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The Journal of Neuroscience : the... Apr 2012Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is...
Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is formed along the dorsal-most region of the caudal rhombic lip, gives rise to the cochlear and precerebellar nuclei. These centers project sensory inputs toward upper-brain regions. The fundamental role of dA1 interneurons in the assembly and function of these brainstem nuclei is well characterized. However, the precise en route axonal patterns and synaptic targets of dA1 interneurons are not clear as of yet. Novel genetic tools were used to label dA1 neurons and trace their axonal trajectories and synaptic connections at various stages of chick embryos. Using dA1-specific enhancers, two contralateral ascending axonal projection patterns were identified; one derived from rhombomeres 6-7 that elongated in the dorsal funiculus, while the other originated from rhombomeres 2-5 and extended in the lateral funiculus. Targets of dA1 axons were followed at later stages using PiggyBac-mediated DNA transposition. dA1 axons were found to project and form synapses in the auditory nuclei and cerebellum. Investigation of mechanisms that regulate the patterns of dA1 axons revealed a fundamental role of Lim-homeodomain (HD) proteins. Switch in the expression of the specific dA1 Lim-HD proteins Lhx2/9 into Lhx1, which is typically expressed in dB1 interneurons, modified dA1 axonal patterns to project along the routes of dB1 subgroup. Together, the results of this research provided new tools and knowledge to the assembly of trajectories and connectivity of hindbrain dA1 interneurons and of molecular mechanisms that control these patterns.
Topics: Age Factors; Animals; Axons; Basic Helix-Loop-Helix Transcription Factors; Body Patterning; Cell Count; Chick Embryo; Ectodysplasins; Electroporation; Functional Laterality; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Humans; Interneurons; LIM-Homeodomain Proteins; Nerve Tissue Proteins; PAX2 Transcription Factor; Rhombencephalon; Synapses
PubMed: 22539838
DOI: 10.1523/JNEUROSCI.4231-11.2012 -
Neural Regeneration Research Feb 2012Fluoro-ruby was injected into the posterior funiculus of the spinal cord in the cervical (C5-T2) and lumbar (L3-6) segments of adult guinea pigs. The spinal cord was cut...
Fluoro-ruby was injected into the posterior funiculus of the spinal cord in the cervical (C5-T2) and lumbar (L3-6) segments of adult guinea pigs. The spinal cord was cut into serial frozen sections. The Fluoro-ruby labeling was clearly delineated from the surrounding structure. The labeling traversed the cervical, thoracic and lumbar segments, and was located on the ventral portion of the posterior funiculus on the injected side, proximal to the intermediate zone of the dorsal gray matter. The fluorescence area narrowed rostro-caudally. The spinal cord, spinal cord gray matter and corticospinal tract were reconstructed using 3D-DOCTOR 4.0 software, resulting in a robust three-dimensional profile. Using functionality provided by the reconstruction software, free multi-angle observation and sectioning could be conducted on the spinal cord and corticospinal tract. Our experimental findings indicate that the Fluoro-ruby retrograde fluorescent tracing technique can accurately display the anatomical location of corticospinal tract in the guinea pig and that three-dimensional reconstruction software can be used to provide a three-dimensional image of the corticospinal tract.
PubMed: 25774176
DOI: 10.3969/j.issn.1673-5374.2012.05.007 -
Neuroscience Research Mar 2012Previous studies showed that ipsilaterally projecting dorsal horn dorsal spinocerebellar tract (dh-DSCT) neurons located outside Clarke's column in mid- and...
Previous studies showed that ipsilaterally projecting dorsal horn dorsal spinocerebellar tract (dh-DSCT) neurons located outside Clarke's column in mid- and caudal-lumbar segments of the spinal cord receive different afferent inputs. Here, we examined, using extracellular recordings in anaesthetized cats, whether there are also input differences to these populations of dh-DSCT neurons from: (a) the spinocervical tract (SCT), estimated by stimulation of the ipsilateral dorsolateral funiculus at cervical cord C3 and rostral C1, below and above the termination of SCT axons in the lateral cervical nucleus (LCN), and (b) descending/ascending fibres activated by electrical stimulation at rostral C1. Seventy percent (21/30) of the lower-lumbar (L6-L7) dh-DSCT neurons received significantly greater excitation from C3 than rostral C1, whereas only 17% (5/30) of the mid-lumbar (L5) dh-DSCT neurons had greater responses from C3 than rostral C1. Inhibition of background activity was seen in 30% of the lower-lumbar neurons, but only in 3% of mid-lumbar neurons. These findings suggest that lower-lumbar dh-DSCT neurons are much more likely, than mid-lumbar ones, to be influenced by the SCT and by systems descending from the brain, LCN and/or ascending systems. The experiments provide further evidence for differences in input to the subpopulations of dh-DSCT neurons.
