Forced Remyelination Promotes Axon Regeneration in a Rat Model of Spinal Cord Injury.

Spinal cord injuries result in the loss of motor and sensory functions controlled by neurons located at the site of the lesion and below. We hypothesized that experimentally enhanced remyelination supports axon preservation and/or growth in the total spinal cord transection in rats. Multifocal demyelination was induced by injection of ethidium bromide (EB), either at the time of transection or twice during transection and at 5 days post-injury. We demonstrated that the number of oligodendrocyte progenitor cells (OPCs) significantly increased 14 days after demyelination. Most OPCs differentiated into mature oligodendrocytes by 60-90 dpi in double-EB-injected rats; however, most axons were remyelinated by Schwann cells. A significant number of axons passed the injury epicenter and entered the distant segments of the spinal cord in the double-EB-injected rats. Moreover, some serotoninergic fibers, not detected in control animals, grew caudally through the injury site. Behavioral tests performed at 60-90 dpi revealed significant improvement in locomotor function recovery in double-EB-injected rats, which was impaired by the blockade of serotonin receptors, confirming the important role of restored serotonergic fibers in functional recovery. Our findings indicate that enhanced remyelination per se, without substantial inhibition of glial scar formation, is an important component of spinal cord injury regeneration.

Treadmill training of rats after sciatic nerve graft does not alter accuracy of muscle reinnervation.

Background and purpose: After peripheral nerve lesions, surgical reconstruction facilitates axonal regeneration and motor reinnervation. However, functional recovery is impaired by aberrant reinnervation. Materials and methods: We tested whether training therapy by treadmill exercise (9 × 250 m/week) before (run-idle), after (idle-run), or both before and after (run-run) sciatic nerve graft improves the accuracy of reinnervation in rats. Female Lewis rats (LEW/SsNHsd) were either trained for 12 weeks (run) or not trained (kept under control conditions, idle). The right sciatic nerves were then excised and reconstructed with 5 mm of a congenic allograft. One week later, training started in the run-run and idle-run groups for another 12 weeks. No further training was conducted in the run-idle and idle-idle groups. Reinnervation was measured using the following parameters: counting of retrogradely labeled motoneurons, walking track analysis, and compound muscle action potential (CMAP) recordings. Results: In intact rats, the common fibular (peroneal) and the soleus nerve received axons from 549 ± 83 motoneurons. In the run-idle group, 94% of these motoneurons had regenerated 13 weeks after the nerve graft. In the idle-run group, 81% of the normal number of motoneurons had regenerated into the denervated musculature and 87% in both run-run and idle-idle groups. Despite reinnervation, functional outcome was poor: walking tracks indicated no functional improvement of motion in any group. However, in the operated hindlimb of run-idle rats, the CMAP of the soleus muscle reached 11.9 mV (normal 16.3 mV), yet only 6.3-8.1 mV in the other groups. Conclusion: Treadmill training neither altered the accuracy of reinnervation nor the functional recovery, and pre-operative training (run-idle) led to a higher motor unit activation after regeneration.

Perspectives in the Cell-Based Therapies of Various Aspects of the Spinal Cord Injury-Associated Pathologies: Lessons from the Animal Models.

Traumatic injury of the spinal cord (SCI) is a devastating neurological condition often leading to severe dysfunctions, therefore an improvement in clinical treatment for SCI patients is urgently needed. The potential benefits of transplantation of various cell types into the injured spinal cord have been intensively investigated in preclinical SCI models and clinical trials. Despite the many challenges that are still ahead, cell transplantation alone or in combination with other factors, such as artificial matrices, seems to be the most promising perspective. Here, we reviewed recent advances in cell-based experimental strategies supporting or restoring the function of the injured spinal cord with a particular focus on the regenerative mechanisms that could define their clinical translation.

Unusual Quadrupedal Locomotion in Rat during Recovery from Lumbar Spinal Blockade of 5-HT7 Receptors.

