Regenerating motor bridge axons refine connections and synapse on lumbar motoneurons to bypass chronic spinal cord injury

To restore motor control after spinal cord injury requires reconnecting the brain with spinal motor circuits below the lesion. A bridge around the injury is an important alternative to promoting axon regeneration through the injury. Previously, we reported a novel motor bridge in rats. The thirteenth thoracic nerve was detached from the muscle it innervates and the cut end implanted caudally into the lumbar gray matter where motor bridge axons regenerate. In this study, we first determined that regenerating bridge axons project to spinal motor circuits. Stable projections were present in ventral motor laminae of the cord, including putative synapses directly on motoneurons, 2 months after insertion in the intact cord. At this time, earlier-forming dorsal horn projections were mostly eliminated. Regenerating axons were effective in evoking leg motor activity as early as 2 weeks. We next determined that bridge axons could regenerate caudal to a chronic injury. We hemisected the spinal cord at L2 and inserted the bridge nerve 1 month later at L5 and found ventral laminae projections similar to those in intact animals, including onto motoneurons directly. Finally, we determined that the bridge circuit could be activated by neural pathways rostral to its origin. For spinally hemisected animals, we electrically stimulated the rostral spinal cord and recorded evoked potentials from the bridge and, in turn, motor responses in the sciatic nerve. Our findings suggests that bridge motoneurons could be used by descending motor pathways as premotor interneurons to transmit neural signals to bypass a chronic spinal injury.     

Bilateral corticospinal projections arise from each motor cortex in the macaque monkey: a quantitative study

The corticospinal projection is considered to influence fine motor function through nearly exclusively contralateral projections from the cortex in primates. However, unilateral lesions to this system in various species are frequently followed by significant functional improvement, raising the possibility that bilateral projections of this pathway may exist or emerge after injury. To examine the detailed anatomy and projections of the corticospinal motor neurons in rhesus monkeys (n = 4), we injected the high-resolution anterograde tracer biotinylated dextran amine (BDA) into 126 sites centered about the right lower extremity (LE) primary motor cortex. Projection and termination patterns were quantified at lumbar levels L1, L4, and L7 and mapped by using serial-section reconstructions. Notably, a mean of 10.1 +/- 0.6% (+/- SEM) of corticospinal tract (CST) axons descended in the lateral CST ipsilateral to the cortical BDA injection, and 87.9 +/- 1.0% of total CST axons projected in the contralateral lateral CST. The ipsilateral ventral CST contained only 1.0 +/- 0% of all projecting CST axons, whereas the contralateral ventral CST contained 0.3 +/- 0.2% of all axons. In addition, a minor dorsal column CST projection was identified. Measurement of BDA-labeled terminals in the spinal cord gray matter revealed that 11.2 +/- 2.2% of CST axons terminated ipsilateral to the side of cortical injection, and the remainder terminated contralaterally. As previously reported, most CST axons terminated in spinal cord laminae V-VIII, as well as the laterodorsal motoneuronal group of lamina IX (which innervates distal extremity muscles). Notably, many CST axons crossed the spinal cord midline (mean 19.9 +/- 4.9 axons per 40-microm-thick section). Detailed single-axon reconstructions revealed that most ipsilaterally projecting lateral CST axons terminated in ipsilateral gray matter. Notably, we found that the bouton-like swellings of many ipsilateral CST axons descending in the dorsolateral tract were located within Rexed's lamina IX, in close proximity to motoneuronal somata. Thus, bilateral projections of corticospinal axons originating from a single motor cortex could contribute to bilateral control of spinal motor neurons and to the highly evolved degree of fine motor control in primates. Furthermore, bilateral CST projections from a single motor cortex could represent a potential source of plasticity after injury, as well as a target of therapeutic effort in neural regeneration strategies.     

