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.

Synthesis, transport, and metabolism of serotonin formed from exogenously applied 5-HTP after spinal cord injury in rats.

Spinal cord transection leads to elimination of brain stem-derived monoamine fibers that normally synthesize most of the monoamines in the spinal cord, including serotonin (5-hydroxytryptamine, 5-HT) synthesized from tryptophan by enzymes tryptophan hydroxylase (TPH, synthesizing 5-hydroxytryptophan, 5-HTP) and aromatic l-amino acid decarboxylase (AADC, synthesizing 5-HT from 5-HTP). Here we examine whether spinal cord caudal to transection remains able to manufacture and metabolize 5-HT. Immunolabeling for AADC reveals that, while most AADC is confined to brain stem-derived monoamine fibers in spinal cords from normal rats, caudal to transection AADC is primarily found in blood vessel endothelial cells and pericytes as well as a novel group of neurons (NeuN positive and GFAP negative), all of which strongly upregulate AADC with injury. However, immunolabeling for 5-HT reveals that there is no detectable endogenous 5-HT synthesis in any structure in the spinal cord caudal to a chronic transection, including in AADC-containing vessels and neurons, consistent with a lack of TPH. In contrast, when we applied exogenous 5-HTP (in vitro or in vivo), AADC-containing vessels and neurons synthesized 5-HT, which contributed to increased motoneuron activity and muscle spasms (long-lasting reflexes, LLRs), by acting on 5-HT2 receptors (SB206553 sensitive) located on motoneurons (TTX resistant). Blocking monoamine oxidase (MAO) markedly increased the sensitivity of the motoneurons (LLR) to 5-HTP, more than it increased the sensitivity of motoneurons to 5-HT, suggesting that 5-HT synthesized from AADC is largely metabolized in AADC-containing neurons and vessels. In summary, after spinal cord injury AADC is upregulated in vessels, pericytes, and neurons but does not endogenously produce 5-HT, whereas when exogenous 5-HTP is provided AADC does produce functional amounts of 5-HT, some of which is able to escape metabolism by MAO, diffuse out of these AADC-containing cells, and ultimately act on 5-HT receptors on motoneurons.

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.

Motoneuron excitability and muscle spasms are regulated by 5-HT2B and 5-HT2C receptor activity.

Immediately after spinal cord injury (SCI), a devastating paralysis results from the loss of brain stem and cortical innervation of spinal neurons that control movement, including a loss of serotonergic (5-HT) innervation of motoneurons. Over time, motoneurons recover from denervation and function autonomously, exhibiting large persistent calcium currents (Ca PICs) that both help with functional recovery and contribute to uncontrolled muscle spasms. Here we systematically evaluated which 5-HT receptor subtypes influence PICs and spasms after injury. Spasms were quantified by recording the long-lasting reflexes (LLRs) on ventral roots in response to dorsal root stimulation, in the chronic spinal rat, in vitro. Ca PICs were quantified by intracellular recording in synaptically isolated motoneurons. Application of agonists selective to 5-HT(2B) and 5-HT(2C) receptors (including BW723C86) significantly increased the LLRs and associated Ca PICs, whereas application of agonists to 5-HT(1), 5-HT(2A), 5-HT(3), or 5-HT(4/5/6/7) receptors (e.g., 8-OH-DPAT) did not. The 5-HT(2) receptor agonist-induced increases in LLRs were dose dependent, with doses for 50% effects (EC(50)) highly correlated with published doses for agonist receptor binding (K(i)) at 5-HT(2B) and 5-HT(2C) receptors. Application of selective antagonists to 5-HT(2B) (e.g., RS127445) and 5-HT(2C) (SB242084) receptors inhibited the agonist-induced increase in LLR. However, antagonists that are known to specifically be neutral antagonists at 5-HT(2B/C) receptors (e.g., RS127445) had no effect when given by themselves, indicating that these receptors were not activated by residual 5-HT in the spinal cord. In contrast, inverse agonists (such as SB206553) that block constitutive activity at 5-HT(2B) or 5-HT(2C) receptors markedly reduced the LLRs, indicating the presence of constitutive activity in these receptors. 5-HT(2B) or 5-HT(2C) receptors were confirmed to be on motoneurons by immunolabeling. In summary, 5-HT(2B) and 5-HT(2C) receptors on motoneurons become constitutively active after injury and ultimately contribute to recovery of motoneuron function and emergence of spasms.

Polysynaptic excitatory postsynaptic potentials that trigger spasms after spinal cord injury in rats are inhibited by 5-HT1B and 5-HT1F receptors.