Coordination of four-limb movements during quadrupedal locomotion is controlled by supraspinal monoaminergic descending pathways, among which serotoninergic ones play a crucial role. Here we investigated the locomotor pattern during recovery from blockade of 5-HT7 or 5-HT2A receptors after intrathecal application of SB269970 or cyproheptadine in adult rats with chronic intrathecal cannula implanted in the lumbar spinal cord. The interlimb coordination was investigated based on electromyographic activity recorded from selected fore- and hindlimb muscles during rat locomotion on a treadmill. In the time of recovery after hindlimb transient paralysis, we noticed a presence of an unusual pattern of quadrupedal locomotion characterized by a doubling of forelimb stepping in relation to unaffected hindlimb stepping (2FL-1HL) after blockade of 5-HT7 receptors but not after blockade of 5-HT2A receptors. The 2FL-1HL pattern, although transient, was observed as a stable form of fore-hindlimb coupling during quadrupedal locomotion. We suggest that modulation of the 5-HT7 receptors on interneurons located in lamina VII with ascending projections to the forelimb spinal network can be responsible for the 2FL-1HL locomotor pattern. In support, our immunohistochemical analysis of the lumbar spinal cord demonstrated the presence of the 5-HT7 immunoreactive cells in the lamina VII, which were rarely 5-HT2A immunoreactive.

Noradrenergic Components of Locomotor Recovery Induced by Intraspinal Grafting of the Embryonic Brainstem in Adult Paraplegic Rats.

Intraspinal grafting of serotonergic (5-HT) neurons was shown to restore plantar stepping in paraplegic rats. Here we asked whether neurons of other phenotypes contribute to the recovery. The experiments were performed on adult rats after spinal cord total transection. Grafts were injected into the sub-lesional spinal cord. Two months later, locomotor performance was tested with electromyographic recordings from hindlimb muscles. The role of noradrenergic (NA) innervation was investigated during locomotor performance of spinal grafted and non-grafted rats using intraperitoneal application of α2 adrenergic receptor agonist (clonidine) or antagonist (yohimbine). Morphological analysis of the host spinal cords demonstrated the presence of tyrosine hydroxylase positive (NA) neurons in addition to 5-HT neurons. 5-HT fibers innervated caudal spinal cord areas in the dorsal and ventral horns, central canal, and intermediolateral zone, while the NA fiber distribution was limited to the central canal and intermediolateral zone. 5-HT and NA neurons were surrounded by each other's axons. Locomotor abilities of the spinal grafted rats, but not in control spinal rats, were facilitated by yohimbine and suppressed by clonidine. Thus, noradrenergic innervation, in addition to 5-HT innervation, plays a potent role in hindlimb movement enhanced by intraspinal grafting of brainstem embryonic tissue in paraplegic rats.

CacyBP/SIP in the rat spinal cord in norm and after transection - Influence on the phosphorylation state of ERK1/2 and p38 kinases.

INTRODUCTION: CacyBP/SIP is a multifunctional protein present in various mammalian tissues, among them in brain. Recently, it has been shown that CacyBP/SIP exhibits phosphatase activity towards ERK1/2 and p38 kinases. OBJECTIVES: The aim of our study was to analyze the localization and level of CacyBP/SIP and its substrates, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated p38 (p-p38) kinases, in an intact and transected rat spinal cord. METHODS: To achieve our goals we have performed Western blot/densitometric analysis and double immunofluorescence staining using rat spinal cord tissue, intact and after total transection at different time points. RESULTS: We have observed a decrease in the level of CacyBP/SIP and an increase in the level of p-ERK1/2 and of p-p38 in fragments of the spinal cord excised 1 and 3 months after transection. Moreover, immunofluorescence staining has shown that CacyBP/SIP, p-ERK1/2 or p-p38 co-localized with a neuronal marker, NeuN, and with an oligodendrocyte marker, Olig2. CONCLUSION: The inverse correlation between CacyBP/SIP and p-ERK1/2 or p-p38 levels suggests that CacyBP/SIP may dephosphorylate p-ERK1/2 and p-p38 kinases and be involved in neural plasticity following spinal cord injury.