Development of the rat corticospinal tract through an altered glial environment

The major corticospinal tract (CST) in the rat is located at the base of the dorsal funiculus. It is a late-developing tract, and the growth of its axons into the lumbosacral region of the spinal cord does not occur until postnatal days 5 and 6. This delay is taken advantage of in this study in order to evaluate the effects of a markedly reduced glial population on ingrowth of the CST axons into the lumbosacral spinal cord. A reduction of the glial population is achieved by exposure of this region of spinal cord to X-radiation at 3 days of age. Growth of CST axons into and through the lumbosacral spinal cord in rats in which this region has undergone a radiation-induced depletion of glial cells is compared with that in their non-irradiated littermate controls by axonal tracing techniques using horseradish peroxidase (HRP). The HRP was applied directly to the motor cortices of normal and irradiated rats, and at all ages studied, there was anterograde filling of CST axons and their growth cones. At 3 days postnatally, the age when the lumbosacral spinal cord was irradiated in the experimental animals, CST axons were present in the more rostral thoracic levels. CST axons were observed in the lumbar region of non-irradiated rats on day 5, and by day 7 they were present at sacral levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of aging on axon sprouting and regenerative growth in spinal cord injury

The demographics of acute spinal cord injury (SCI) are changing with an increased incidence in older age. However, the influence of aging on the regenerative growth potential of central nervous system (CNS) axons following SCI is not known. We investigated axonal sprouting along with the efficiency of the infusion of the stromal cell-derived growth factor-1 (SDF-1/CXCL12) and regenerative growth along with the anti-scarring treatment (AST) in young (2–3 months) and geriatric (22–28 months) female rats following SCI. AST included local injection of iron chelator (2,2′-dipyridine-5,5′-dicarboxylic acid) and 8-bromo-cyclic adenosine monophosphate solution into the lesion core. Axon outgrowth was investigated by immunohistological methods at 5 weeks after a partial dorsal hemisection at thoracic level T8. We found that aging significantly reduces spontaneous axon sprouting of corticospinal (CST), serotonergic (5-HT) raphespinal and catecholaminergic (TH) coerulospinal tracts in distinct regions of the spinal cord rostral to the lesion. However, impairment of axon sprouting could be markedly attenuated in geriatric animals by local infusion of SDF-1. Unexpectedly and in contrast to rostral sprouting, aging does not diminish the regenerative growth capacity of 5-HT-, TH- and calcitonin gene-related peptide (CGRP)-immunoreactive axons at 5 weeks after SCI. Moreover, 5-HT and TH axons maintain the ability to react upon AST with significantly enhanced regeneration in aged animals. These data are the first to demonstrate, that old age compromises axonal plasticity, but not regenerative growth, after SCI in a fiber tract-specific manner. Furthermore, AST and SDF-1 infusions remain efficient, which implicates that therapy in elderly patients is still feasible.     

Cholecystokinin-like immunoreactivity in the rat spinal cord: Effects of thoracic transection

A study of cholecystokinin-like immunoreactivity in the lumbar (L1-L5) spinal cord segments of rats was realised 24-48 hours after complete thoracic transection (T6-T8). A comparison was made with corresponding spinal cord segments from control and sham-operated animals. The immunocytochemical study with light microscopy showed cholecystokinin-like immunoreactive cell bodies in laminae VII and X at L1-L5, caudal to the transection. In addition, the immunoreactivity was greatly enhanced in bundles of the dorsolateral funiculus compared to sham-operated animals. Our results suggest that part of cholecystokinin-like cell bodies of laminae VII and X send projections to supraspinal sites. Some of these supraspinal projections would go through the dorsolateral funiculus. In the lumbar dorsal horn of operated animals, the immunoreactivity was greatly enhanced in lamina I, while it was slightly decreased in lamina II, compared to control animals. Using electron microscopy, in lamina I, the immunoreactivity localized in different neurites was generally very intense. Moreover, axon terminals showed swelling: their mean size was 0.8-1.8 microns (0.5-1.2 in control animals). This result suggests that some cholecystokinin-like neurons also project to lamina I of rostral cervical segments. In lamina II, numerous degenerating axons were observed (24 hours after thoracic spinal transection). This would suggest that part of descending cholecystokinin-like projections terminate in lamina II.     