Topics: Animals; Cats; Cerebellum; Cervical Vertebrae; Electric Stimulation; Nerve Net; Neural Pathways; Posterior Horn Cells; Spinal Cord; Spinocerebellar Tracts
PubMed: 22192466
DOI: 10.1016/j.neures.2011.12.007 -
Journal of Neurotrauma Feb 2012Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury...
Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury (SCI). Docosahexaenoic acid (DHA, C22:6 n-3) is neuroprotective when administered following SCI, but its potential as a pretreatment modality has not been addressed. This study used a novel DHA pretreatment experimental paradigm that targets acute cellular and molecular events during the first week after SCI in rats. We found that DHA pretreatment reduced functional deficits during the acute phase of injury, as shown by significant improvements in Basso-Beattie-Bresnahan (BBB) locomotor scores, and the detection of transcranial magnetic motor evoked potentials (tcMMEPs) compared to vehicle-pretreated animals. We demonstrated that, at 7 days post-injury, DHA pretreatment significantly increased the percentage of white matter sparing, and resulted in axonal preservation, compared to the vehicle injections. We found a significant increase in the survival of NG2+, APC+, and NeuN+ cells in the ventrolateral funiculus (VLF), dorsal corticospinal tract (dCST), and ventral horns, respectively. Interestingly, these DHA protective effects were observed despite the lack of inhibition of inflammatory markers for monocytes/macrophages and astrocytes, ED1/OX42 and GFAP, respectively. DHA pretreatment induced levels of Akt and cyclic AMP responsive element binding protein (CREB) mRNA and protein. This study shows for the first time that DHA pretreatment ameliorates functional deficits, and increases tissue sparing and precursor cell survival. Further, our data suggest that DHA-mediated activation of pro-survival/anti-apoptotic pathways may be independent of its anti-inflammatory effects.
Topics: Animals; Cell Death; Cyclic AMP Response Element-Binding Protein; Docosahexaenoic Acids; Evoked Potentials, Motor; Female; Fluorescent Antibody Technique; Hindlimb; In Situ Nick-End Labeling; Inflammation; Injections, Intravenous; Locomotion; Macrophage Activation; Microglia; Myelin Sheath; Neural Conduction; Neural Stem Cells; Neuroprotective Agents; Oncogene Protein v-akt; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Spinal Cord Injuries
PubMed: 21970623
DOI: 10.1089/neu.2011.2141 -
Brain Structure & Function Apr 2012We studied the organization and spinal projection of the mouse red nucleus with a range of techniques (Nissl stain, immunofluorescence, retrograde tracer injections into...
We studied the organization and spinal projection of the mouse red nucleus with a range of techniques (Nissl stain, immunofluorescence, retrograde tracer injections into the spinal cord, anterograde tracer injections into the red nucleus, and in situ hybridization) and counted the number of neurons in the red nucleus (3,200.9 ± 230.8). We found that the rubrospinal neurons were mainly located in the parvicellular region of the red nucleus, more lateral in the rostral part and more medial in the caudal part. Labeled neurons were least common in the rostral and caudal most parts of the red nucleus. Neurons projecting to the cervical cord were predominantly dorsomedially placed and neurons projecting to the lumbar cord were predominantly ventrolaterally placed. Immunofluorescence staining with SMI-32 antibody showed that ~60% of SMI-32-positive neurons were cervical cord-projecting neurons and 24% were lumbar cord-projecting neurons. SMI-32-positive neurons were mainly located in the caudomedial part of the red nucleus. A study of vGluT2 expression showed that the number and location of glutamatergic neurons matched with those of the rubrospinal neurons. In the anterograde tracing experiments, rubrospinal fibers travelled in the dorsal portion of the lateral funiculus, between the lateral spinal nucleus and the calretinin-positive fibers of the lateral funiculus. Rubrospinal fibers terminated in contralateral laminae 5, 6, and the dorsal part of lamina 7 at all spinal cord levels. A few fibers could be seen next to the neurons in the dorsolateral part of lamina 9 at levels of C8-T1 (hand motor neurons) and L5-L6 (foot motor neurons), which is consistent with a view that rubrospinal fibers may play a role in distal limb movement in rodents.