Contribution of 5-HT2 Receptors to the Control of the Spinal Locomotor System in Intact Rats.

Applying serotonergic (5-HT) agonists or grafting of fetal serotonergic cells into the spinal cord improves locomotion after spinal cord injury. Little is known about the role of 5-HT receptors in the control of voluntary locomotion, so we administered inverse agonists of 5-HT2 (Cyproheptadine; Cypr), 5-HT2A neutral antagonist (Volinanserin; Volin), 5-HT2C neutral antagonist (SB 242084), and 5-HT2B/2C inverse agonist (SB 206553) receptors intrathecally in intact rats and monitored their effects on unrestrained locomotion. An intrathecal cannula was introduced at the low thoracic level and pushed caudally until the tip reached the L2/L3 or L5/L6 spinal segments. Locomotor performance was evaluated using EMG activity of hindlimb muscles during locomotion on a 2 m long runway. Motoneuron excitability was estimated using EMG recordings during dorsi- and plantar flexion at the ankle. Locomotion was dramatically impaired after the blockage of 5-HT2A receptors. The effect of Cypr was more pronounced than that of Volin since in the L5/L6 rats Cypr (but not Volin) induced significant alteration of the strength of interlimb coordination followed by total paralysis. These agents significantly decreased locomotor EMG amplitude and abolished or substantially decreased stretch reflexes. Blocking 5-HT2B/2C receptors had no effect either on locomotion or reflexes. We suggest that in intact rats serotonin controls timing and amplitude of muscle activity by acting on 5-HT2A receptors on both CPG interneurons and motoneurons, while 5-HT2B/2C receptors are not involved in control of the locomotor pattern in lumbar spinal cord.

Intraspinal Grafting of Serotonergic Neurons Modifies Expression of Genes Important for Functional Recovery in Paraplegic Rats.

Serotonin (5-hydroxytryptamine; 5-HT) plays an important role in control of locomotion, partly through direct effects on motoneurons. Spinal cord complete transection (SCI) results in changes in 5-HT receptors on motoneurons that influence functional recovery. Activation of 5-HT2A and 5-HT7 receptors improves locomotor hindlimb movements in paraplegic rats. Here, we analyzed the mRNA of 5-HT2A and 5-HT7 receptors (encoded by Htr2a and Htr7 genes, resp.) in motoneurons innervating tibialis anterior (TA) and gastrocnemius lateralis (GM) hindlimb muscles and the tail extensor caudae medialis (ECM) muscle in intact as well as spinal rats. Moreover, the effect of intraspinal grafting of serotonergic neurons on Htr2a and Htr7 gene expression was examined to test the possibility that the graft origin 5-HT innervation in the spinal cord of paraplegic rats could reverse changes in gene expression induced by SCI. Our results indicate that SCI at the thoracic level leads to changes in Htr2a and Htr7 gene expression, whereas transplantation of embryonic serotonergic neurons modifies these changes in motoneurons innervating hindlimb muscles but not those innervating tail muscles. This suggests that the upregulation of genes critical for locomotor recovery, resulting in limb motoneuron plasticity, might account for the improved locomotion in grafted animals.

Critical period of neuromuscular development: Importance for a new treatment of SMA.

Findings from mice that had their Smn gene deleted and some copies of the human SMN2 gene introduced to produce SMN protein are summarized. Symptoms due to this manipulation can be corrected only by restoring the SMN protein expression in neurones and not in muscle. The changes in muscle and neuromuscular junction (NMJ) in these mutant mice are probably due to the malfunction of the neuronal component of the NMJ i.e. the nerve terminal. The reduction of transmitter release by nerve terminals in animals with reduced SMN protein supports this notion. There is a critical period during which the presence of the SMN protein is mandatory for the survival of the motor unit and the individual. This period coincides with the most important events involved in the development of the motor unit. Results from normal genetically unaffected rats and mice show that during a critical period of development the function of the nerve terminal and the release of transmitter play a crucial role in the development of the motor neurone and muscle. The possibility that targeting the function of the nerve terminal to overcome its inability to release transmitter could benefit patients with the deletion of the SMN gene.