Both dorsal and ventral spinal cord pathways contribute to overground locomotion in the adult rat

Identification of long tracts responsible for spontaneous locomotion is critical for spinal cord injury (SCI) repair strategies. We recently demonstrated that extensive demyelination of adult rat thoracic ventral columns, ventromedial, and ventrolateral white matter produces persistent, significant open-field hindlimb locomotor deficits. Locomotor movements resulting from stimulation of the pontomedullary locomotor region are inhibited by dorsolateral funiculus (DLF) lesions suggesting that important pathways for locomotion may also exist in the dorsal white matter. However, dorsal hemisections that interrupt dorsal columns/dorsal corticospinal tract (DC/CST) and DLF pathways do not produce persistent, severe locomotor deficits in the adult rat. We studied the contributions of myelinated tracts in the DLF and DC/CST to overground locomotion following complete conduction blockade of axons in the ventrolateral funiculus (VLF), a region important for locomotor movements and for transcranial magnetic motor-evoked potentials (tcMMEP). Animals received ethidium bromide plus photon irradiation to produce discrete demyelinating lesions sufficient to stop axonal conduction in the VLF, combined VLF + DLF, or combined VLF + DC/CST. Open-field BBB scores and tcMMEPs were studied at 1, 2, 3, and 4 weeks postlesion. VLF lesions resulted in mean BBB scores of 17 at 4 weeks. VLF + DC/CST and VLF + DLF lesions resulted in mean BBB scores of 15.9 and 11.1, respectively. TcMMEPs were absent in all lesion types confirming VLF conduction blockade throughout the study. Our data indicate that significant contributions to locomotion from myelinated pathways within the rat DLF can be revealed when combined with simultaneous compromise of the VLF.     

Pathophysiological activity in rat dorsal horn neurones in segments rostral to a chronic spinal cord injury

As a sequel of complete spinal cord injury (SCI), patients often develop chronic pain which is perceived at or just below the level of the lesion. Likewise, in animal models of SCI, spontaneous and evoked pain-related behaviour can be observed. In the present study, the hypothesis was tested that pain related behaviour after SCI in animals is at least partly due to neuronal hyperactivity in spinal segments rostral to the site of injury. In rats with a chronic transected spinal cord, the impulse activity of single dorsal horn neurones was recorded in two locations: (1) directly rostrally adjacent to the lesion, and (2) 2-3 segments more rostrally. Cord transections were made either at the thoracic or at the lumbar level. Sham-operated rats and rats which underwent no surgical interventions served as controls. Compared with both controls, in SCI animals the background activity of the neurones had a significantly higher level in both series. Often the activity showed a pathophysiological altered discharge pattern. Following SCI, there was a general increase in the mechanical responsiveness of neurones that were recorded 2-3 segments rostrally to the lesion. The results suggest that neuronal hyperactivity in spinal segments just rostral to the lesion may contribute to chronic spontaneous SCI pain. Further, there is some indication that the allodynia perceived in body regions near and above the level of the SCI may be due to increased responsiveness to weak stimuli of neurones located more rostrally to the lesion.     

Plasticity of the corticospinal tract following midthoracic spinal injury in the postnatal rat

Rats received a midthoracic spinal cord "overhemisection" including right hemicord and left dorsal funiculus at birth (neonatal operates, N = 15) or 21 days of age (weanling operates, N = 14). In a second experiment neonatal (N = 6), 6-day (N = 3), and 12-day (N = 7) rats sustained a right sensorimotor cortex (SmI) ablation to destroy the left corticospinal tract (CST) at the same time as the spinal injury (double lesion operates). Later (3-12 months) injections of 3H-proline and autoradiography were used to label the left or right CST. The results of the first experiment showed that most right CST axons failed to grow around the spinal lesion in neonatal operates (N = 9). There was an increase in the density of label, mainly to CST projection areas, in a 1-mm zone rostral to the lesion. However, left CST axons bypassed the lesion by growing through the intact tissue in neonatal operates (N = 6). These displaced axons were consistently located within the dorsal portion of the lateral funiculus (dLF) and remained within that location caudal to the lesion, an area normally containing only a few CST axons. In spite of this abnormal position, these axons terminated bilaterally throughout the remainder of the cord in normal CST sites. In weanling operates, CST axons severed by the lesion did not regenerate around the lesion site. An increased density of label over the few spared axons within the left dLF and in CST projection zones immediately caudal to the lesion site suggested axonal sprouting by these axons. The results of the second experiment showed that the lack of growth of right CST axons around this injury in neonatal operates was, at least partially, due to an interaction with left CST axons. In neonatal double lesion operates, right CST axons grew around the spinal injury for a varying distance within the left dLF and distributed bilaterally to normal CST sites. The number of right CST axons bypassing the lesion was related to the configuration of the lesion site. A smaller number of right CST axons bypassed the lesion in 6-day double lesion operates and most terminated within 2-3 mm of the lesion site. Right CST axons failed to grow around this injury in 12-day double lesion operates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonspecific labeling limits the utility of Cre‐Lox bred CST‐YFP mice for studies of corticospinal tract regeneration

