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Fukushima Journal of Medical Science Dec 2004Localization and ultrastructural features of immunoreactive fibers and terminals against RT-97, a mouse monoclonal antibody that recognizes subunit of a 200-kD...
Histological distribution and ultrastructural features of immunoreactive terminals against RT97, a monoclonal antibody to a 200 kD neurofilament, in the spinal dorsal horn of a rat.
Localization and ultrastructural features of immunoreactive fibers and terminals against RT-97, a mouse monoclonal antibody that recognizes subunit of a 200-kD neurofilament, were examined in the spinal dorsal horn of adult rats. Under a light-microscope, many RT-97 immunoreactive fibers were detected in the dorsal root, collaterals of the dorsal root in the dorsal funiculus, and laminae III and IV in the dorsal horn. Few immunoreactive fibers were found in laminae I and II. Electron microscopic observation demonstrated that almost all RT-97 immunoreactive fibers in the dorsal root were myelinated, and unmyelinated fibers immunonegative. The immunoreactive fibers entered into the dorsal horn passing through the collaterals of the dorsal root along the superficial gray lamina. In the dorsal horn, these fibers ascended into and then terminated in lamina II. RT-97 immunoreactive central terminals were semicircular or ellipsoid in appearance and contained many flat-type presynaptic vesicles. Some terminals made synaptic contact with dendritic profiles in lamina II. Our present results indicate that RT-97 is a useful marker for ultrastructural examination of terminals served by non-nociceptive A-fibers.
Topics: Animals; Antibodies, Monoclonal; Immunohistochemistry; Male; Mice; Microscopy, Immunoelectron; Nerve Fibers; Neurofilament Proteins; Posterior Horn Cells; Presynaptic Terminals; Rats; Rats, Wistar
PubMed: 15779572
DOI: 10.5387/fms.50.65 -
The Journal of Neuroscience : the... Sep 2004Olfactory ensheathing cells (OECs) prepared from the olfactory bulbs of adult transgenic Sprague Dawley (SD) rats expressing green fluorescent protein (GFP) were...
Olfactory ensheathing cells (OECs) prepared from the olfactory bulbs of adult transgenic Sprague Dawley (SD) rats expressing green fluorescent protein (GFP) were transplanted into a dorsal spinal cord transection lesion of SD rats. Five weeks after transplantation, the cells survived within the lesion zone and oriented longitudinally along axons that bridged the transection site. Although the highest density of GFP cells was within the lesion zone, some cells distributed longitudinally outside of the lesion area. Myelinated axons spanning the lesion were observed in discrete bundles encapsulated by a cellular element. Electron micrographs of spinal cords immunostained with an anti-GFP antibody indicated that a majority of the peripheral-like myelinated axons were derived from donor OECs. Open-field locomotor behavior was significantly improved in the OEC transplantation group. Thus, transplanted OECs derived from the adult olfactory bulb can survive and orient longitudinally across a spinal cord transection site and form myelin. This pattern of repair is associated with improved locomotion.
Topics: Animals; Animals, Genetically Modified; Axons; Female; Green Fluorescent Proteins; Locomotion; Microscopy, Immunoelectron; Myelin Sheath; Neuroglia; Olfactory Bulb; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Spinal Cord; Spinal Cord Injuries
PubMed: 15456822
DOI: 10.1523/JNEUROSCI.1998-04.2004 -
Journal of Neurophysiology Feb 2005Selective ablation of spinal neurons possessing substance P receptors (NK-1 receptors) using the selective cytotoxin conjugate substance P-saporin (SP-SAP) decreases... (Comparative Study)
Comparative Study
Selective ablation of spinal neurons possessing substance P receptors (NK-1 receptors) using the selective cytotoxin conjugate substance P-saporin (SP-SAP) decreases hyperalgesia and central sensitization. The mechanisms by which NK-1 expressing neurons modulate the excitability of other dorsal horn neurons are unclear. Because the majority of NK-1 expressing spinal neurons project rostrally, it is possible that they are part of a spinal-supraspinal circuitry that contributes to descending modulation of excitability of spinal nociceptive neurons. We therefore determined whether ablation of spinal neurons that possess the NK-1 receptor altered descending systems that travel via the dorsolateral funiculus (DLF). Spontaneous activity and responses of dorsal horn neurons evoked by mechanical (von Frey monofilaments) and heat (35-51 degrees C) stimuli were determined before and after transection of the DLF and were compared in rats pretreated with intrathecal application of vehicle or SP-SAP. In vehicle-treated rats, transection of the DLF caused a 233% increase in mean spontaneous activity of neurons and enhanced their responses to mechanical and heat stimuli, whereas these increases in excitation were blocked in rats pretreated with SP-SAP. Importantly, SP-SAP alone had no effect on spontaneous or evoked activity in the absence of DLF transection. These results demonstrate that spinal neurons expressing the NK-1 receptor appear to play a pivotal role in regulating descending systems that modulate activity of nociceptive dorsal horn neurons.