LFP Oscillations in the Mesencephalic Locomotor Region during Voluntary Locomotion.

Oscillatory rhythms in local field potentials (LFPs) are thought to coherently bind cooperating neuronal ensembles to produce behaviors, including locomotion. LFPs recorded from sites that trigger locomotion have been used as a basis for identification of appropriate targets for deep brain stimulation (DBS) to enhance locomotor recovery in patients with gait disorders. Theta band activity (6-12 Hz) is associated with locomotor activity in locomotion-inducing sites in the hypothalamus and in the hippocampus, but the LFPs that occur in the functionally defined mesencephalic locomotor region (MLR) during locomotion have not been determined. Here we record the oscillatory activity during treadmill locomotion in MLR sites effective for inducing locomotion with electrical stimulation in rats. The results show the presence of oscillatory theta rhythms in the LFPs recorded from the most effective MLR stimulus sites (at threshold ≤60 µA). Theta activity increased at the onset of locomotion, and its power was correlated with the speed of locomotion. In animals with higher thresholds (>60 µA), the correlation between locomotor speed and theta LFP oscillations was less robust. Changes in the gamma band (previously recorded in vitro in the pedunculopontine nucleus (PPN), thought to be a part of the MLR) were relatively small. Controlled locomotion was best achieved at 10-20 Hz frequencies of MLR stimulation. Our results indicate that theta and not delta or gamma band oscillation is a suitable biomarker for identifying the functional MLR sites.

Correction: Treatment with Riluzole Restores Normal Control of Soleus and Extensor Digitorum Longus Muscles during Locomotion in Adult Rats after Sciatic Nerve Crush at Birth.

[This corrects the article DOI: 10.1371/journal.pone.0170235.].

Treatment with Riluzole Restores Normal Control of Soleus and Extensor Digitorum Longus Muscles during Locomotion in Adult Rats after Sciatic Nerve Crush at Birth.

The effects of sciatic nerve crush (SNC) and treatment with Riluzole on muscle activity during unrestrained locomotion were identified in an animal model by analysis of the EMG activity recorded from soleus (Sol) and extensor digitorum longus (EDL) muscles of both hindlimbs; in intact rats (IN) and in groups of rats treated for 14 days with saline (S) or Riluzole (R) after right limb nerve crush at the 1st (1S and 1R) or 2nd (2S and 2R) day after birth. Changes in the locomotor pattern of EMG activity were correlated with the numbers of survived motor units (MUs) identified in investigated muscles. S rats with 2-8 and 10-28 MUs that survived in Sol and EDL muscles respectively showed increases in the duration and duty factor of muscle EMG activity and a loss of correlation between the duty factors of muscle activity, and abnormal flexor-extensor co-activation 3 months after SNC. R rats with 5, 6 (Sol) and 15-29 MUs (EDL) developed almost normal EMG activity of both Sol and control EDL muscles, whereas EDL muscles with SNC showed a lack of recovery. R rats with 8 (Sol) and 23-33 (EDL) MUs developed almost normal EMG activities of all four muscles. A subgroup of S rats with a lack of recovery and R rats with almost complete recovery that had similar number of MUs (8 and 24-28 vs 8 and 23-26), showed that the number of MUs was not the only determinant of treatment effectiveness. The results demonstrated that rats with SNC failed to develop normal muscle activity due to malfunction of neuronal circuits attenuating EDL muscle activity during the stance phase, whereas treatment with Riluzole enabled almost normal EMG activity of Sol and EDL muscles during locomotor movement.

Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats.