Studies of axon regeneration in the spinal cord often assess regeneration of the corticospinal tract (CST). Emx1‐Cre x Thy1‐STOP‐YFP mice have been reported to have yellow fluorescent protein (YFP) selectively expressed in forebrain neurons leading to genetic labeling of CST axons in the spinal cord, and it was suggested that these CST‐YFP mice would be useful for studies of CST regeneration. Because regeneration past a lesion may involve only a few axons, the presence of labeled non‐CST axons compromises interpretation. We show here that in CST‐YFP mice, some YFP‐labeled axons are not from the CST. Specifically, YFP‐labeled axons are present in regions beyond those with anterogradely labeled CST axons, most YFP‐labeled axons beyond established CST locations do not undergo Wallerian degeneration following a large lesion of the sensorimotor cortex, some rubrospinal and reticulospinal neurons are labeled with YFP, and some YFP‐labeled cells in the spinal gray matter have YFP‐labeled projections into the spinal cord white matter. We further demonstrate that the density of YFP‐labeled axon arbors hinders tracing of single axons to their point of origin in the main descending tracts. In light of recent advances in 3D imaging for visualizing axons in unsectioned blocks of spinal cord, we also assessed CST‐YFP mice for 3D imaging and found that YFP fluorescence in CST‐YFP mice is faint for clearing‐based 3D imaging in comparison with fluorescence in Thy1‐YFP‐H mice and fluorescence of mini‐ruby biotinylated dextran amine (BDA). Overall, the nonspecific and faint YFP labeling in CST‐YFP mice limits their utility for assessments of CST axon regeneration. J. Comp. Neurol. 523:2665–2682, 2015. © 2015 Wiley Periodicals, Inc.     

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.     

Suppression of fibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons, rescue of primary motoneurons in somatosensory cortex and significant functional recovery

Traumatic injury of the central nervous system results in formation of a collagenous basement membrane-rich fibrous scar in the lesion centre. Due to accumulation of numerous axon-growth inhibitory molecules the lesion scar is considered a major impediment for axon regeneration. Following transection of the dorsal corticospinal tract (CST) at thoracic level 8 in adult rats, transient suppression of collagenous scarring in the lesion zone by local application of a potent iron chelator and cyclic adenosine monophosphate resulted in the delay of fibrous scarring. Treated animals displayed long-distance growth of CST axons through the lesion area extending for up to 1.5-2 cm into the distal cord. In addition, the treatment showed a strong neuroprotective effect, rescuing cortical motoneurons projecting into the CST that normally die (30%) after thoracic axotomy. Further, anterogradely traced CST axons regenerated through both grey and white matter and developed terminal arborizations in grey matter regions. In contrast to controls, injured animals receiving treatment showed significant functional recovery in the open field, in the horizontal ladder and in CatWalk locomotor tasks. We conclude that the fibrous lesion scar plays a pivotal role as a growth barrier for regenerating axons in adult spinal cord and that a delay in fibrotic scarring by local inhibition of collagen biosynthesis and basement membrane deposition is a promising and unique therapeutic strategy for treating human spinal trauma.     