Topics: Animals; Gene Expression Regulation; Immunotoxins; Male; Medulla Oblongata; Posterior Horn Cells; Pyramidal Tracts; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Ribosome Inactivating Proteins, Type 1; Saporins; Substance P
PubMed: 15456795
DOI: 10.1152/jn.01160.2003 -
Journal of Neurophysiology Oct 2004N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip...
N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.
Topics: Animals; Animals, Newborn; Critical Period, Psychological; Electrophysiology; Genetic Vectors; Immunohistochemistry; In Vitro Techniques; Magnesium; Motor Neurons; Neurotrophin 3; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; Simplexvirus; Spinal Cord; Synapses
PubMed: 15152019
DOI: 10.1152/jn.00278.2004 -
The Journal of Neuroscience : the... Nov 2003The normal development of the somatosensory system requires intact sensory inputs from the periphery during a critical window of time early in development. Here we...
The normal development of the somatosensory system requires intact sensory inputs from the periphery during a critical window of time early in development. Here we determined how the removal of only part of the ascending spinal inputs early in development affects the anatomical and neurophysiological development of the somatosensory system. We performed spinal overhemisections in rat pups at C3/C4 levels on the third day after birth. This procedure hemisects the spinal cord on one side and transects the dorsal funiculus on the other side. When the rats were 6-8 months old, the responsiveness and somatotopy of the primary somatosensory cortex (S1) contralateral to the hemisection were determined using standard multiunit mapping techniques. Sections of the flattened cortex were processed for cytochrome oxidase activity, Nissl substance, or myelin. We found that histologically apparent modules that are normally present in the regions of the forepaw and the hindpaw representations were absent, whereas the lateral barrel field representing the face was completely normal. The neurons in the forepaw regions of S1 either did not respond to the stimulation of the skin of any region of the body or responded to the stimulation of the upper arm afferents that enter the spinal cord rostral to the site of the lesion. The results show that a lack of normal sensory inputs via ascending pathways in the dorsal spinal cord during early development results in massive anatomical and neurophysiological abnormalities in the cortex. Intact crossed spinothalamic pathways are unable to support the normal development of the forepaw barrels.
Topics: Afferent Pathways; Animals; Animals, Newborn; Brain Mapping; Brain Stem; Critical Period, Psychological; Female; Forelimb; Hindlimb; Male; Motor Activity; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Somatosensory Cortex; Spinal Cord; Spinal Cord Injuries
PubMed: 14614091
DOI: 10.1523/JNEUROSCI.23-32-10321.2003 -
The Journal of Neuroscience : the... Oct 2003To determine what neural pathways trigger opioid release in the dorsal horn, we stimulated the dorsal root, the dorsal horn, or the dorsolateral funiculus (DLF) in...
Dorsal horn neurons firing at high frequency, but not primary afferents, release opioid peptides that produce micro-opioid receptor internalization in the rat spinal cord.