KEY POINTS: Experiments on neonatal rodent spinal cord showed that serotonin (5-HT), acting via 5-HT7 receptors, is required for initiation of locomotion and for controlling the action of interneurons responsible for inter- and intralimb coordination, but the importance of the 5-HT system in adult locomotion is not clear. Blockade of spinal 5-HT7 receptors interfered with voluntary locomotion in adult rats and fictive locomotion in paralysed decerebrate rats with no afferent feedback, consistent with a requirement for activation of descending 5-HT neurons for production of locomotion. The direct control of coordinating interneurons by 5-HT7 receptors observed in neonatal animals was not found during fictive locomotion, revealing a developmental shift from direct control of locomotor interneurons in neonates to control of afferent input from the moving limb in adults. An understanding of the afferents controlled by 5-HT during locomotion is required for optimal use of rehabilitation therapies involving the use of serotonergic drugs. ABSTRACT: Serotonergic pathways to the spinal cord are implicated in the control of locomotion based on studies using serotonin type 7 (5-HT7 ) receptor agonists and antagonists and 5-HT7 receptor knockout mice. Blockade of these receptors is thought to interfere with the activity of coordinating interneurons, a conclusion derived primarily from in vitro studies on isolated spinal cord of neonatal rats and mice. Developmental changes in the effects of serotonin (5-HT) on spinal neurons have recently been described, and there is increasing data on control of sensory input by 5-HT7 receptors on dorsal root ganglion cells and/or dorsal horn neurons, leading us to determine the effects of 5-HT7 receptor blockade on voluntary overground locomotion and on locomotion without afferent input from the moving limb (fictive locomotion) in adult animals. Intrathecal injections of the selective 5-HT7 antagonist SB269970 in adult intact rats suppressed locomotion by partial paralysis of hindlimbs. This occurred without a direct effect on motoneurons as revealed by an investigation of reflex activity. The antagonist disrupted intra- and interlimb coordination during locomotion in all intact animals but not during fictive locomotion induced by stimulation of the mesencephalic locomotor region (MLR). MLR-evoked fictive locomotion was transiently blocked, then the amplitude and frequency of rhythmic activity were reduced by SB269970, consistent with the notion that the MLR activates 5-HT neurons, leading to excitation of central pattern generator neurons with 5-HT7 receptors. Effects on coordination in adults required the presence of afferent input, suggesting a switch to 5-HT7 receptor-mediated control of sensory pathways during development.

Thoracic Hemisection in Rats Results in Initial Recovery Followed by a Late Decrement in Locomotor Movements, with Changes in Coordination Correlated with Serotonergic Innervation of the Ventral Horn.

Lateral thoracic hemisection of the rodent spinal cord is a popular model of spinal cord injury, in which the effects of various treatments, designed to encourage locomotor recovery, are tested. Nevertheless, there are still inconsistencies in the literature concerning the details of spontaneous locomotor recovery after such lesions, and there is a lack of data concerning the quality of locomotion over a long time span after the lesion. In this study, we aimed to address some of these issues. In our experiments, locomotor recovery was assessed using EMG and CatWalk recordings and analysis. Our results showed that after hemisection there was paralysis in both hindlimbs, followed by a substantial recovery of locomotor movements, but even at the peak of recovery, which occurred about 4 weeks after the lesion, some deficits of locomotion remained present. The parameters that were abnormal included abduction, interlimb coordination and speed of locomotion. Locomotor performance was stable for several weeks, but about 3-4 months after hemisection secondary locomotor impairment was observed with changes in parameters, such as speed of locomotion, interlimb coordination, base of hindlimb support, hindlimb abduction and relative foot print distance. Histological analysis of serotonergic innervation at the lumbar ventral horn below hemisection revealed a limited restoration of serotonergic fibers on the ipsilateral side of the spinal cord, while on the contralateral side of the spinal cord it returned to normal. In addition, the length of these fibers on both sides of the spinal cord correlated with inter- and intralimb coordination. In contrast to data reported in the literature, our results show there is not full locomotor recovery after spinal cord hemisection. Secondary deterioration of certain locomotor functions occurs with time in hemisected rats, and locomotor recovery appears partly associated with reinnervation of spinal circuitry by serotonergic fibers.

The expression and function of gelatinolytic activity at the rat neuromuscular junction upon physical exercise.