Embryonic spinal cord neurons develop preferential cholinergic projections to sympathetic ganglia explanted from appropriate levels of the neuraxis

To determine whether cholinergic spinal cord neurons can develop preferential projections in vitro within sympathetic ganglia (SGs) of appropriate levels of the neuraxis, organotypic explants of fetal mouse spinal cord (E13) from cervical, thoracic (upper and lower) and lumbar segments were co-cultured with either pairs of neonatal SGs: the rostral superior cervical ganglion (SCG) or a caudally located upper lumbar ganglion (LG). After 3.5-4 weeks of co-culture, levels of the enzyme, choline acetyltransferase (ChAT), were measured in individual spinal cord explants and SCGs or double LGs (to match the target mass of a single SCG). Interaction was assumed to occur primarily between an SCG or LG doublet and the adjacent ipsilateral half of the co-cultured cord segment. An index of cholinergic interaction was defined as the ganglion ChAT activity per unit ChAT activity in half co-cultured cord segment. The index of interaction with the SCG was highest with the T1/T2 (1.4) as compared with the T10/T11 (0.79), L1/L2 (0.38) and C2/C3 (0.11) segments. In contrast, the index of cholinergic interaction with double LGs was highest with the more caudally located T10/T11 (0.62) cord segment as compared with the rostral T1/T2 (0.33), cervical C2/C3 (0.2) and lumbar L1/L2 (0.17) segments. Ganglion compound action potentials evoked in LGs by stimulation of the ipsilateral portion of T10/T11 cord were blocked by the ganglionic antagonist, hexamethonium, as previously observed in co-cultures of SCGs with T1/T2 cord. These results indicate that pools of preganglionic neurons in thoracic cord segments can develop in vitro preferential cholinergic projections within SGs of appropriate position. Cervical and lumbar cord segments which contain a preponderance of somatic motoneurons over preganglionic neurons did not interact as effectively with either type of SG. The preferential cholinergic projections from rostral thoracic cord explants within co-cultured SCGs and from caudal thoracic cord explants within co-cultured SCGs and from caudal thoracic cord explants within LGs may reflect some degree of positional preference intrinsic to embryonic spinal cord neurons and/or their appropriate target SGs, consistent with the positional specificity expressed by preganglionic neurons and SGs in situ.     

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.     

Neuronal overexpression of tissue-type plasminogen activator does not enhance sensory axon regeneration or locomotor recovery following dorsal hemisection of adult mouse thoracic spinal cord

CNS axons rarely regenerate spontaneously back to original targets following spinal cord injury (SCI). Neuronal expression of the serine protease tissue-type plasminogen activator (tPA) enhances axon growth in vitro and following PNS injury. Here we test the hypothesis that neuronal overexpression of tPA in adult transgenic mice promotes CNS axon regeneration and functional recovery following SCI. Adult wild-type and transgenic mouse spinal cords were subjected to dorsal hemisection at the level of the T10/T11 vertebrae. PCR confirmed incorporation of the transgene. Immunolabeling revealed overexpression of tPA in transgenic mice in neurons, including large-diameter neurons in lumbar dorsal root ganglia that contribute axons to the dorsal columns. Immunolabeling also revealed the presence of tPA protein within axons juxtaposing the injury site in transgenics but not wild types. In situ zymography revealed abundant enzymatic activity of tPA in gray matter of thoracic spinal cords of transgenics but not wild types. Rotorod locomotor testing revealed no differences between groups in locomotor function up to 21 days postinjury. Transganglionic tracer was injected into the crushed right sciatic nerve 28 days postinjury, and mice were killed 3 days later. There was no evidence for regrowth of ascending dorsal column sensory axons through or beyond the injury site. In conclusion, despite neuronal overexpression of tPA in injured neurons of transgenics, neither locomotor recovery nor regeneration of ascending sensory axons was observed following thoracic dorsal hemisection.     