Topics: Animals; Calbindin 2; Cell Count; Cell Surface Extensions; Fluorescent Antibody Technique; Mice; Mice, Inbred C57BL; Motor Neurons; Nerve Fibers; Red Nucleus; S100 Calcium Binding Protein G; Spinal Cord
PubMed: 21927901
DOI: 10.1007/s00429-011-0348-3 -
Neuroscience Sep 2011Progesterone treatment of mice with experimental autoimmune encephalomyelitis has shown beneficial effects in the spinal cord according to enhanced clinical, myelin and...
Progesterone treatment of mice with experimental autoimmune encephalomyelitis has shown beneficial effects in the spinal cord according to enhanced clinical, myelin and neuronal-related parameters. In the present work, we report progesterone effects in a model of primary demyelination induced by the intraspinal injection of lysophospatidylcholine (LPC). C57Bl6 adult male mice remained steroid-untreated or received a single 100 mg progesterone implant, which increased circulating steroid levels to those of mouse pregnancy. Seven days afterwards mice received a single injection of 1% LPC into the dorsal funiculus of the spinal cord. A week after, anesthetized mice were perfused and paraffin embedded sections of the spinal cord stained for total myelin using Luxol Fast Blue (LFB) histochemistry, for myelin basic protein (MBP) immunohistochemistry and for determination of OX-42+ microglia/macrophages. Cryostat sections were also prepared and stained for oligodendrocyte precursors (NG2+ cells) and mature oligodendrocytes (CC1+ cells). A third batch of spinal cords was prepared for analysis of the microglial marker CD11b mRNA using qPCR. Results showed that progesterone pretreatment of LPC-injected mice decreased by 50% the area of demyelination, evaluated by either LFB staining or MBP immunostaining, increased the density of NG2+ cells and of mature, CC1+ oligodendrocytes and decreased the number of OX-42+ cells, respect of steroid-untreated LPC mice. CD11b mRNA was hyperexpressed in LPC-treated mice, but significantly reduced in LPC-mice receiving progesterone. These results indicated that progesterone antagonized LPC injury, an effect involving (a) increased myelination; (b) stimulation of oligodendrocyte precursors and mature oligodendrocytes, and (c) attenuation of the microglial/macrophage response. Thus, use of a focal demyelination model suggests that progesterone exerts promyelinating and anti-inflammatory effects at the spinal cord level.
Topics: Animals; Demyelinating Diseases; Disease Models, Animal; Immunohistochemistry; Lysophosphatidylcholines; Male; Mice; Mice, Inbred C57BL; Microglia; Progesterone; Spinal Cord
PubMed: 21736923
DOI: 10.1016/j.neuroscience.2011.06.065 -
Neurotoxicology Dec 2011Epidural injection of cyclooxygenase-2 inhibitors has been suggested as a useful therapeutic modality in pain management in animal studies and clinical settings. Direct...
Epidural injection of cyclooxygenase-2 inhibitors has been suggested as a useful therapeutic modality in pain management in animal studies and clinical settings. Direct epidural administration of parecoxib, a highly selective cyclooxygenase-2 inhibitor, may have advantages over its parenteral administration regarding required dose, side effects, and efficacy. However, no animal studies have been performed to investigate the possible neurotoxicity of epidurally injected parecoxib. Therefore, the present study was performed to assess the neurotoxicity of epidurally injected parecoxib in rats. Rats (n=45) were randomly divided into three groups: normal saline group (group N, n=15), ethanol group (group E, n=15), and parecoxib group (group P, n=15). 0.3 mL of epidural parecoxib (6 mg) and the same volume of epidural ethanol or normal saline were injected into the epidural space. Neurologic assessment was performed 3, 7 and 21 days after the injection by pinch toe testing. Histologic changes were evaluated for vacuolation of the dorsal funiculus, chromatolytic changes of the motor neurons, neuritis, and meningeal inflammation. All rats in groups N and P showed normal response to pinch-toe testing and had a normal gait at each observation point. Histological examination showed no evidence suggestive of neuronal body or axonal lesions, gliosis, or myelin sheet damage in group N or P at any time. However, all rats in group E showed sensory-motor dysfunction, behavioral change, or histopathological abnormalities. No neurotoxicity on the spinal cord or abnormalities in sensorimotor function or behavior was noted in rats that received epidural parecoxib.