The gelatinases MMP-9 and MMP-2 have been implicated in skeletal muscle adaptation to training; however, their specific role(s) in the different muscle types are only beginning to be unraveled. Recently, we found that treadmill running increased the activity and/or expression of these enzymes in myonuclei and in activated satellite cells of the soleus (Sol), but not extensor digitorum longus (EDL) muscles on the fifth day of training of adult rats. Here, we asked whether the gelatinases can be involved in physical exercise-induced adaptation of the neuromuscular compartment. To determine the subcellular localization of the gelatinolytic activity, we used high-resolution in situ zymography and immunofluorescence techniques. In both control and trained muscles, strong gelatinolytic activity was associated with myelin sheaths within intramuscular nerve twigs. In EDL, but not Sol, there was an increase in the gelatinolytic activity at the postsynaptic domain of the neuromuscular junction (NMJ). The increased activity was found within punctate structures situated in the vicinity of synaptic cleft of the NMJ, colocalizing with a marker of endoplasmic reticulum. Our results support the hypothesis that the gelatinolytic activity at the NMJ may be involved in NMJ plasticity.

Cholinergic mechanisms in spinal locomotion-potential target for rehabilitation approaches.

Previous experiments implicate cholinergic brainstem and spinal systems in the control of locomotion. Our results demonstrate that the endogenous cholinergic propriospinal system, acting via M2 and M3 muscarinic receptors, is capable of consistently producing well-coordinated locomotor activity in the in vitro neonatal preparation, placing it in a position to contribute to normal locomotion and to provide a basis for recovery of locomotor capability in the absence of descending pathways. Tests of these suggestions, however, reveal that the spinal cholinergic system plays little if any role in the induction of locomotion, because MLR-evoked locomotion in decerebrate cats is not prevented by cholinergic antagonists. Furthermore, it is not required for the development of stepping movements after spinal cord injury, because cholinergic agonists do not facilitate the appearance of locomotion after spinal cord injury, unlike the dramatic locomotion-promoting effects of clonidine, a noradrenergic α-2 agonist. Furthermore, cholinergic antagonists actually improve locomotor activity after spinal cord injury, suggesting that plastic changes in the spinal cholinergic system interfere with locomotion rather than facilitating it. Changes that have been observed in the cholinergic innervation of motoneurons after spinal cord injury do not decrease motoneuron excitability, as expected. Instead, the development of a "hyper-cholinergic" state after spinal cord injury appears to enhance motoneuron output and suppress locomotion. A cholinergic suppression of afferent input from the limb after spinal cord injury is also evident from our data, and this may contribute to the ability of cholinergic antagonists to improve locomotion. Not only is a role for the spinal cholinergic system in suppressing locomotion after SCI suggested by our results, but an obligatory contribution of a brainstem cholinergic relay to reticulospinal locomotor command systems is not confirmed by our experiments.

5-HT2 and 5-HT7 receptor agonists facilitate plantar stepping in chronic spinal rats through actions on different populations of spinal neurons.

There is considerable evidence from research in neonatal and adult rat and mouse preparations to warrant the conclusion that activation of 5-HT2 and 5-HT1A/7 receptors leads to activation of the spinal cord circuitry for locomotion. These receptors are involved in control of locomotor movements, but it is not clear how they are implicated in the responses to 5-HT agonists observed after spinal cord injury. Here we used agonists that are efficient in promoting locomotor recovery in paraplegic rats, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT) (acting on 5-HT1A/7 receptors) and quipazine (acting on 5-HT2 receptors), to examine this issue. Analysis of intra- and interlimb coordination confirmed that the locomotor performance was significantly improved by either drug, but the data revealed marked differences in their mode of action. Interlimb coordination was significantly better after 8-OHDPAT application, and the activity of the extensor soleus muscle was significantly longer during the stance phase of locomotor movements enhanced by quipazine. Our results show that activation of both receptors facilitates locomotion, but their effects are likely exerted on different populations of spinal neurons. Activation of 5-HT2 receptors facilitates the output stage of the locomotor system, in part by directly activating motoneurons, and also through activation of interneurons of the locomotor central pattern generator (CPG). Activation of 5-HT7/1A receptors facilitates the activity of the locomotor CPG, without direct actions on the output components of the locomotor system, including motoneurons. Although our findings show that the combined use of these two drugs results in production of well-coordinated weight supported locomotion with a reduced need for exteroceptive stimulation, they also indicate that there might be some limitations to the utility of combined treatment. Sensory feedback and some intraspinal circuitry recruited by the drugs can conflict with the locomotor activation.