Sensory afferent transmission and associated spinal reflexes are normally inhibited by serotonin (5-HT) derived from the brain stem. Spinal cord injury (SCI) that eliminates this 5-HT innervation leads to a disinhibition of sensory transmission and a consequent emergence of unusually long polysynaptic excitatory postsynaptic potentials (EPSPs) in motoneurons. These EPSPs play a critical role in triggering long polysynaptic reflexes (LPRs) that initiate muscles spasms. In the present study we examined which 5-HT receptors modulate the EPSPs and whether these receptors adapt to a loss of 5-HT after chronic spinal transection in rats. The EPSPs and associated LPRs recorded in vitro in spinal cords from chronic spinal rats were consistently inhibited by 5-HT(1B) or 5-HT(1F) receptor agonists, including zolmitriptan (5-HT(1B/1D/1F)) and LY344864 (5-HT(1F)), with a sigmoidal dose-response relation, from which we computed the 50% inhibition (EC(50)) and potency (-log EC(50)). The potencies of 5-HT receptor agonists were highly correlated with their binding affinity to 5-HT(1B) and 5-HT(1F) receptors, and not to other 5-HT receptors. Zolmitriptan also inhibited the LPRs and general muscle spasms recorded in vivo in the awake chronic spinal rat. The 5-HT(1B) receptor antagonists SB216641 and GR127935 and the inverse agonist SB224289 reduced the inhibition of LPRs by 5-HT(1B) agonists (zolmitriptan). However, when applied alone, SB224289, SB216641, and GR127935 had no effect on the LPRs, indicating that 5-HT(1B) receptors do not adapt to chronic injury, remaining silent, without constitutive activity. The reduction in EPSPs with zolmitriptan unmasked a large glycine-mediated inhibitory postsynaptic current (IPSC) after SCI. This IPSC and associated chloride current reversed at -73 mV, slightly below the resting membrane potential. Zolmitriptan did not change motoneuron properties. Our results demonstrate that 5-HT(1B/1F) agonists, such as zolmitriptan, can restore inhibition of sensory transmission after SCI without affecting general motoneuron function and thus may serve as a novel class of antispastic drugs.

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.

The effects of rotational platform training on balance and ADLs.

Patients with vestibular dysfunction complain of postural instability and disorientation long after the central compensation is thought to be complete. Previously it has been demonstrated that patients with unilateral vestibular loss who orient more to vertical have better perceived functional status. We proposed that performing balance training with surface perturbations at velocities that target the vestibular system would lead to increased reliance on vestibular information, and therefore improve function. The purpose of this study was to determine whether patients who train using repetitive platform perturbations at these vestibular dependent velocities demonstrate improved postural stability and greater functional abilities than patients who perform traditional balance therapy. Twelve subjects with chronic vestibular and balance dysfunction (age 58 ± 15 years; 3 males, 8 females) and 4 healthy control subjects (age 62 ± 23 years; 4 females) participated. Patients were randomized into 3 groups: clinical balance training (CBT n=3) and training with ramp platform perturbations (4 deg amplitude) either at vestibular (1, 2, 4 deg/sec; VESTIB n=6) or at non-vestibular velocities (0.5, 8, 16 deg/sec; Non-VESTIB n=3). The healthy control subjects completed training at vestibular velocities. Subjects' kinematic and kinetic responses to ramp rotational platform perturbations (0.5, 1, 2, 4, 8, 16 deg/sec at 6 deg amplitude), and scores on the Activities-specific Balance Confidence Scale (ABC), Dizziness Handicap Inventory (DHI), Vestibular Activities of Daily Living Scale (VADL) and Functional Gait Assessment (FGA) were compared before and after the 2 week, 3x/week training sessions. Control subjects demonstrated minimal change in orientation to vertical during platform rotations following training. The VESTIB group demonstrated greater improvements in orientation to vertical during ramp perturbations following training than the Non-VESTIB or CBT groups. Both the CBT and VESTIB groups demonstrated improvements on a composite clinical score incorporating the ABC, DHI, VADL, and FGA following training whereas the Non-VESTIB group did not demonstrate improvement. These preliminary results indicate that training using platform rotations may be an effective intervention for improving postural control following vestibular loss. Further research is needed to explore the efficacy of incorporating rotational platform training with clinical balance training.

Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors.

Muscle paralysis after spinal cord injury is partly caused by a loss of brainstem-derived serotonin (5-HT), which normally maintains motoneuron excitability by regulating crucial persistent calcium currents. Here we examine how over time motoneurons compensate for lost 5-HT to regain excitability. We find that, months after a spinal transection in rats, changes in post-transcriptional editing of 5-HT2C receptor mRNA lead to increased expression of 5-HT2C receptor isoforms that are spontaneously active (constitutively active) without 5-HT. Such constitutive receptor activity restores large persistent calcium currents in motoneurons in the absence of 5-HT. We show that this helps motoneurons recover their ability to produce sustained muscle contractions and ultimately enables recovery of motor functions such as locomotion. However, without regulation from the brain, these sustained contractions can also cause debilitating muscle spasms. Accordingly, blocking constitutively active 5-HT2C receptors with SB206553 or cyproheptadine, in both rats and humans, largely eliminates these calcium currents and muscle spasms, providing a new rationale for antispastic drug therapy.

Learning effects of repetitive administrations of the sensory organization test in healthy young adults.

OBJECTIVES: To evaluate the learning effect of multiple administrations of the Sensory Organization Test (SOT) on performance and to begin to establish clinical meaningful change scores for the SOT. DESIGN: Descriptive case series. SETTING: University-affiliated clinic. PARTICIPANTS: Healthy young adults (6 men, 7 women; mean age, 24+/-4y). INTERVENTION: All subjects performed the standardized SOT using the SMART EquiTest 5 times over a 2-week period, and 1 month later. MAIN OUTCOME MEASURE: Composite and individual SOT test condition standardized equilibrium scores. RESULTS: Test-retest reliability (intraclass correlation coefficient model 2,3) of the composite (.67) and equilibrium score (range, .35-.79) were fair to good. Repeated-measures analysis of variance revealed a significant (P<.05) increase in the composite and equilibrium scores for conditions 4, 5, and 6 over the 5 sessions that plateaued after the third session, and were retained at 1 month. The 95% confidence interval for the composite score change from session 1 to session 4, the plateau of the learning effect, was 3.9 to 8.1. CONCLUSIONS: Although the findings of this study would indicate that multiple baseline measures are desirable for the more challenging conditions, a composite change of greater than 8 points would indicate change due to rehabilitation.