Nerve Growth Factor Promotes Regeneration of Sensory Axons into Adult Rat Spinal Cord

Injured adult mammalian axons are unable to regenerate spontaneously in the central nervous tissue. This study investigated in two adult rat models the effects of nerve growth factor (NGF) on the capacity of central primary sensory axons to regenerate back into the spinal cord. Sensory fibers were conditioned by transection of the peripheral nerve 1 week prior to the experiment and identified by anterograde tracing with cholera toxin B subunit injected in the sciatic nerve. In the first model, a predegenerated autologous peripheral nerve graft was implanted as a bridge for the transected sensory fibers into a resection gap in the dorsal columns at the tenth thoracic (T10) spinal cord segment. Vehicle or vehicle with purified mouse or recombinant human NGF was continuously infused for 2 weeks directly into the dorsal column at T9, 3 mm from the rostral border of the nerve graft. With vehicle infusion many ascending sensory axons had grown across the nerve bridge, but essentially none had grown back into the rostral cord. In sharp contrast, NGF promoted the reentry into the denervated dorsal columns of 51% of the sensory axons that had reached the rostral level of the nerve graft. Twenty-six percent had grown 2 mm into the spinal tissue and 10% had reached the NGF-infusion site at 3 mm from the nerve graft. A few fibers were found circling around, but not beyond, the infusion site, perhaps due to the chemoattractant action of NGF. In a second model, the fourth lumbar (L4) dorsal root was crushed 2 mm from its insertion point into the spinal cord and the dorsal roots L2, L3, L5, and L6 were transected. Vehicle or vehicle with purified mouse NGF was infused for 2 weeks directly into the lumbar spinal cord, 2.5 mm rostral to the transition zone of the crushed L4 root. With vehicle, only 6% of the regenerating fibers at the transition zone had crossed the root–spinal cord barrier, but not farther than 0.5 mm into the spinal tissue. With NGF, 18% of the fibers at the transition zone were found at 0.5 mm, 9% at 1.5 mm, and 5% at 2.5 mm (the infusion site) from the transition zone. The present results demonstrate that NGF can promote the regeneration of adult sensory fibers into the otherwise nonpermissive spinal cord white matter.     

Semaphorin7A: its role in the control of serotonergic circuits and functional recovery following spinal cord injury

Serotonin is a monoamine neurotransmitter synthetized in various populations of brainstem neurons.In the spinal cord,descending serotonergic projections regulate postural muscle tone,locomotion and rhythm and coordination of movements via the Central Pattern Generator.Following a spinal cord injury,serotonergic projections to the lumbar spinal cord,where the Central Pattern Generators are located,are interrupted resulting in devastating locomotor impairments and changes in the expression and activation of serotonin and its spinal receptors.The molecular cues that control the precise patterning and targeting of serotonergic inputs onto Central Pattern Generator networks in healthy animals or after injury are still unknown.In our recent research work,we have been particularly interested in Semaphorin7A,which belongs to the Semaphorins family involved in guiding growing axons and controlling plasticity of synaptic connections.In this review,we discuss the role of Semaphorin7A signaling as an important molecular actor that instructs the patterning of serotonin inputs to spinal Central Pattern Generator networks.We show that Semaphorin7A controls the wiring of descending serotonin axons in the spinal cord.Our results reveal that mistargetting of serotonin fibers in the spinal cord is compensated in healthy uninjured Semaphorin7A deficient mice so that their gross locomotion proceeds accurately.We also demonstrate that when the system is challenged with a spinal lesion,the pattern of post-injury serotonin expression is significantly altered in Semaphorin7A deficient mice with specific ectopic targeting of serotonin fibers in the lumbar spinal cord.Compensatory mechanisms in place in uninjured Semaphorin7A deficient mice are lost and injured Semaphorin7A deficient mice exhibit a worsening of their post-injury locomotor abilities.Our findings identify Semaphorin7A as a critical determinant of serotonergic circuit formation in healthy or spinal cord injured mice.     

Projections from upper cervical inspiratory neurons to thoracic and lumbar expiratory motor nuclei in the cat