The role of serotonin in the control of locomotor movements and strategies for restoring locomotion after spinal cord injury.

In this review we will discuss different ways for re-establishing serotonergic activity that can enhance recovery of coordinated plantar stepping after spinal cord injury in adult rats. It is well known that serotoninergic neurons located in the medulla are able to initiate locomotor activity. This effect is exerted by actions on motoneurons and on neurons of the locomotor CPG (Central Pattern Generator). Motoneuron and interneuron excitability is increased, and putative CPG interneurons display oscillatory behaviour in response to serotonin receptor activation. The medullary serotonergic nuclei play multiple roles in the control of locomotion, and they terminate on specific target neurons with different types of serotonergic receptors in the spinal cord. Activation of these serotonergic receptors can restore locomotor movements after spinal cord injury. Specifically, using defined serotonergic agonists the 5-HT2 receptors can be stimulated to control CPG activation as well as motoneuron output, while 5-HT7 receptors to control activity of the locomotor CPG. These results are consistent with the roles for these receptors during locomotion in intact rodents and in rodent brainstem-spinal cord in vitro preparations. The other possibility to encourage the remaining spinal cord circuitry below the total transection to control recovery of plantar hindlimb stepping is restoration of serotonergic innervation by intraspinal grafting of embryonic 5-HT neurons. Our data show that grafting of different populations of 5-HT neurons dissected from embryonic brainstem provides differential control over multiple components of the spinal locomotor circuitry through specific serotonin receptors. Moreover, we demonstrated that the best effect of motor recovery is obtained after grafting of neurons destined to form the B1, B2 and B3 descending 5-HT systems. Using only one of the subpopulations for intraspinal grafting, for example, B3 or the lateral group of 5-HT neurons, induces only partial recovery of plantar stepping with a clear lack of proper interlimb coordination. This confirms the hypothesis that transplantation of 5-HT neurons from specific embryonic sources is necessary to obtain optimal recovery of locomotor hindlimb movement.

Grafting of fetal brainstem 5-HT neurons into the sublesional spinal cord of paraplegic rats restores coordinated hindlimb locomotion.

In rodent models of spinal cord injury, there is increasing evidence that activation of the locomotor central pattern generator (CPG) below the site of injury with 5-hydroxytryptamine (5-HT) agonists improves locomotor recovery and restores coordination. A promising means of replacing 5-HT control of locomotion is to graft brainstem 5-HT neurons into the spinal cord below the level of the spinal cord injury. However, it is not known whether this approach improves limb coordination because recovery of coordinated stepping has not been documented in detail in previous studies employing this transplantation strategy. Here, adult rats with complete spinal cord transections at the T9/10 level were grafted with E14 fetal neurons from the medulla at the T10/11 vertebra level one month after injury. The B1, B2 and B3 fetal anlagen of brainstem 5-HT neurons, a grouping that included the presumed precursors of recently described 5-HT locomotor command neurons, were used in these grafts. EMG and video recordings of treadmill locomotion evoked by tail stimulation showed full recovery of inter- and intralimb coordination in the grafted rats. We showed, using systemically applied antagonists, that 5-HT2 and 5-HT7 receptors mediate the improved locomotion after grafting, but through actions on different populations of spinal locomotor neurons. Specifically, 5-HT2 receptors control CPG activation as well as motoneuron output, while 5-HT7 receptors contribute primarily to activity of the locomotor CPG. These results are consistent with the roles for these receptors during locomotion in intact rodents and in rodent brainstem-spinal cord in vitro preparations.

Myosin VI in skeletal muscle: its localization in the sarcoplasmic reticulum, neuromuscular junction and muscle nuclei.

Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 µm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.