Topics: Animals; Cyclooxygenase 2 Inhibitors; Ethanol; Gait; Injections, Epidural; Isoxazoles; Male; Neurologic Examination; Neurotoxicity Syndromes; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Risk Assessment; Spinal Cord; Time Factors
PubMed: 21669221
DOI: 10.1016/j.neuro.2011.05.011 -
Neuroscience Research Sep 2011Spina bifida aperta (SBA) is an open neural tube defect that occurs during the embryonic period. We created SBA chicks by incising the roof plate of the neural tube in...
Spina bifida aperta (SBA) is an open neural tube defect that occurs during the embryonic period. We created SBA chicks by incising the roof plate of the neural tube in the embryo. The area of the dorsal funiculus was smaller in the SBA chicks than in the normal controls. Additionally, the SBA group had fewer nerve fibres in the dorsal funiculus than the normal controls. The pathway of the ascending sensory nerves was revealed by tracing the degenerated nerve fibres using osmification. We cut the sciatic nerve (L5) of the control and SBA chicks at the central end of the dorsal root ganglion 1 day after hatching and fixed the tissue 3 days later. Degenerated sensory nerve fibres were observed in the ipsilateral dorsal funiculus in the control chicks. In contrast, degenerated sensory nerve fibres were observed in the ipsilateral and contralateral dorsal, ventral and lateral funiculi of the spinal cord in the SBA chicks. Consequently, fewer sensory nerve fibres ascended to the thoracic dorsal funiculus in the SBA chicks than in the normal controls. This is the first report of abnormal changes in the ascending sensory nerve fibres in SBA.
Topics: Afferent Pathways; Animals; Axons; Chick Embryo; Chickens; Disease Models, Animal; Gait Disorders, Neurologic; Growth Cones; Hindlimb; Rhizotomy; Sensory Receptor Cells; Spina Bifida Cystica; Spinal Cord; Spinal Nerve Roots; Wallerian Degeneration
PubMed: 21658418
DOI: 10.1016/j.neures.2011.05.017 -
PloS One 2011Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we...
Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.
Topics: Animals; Axons; Female; GTPase-Activating Proteins; Immunohistochemistry; Immunoprecipitation; In Situ Hybridization; Mice; Mice, Knockout; Nerve Tissue Proteins; Pregnancy; Protein Binding; Receptors, Immunologic; Spinal Cord; Roundabout Proteins
PubMed: 21655271
DOI: 10.1371/journal.pone.0019887 -
The Journal of Comparative Neurology Oct 2011Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent...
Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent study reported an approximately 20% loss of retrogradely labeled cortical motoneurons following damage to their axons resulting from SCI at T9 (Hains et al. [2003] J. Comp. Neurol. 462:328-341). In follow-up studies, however, we failed to find any evidence of loss of CST axons in the medullary pyramid, which must occur if CST neurons die. Here, we seek to resolve the discrepancy by re-evaluating possible loss of CST neurons using the same techniques as Hains et al. (quantitative analysis of retrograde labeling and staining for cell death markers including TUNEL and Hoechst labeling of the nuclei). Following either dorsal funiculus lesions at thoracic level 9 (T9) or lateral hemisection at cervical level 5 (C5), our results reveal no evidence for a loss of retrogradely labeled neurons and no evidence for TUNEL staining of axotomized cortical motoneurons. These results indicate that CST cell bodies do not undergo retrograde cell death following SCI, and therefore targeting such cell death is not a valid therapeutic target.
Topics: Animals; Apoptosis; Cerebral Cortex; Female; In Situ Nick-End Labeling; Motor Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries
PubMed: 21618218
DOI: 10.1002/cne.22661