Previous studies have demonstrated the existence of axonal projections from propriospinal respiratory neurons in the rostral cervical cord of the cat (upper cervical inspiratory neurons) to the vicinity of the phrenic and rostral thoracic inspiratory (external) intercostal motoneurons. However, no synaptic targets of the upper cervical inspiratory neurons have been identified. This study investigated the axonal projections to the caudal thoracic and upper lumbar cord and the possible existence of inhibitory connections to the expiratory intercostal and abdominal motoneurons. The connections from upper cervical inspiratory neurons to expiratory motoneurons in the lower thoracic cord were examined using the methods of antidromic mapping and the spike-triggered averaging of intracellular potentials. Of the 70 upper cervical inspiratory neurons examined, only four (5.7%) could not be antidromically activated from the T9 segment of the spinal cord. The axons of 66 upper cervical inspiratory antidromic activation at less than 5 microV, and the presence of collaterals was confirmed by antidromic mapping in 30 cases (49.2%). Of 21 axons tested for lumbar projections, 13 (61.9%) projected as far as T12, seven (33.3%) as far as L1, three (14.3%) as far as L2, and one (4.8%) was antidromically activated from L3. Spike-triggered averaging of the synaptic potentials recorded intracellularly from expiratory intercostal motoneurons in T9 and T10 spinal segments was done for 27 upper cervical inspiratory neurons, 17 of these with 4 or more motoneurons, for a total of 111 expiratory motoneurons. In 16 cases the motoneurons were injected with Cl- to reverse IPSPs and the spike-triggered averaging was repeated. No monosynaptic or disynaptic post-synaptic potentials were seen in any of the spike-triggered averages. We concluded that the upper cervical inspiratory neurons may provide inspiratory inhibition to expiratory motoneurons via a di- or oligosynaptic pathway involving segmental inhibitory interneurons and that either the spike-triggered averaging technique was not sensitive enough to detect the ipsilateral connections or the interneuron pathway was to the contralateral side of the spinal cord.     

The central nervous system in canine giant axonal neuropathy

Summary The pathology of the central nervous system (CNS) in a dog with giant axonal neuropathy (GAN) is presented. Swollen axons containing excessive and disorganised neurofilaments were present in the spinal cord, mainly at the distal portions of long tracts. The fasciculus gracilis and dorsal spinocerebellar tracts were affected only in the rostral cervical cord while the lateral cortico spinal tract was principally involved in the lower thoracic and lumbar cord. Occasional swellings were also found in the central dorsal columns of the rostral lumbar segments and in the dorsal and intermediate grey matter. The nuclei gracilis and cuneatus, restiform body and ventral spinocerebellar tracts were all involved in the brain stem. Spheroids were seen in the white matter of the rostral cerebellar vermis and in the granule cell layer. The brachium of the superior colliculus contained swollen axons and the cortex was diffusely involved with spheroids. The distribution was of a distal axonopathy and the cortical changes provided an explanation for the abnormal EEG and mental retardation found in some human patients.     

A re-assessment of the effects of a Nogo-66 receptor antagonist on regenerative growth of axons and locomotor recovery after spinal cord injury in mice

This study was undertaken as part of the NIH "Facilities of Research-Spinal Cord Injury" project to support independent replication of published studies. Here, we repeated a study reporting that treatment with the NgR antagonist peptide NEP1-40 results in enhanced growth of corticospinal and serotonergic axons and enhanced locomotor recovery after thoracic spinal cord injury. Mice received dorsal hemisection injuries at T8 and then received either NEP1-40, Vehicle, or a Control Peptide beginning 4-5 h (early treatment) or 7 days (delayed treatment) post-injury. CST axons were traced by injecting BDA into the sensorimotor cortex. Serotonergic axons were assessed by immunocytochemistry. Hindlimb motor function was assessed using the BBB and BMS scales, kinematic and footprint analyses, and a grid climbing task. There were no significant differences between groups in the density of CST axon arbors in the gray matter rostral to the injury or in the density of serotonergic axons caudal to the injury. Tract tracing revealed that a small number of CST axons extended past the lesion in the ventral column in some mice in all treatment groups. The proportion of mice with such axons was higher in the NEP1-40 groups that received early treatment. In one experiment, mice treated with either NEP1-40 or a Control Peptide (reverse sequence) had higher BBB and BMS scores than Vehicle-treated controls at the early post-injury testing intervals, but scores converged at later intervals. There were no statistically significant differences between groups on other functional outcome measures. In a second experiment comparing NEP-treated and Vehicle controls, there were no statistically significant differences on any of the functional outcome measures. Together, our results suggest that treatment with NEP1-40 created a situation that was slightly more conducive to axon regeneration or sprouting. Enhanced functional recovery was not seen consistently with the different functional assessments, however.