 dorsiflexor motoneurons simultaneously recorded during ramp contractions was estimated from firing profiles of motor units decomposed from high-density surface electromyography (EMG). To avoid errors introduced when using high-threshold units firing in their nonlinear range, we developed methods where the lowest threshold units firing linearly with force were used to construct a composite (control) unit firing rate profile to estimate synaptic input to higher threshold (test) units. The difference in the composite firing rate (synaptic input) at the time of test unit recruitment and derecruitment (ΔF = Frecruit - Fderecruit) was used to measure IP amplitude that sustained firing. Test units with recruitment thresholds 1-30% of maximum had similar ΔF values, which likely included both slow and fast motor units activated by small and large motoneurons, respectively. This suggests that the portion of the IP that sustains firing is similar across a wide range of motoneuron sizes.NEW & NOTEWORTHY A new method of estimating synaptic drive to multiple, simultaneously recorded motor units provides evidence that the portion of the depolarizing drive from persistent inward currents that contributes to self-sustained firing is similar across motoneurons of different sizes.

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.

Bursting interneurons in the deep dorsal horn develop increased excitability and sensitivity to serotonin after chronic spinal injury.

The loss of descending serotonin (5-HT) to the spinal cord contributes to muscle spasms in chronic spinal cord injury (SCI). Hyperexcitable motoneurons receive long-lasting excitatory postsynaptic potentials (EPSPs), which activate their persistent inward currents to drive muscle spasms. Deep dorsal horn (DDH) neurons with bursting behavior could be involved in triggering the EPSPs due to loss of inhibition in the chronically 5-HT-deprived spinal cord. Previously, in an acutely transected preparation, we found that bursting DDH neurons were affected by administration of the 5-HT1B/1D receptor agonist zolmitriptan, which suppressed their bursts, and by N-methyl-d-aspartate (NMDA), which enhanced their bursting behavior. Nonbursting DDH neurons were not influenced by these agents. In the present study, we investigate the firing characteristics of bursting DDH neurons following chronic spinal transection at T10 level in adult mice and examine the effects of replacing lost endogenous 5-HT with zolmitriptan. Terminal experiments using our in vitro preparation of the sacral cord were carried out ~10 wk postransection. Compared with the acute spinal stage of our previous study, DDH neurons in the chronic stage became more responsive to dorsal root stimulation, with burst duration doubling with chronic injury. The suppressive effects of zolmitriptan were stronger overall, but the facilitative effects of NMDA were weaker. In addition, the onset of DDH neuron activity preceded ventral root output and the firing rates of DDH interneurons correlated with the integrated long-lasting ventral root output. These results support a contribution of the bursting DDH neurons to muscle spasms following SCI and inhibition by 5-HT.NEW & NOTEWORTHY We investigate the firing characteristics of bursting deep dorsal horn (DDH) neurons following chronic spinal transection. DDH neurons in the chronic stage are different from those in the acute stage as noted by their increase in excitability overall and their differing responses serotonin (5-HT) and N-methyl-d-aspartate (NMDA) receptor agonists. Also, there is a strong relationship between DDH neuron activity and ventral root output. These results support a contribution of the bursting DDH neurons to muscle spasms following chronic spinal cord injury (SCI).

A Selective Synthesis of 2,2-Difluorobicyclo[1.1.1]pentane Analogues: "BCP-F2".

The bicyclo[1.1.1]pentane (BCP) motif has been utilized as bioisosteres in drug candidates to replace phenyl, tert-butyl, and alkynyl fragments in order to improve physicochemical properties. However, bceause of the difficulty of synthesis, most BCP analogues prepared only bear 1,3-"para"-substituents. We report the first selective synthesis of 2,2-difluorobicyclo[1.1.1]pentanes via difluorocarbene insertion into bicyclo[1.1.0]butanes. Moreover, this methodology should inspire future studies on synthesis of other "ortho/meta-substituted" BCPs via similar mechanisms.

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.