To determine what neural pathways trigger opioid release in the dorsal horn, we stimulated the dorsal root, the dorsal horn, or the dorsolateral funiculus (DLF) in spinal cord slices while superfusing them with peptidase inhibitors to prevent opioid degradation. Internalization of mu-opioid receptors (MOR) and neurokinin 1 receptors (NK1R) was measured to assess opioid and neurokinin release, respectively. Dorsal root stimulation at low, high, or mixed frequencies produced abundant NK1R internalization but no MOR internalization, indicating that primary afferents do not release opioids. Moreover, capsaicin and NMDA also failed to produce MOR internalization. In contrast, dorsal horn stimulation elicited MOR internalization that increased with the frequency, being negligible at <10 Hz and maximal at 500 Hz. The internalization was abolished by the MOR antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), in the presence of low Ca2+ and by the Na+ channel blocker lidocaine, confirming that it was caused by opioid release and neuronal firing. DLF stimulation in "oblique" slices (encompassing the DLF and the dorsal horn of T11-L4) produced MOR internalization, but only in areas near the stimulation site. Moreover, cutting oblique slices across the dorsal horn (but not across the DLF) eliminated MOR internalization in areas distal to the cut, indicating that it was produced by signals traveling in the dorsal horn and not via the DLF. These findings demonstrate that some dorsal horn neurons release opioids when they fire at high frequencies, perhaps by integrating signals from the rostral ventromedial medulla, primary afferents, and other areas of the spinal cord.
Topics: Anesthetics, Local; Animals; Calcium; Capsaicin; Electric Stimulation; Enzyme Inhibitors; GABA Antagonists; Glycine Agents; In Vitro Techniques; N-Methylaspartate; Neural Pathways; Neurons, Afferent; Opioid Peptides; Peptide Fragments; Peptides; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin; Spinal Cord; Spinal Nerve Roots
PubMed: 14534251
DOI: 10.1523/JNEUROSCI.23-27-09171.2003 -
Mechanisms of Development Dec 2002Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including...
Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.
Topics: Animals; Brain; Central Nervous System; Gene Expression Regulation, Developmental; Mice; Receptor Protein-Tyrosine Kinases; Receptor, EphA4; Spinal Cord
PubMed: 14516691
DOI: 10.1016/s0925-4773(03)00122-9 -
Glia Nov 2003The remyelinating potential of autologous bone marrow cells was studied after direct injection and following intravenous injection into rats with a demyelinated lesion... (Comparative Study)
Comparative Study
The remyelinating potential of autologous bone marrow cells was studied after direct injection and following intravenous injection into rats with a demyelinated lesion in the spinal cord. Both focal and intravenous injections of acutely isolated mononuclear bone marrow cell fractions resulted in varying degrees of remyelination. Suspensions of bone marrow cells collected from the same rat were delivered at varied concentrations (10(2) to 10(5) for direct injection and 10(4) to 10(7) for i.v. injections). The lesions were examined histologically 3 weeks after transplantation. Light microscopic examination revealed remyelination in the dorsal funiculus with both injection protocols, but the extent of remyelination was proportional to the number of injected cells. To attain the same relative density of remyelination achieved by direct injection, intravenous administration of cells required delivery of substantially more cells (two orders of magnitude). However, the availability of autologous bone marrow cells in large number and the potential for systemically delivering cells to target lesion areas without neurosurgical intervention suggest the potential utility of intravenous cell delivery as a prospective therapeutic approach in demyelinating disease.
Topics: Animals; Bone Marrow Transplantation; Cell Transplantation; Demyelinating Diseases; Female; Injections, Intravenous; Nerve Fibers, Myelinated; Rats; Rats, Wistar; Spinal Cord; Transplantation, Autologous
PubMed: 14515327
DOI: 10.1002/glia.10285 -
The Journal of Neuroscience : the... Sep 2003We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecally into neonatal rats, can facilitate synaptic transmission in the...
We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecally into neonatal rats, can facilitate synaptic transmission in the spinal cord. A small collagen plug containing NT-3-secreting fibroblasts was placed on the exposed dorsal surface of the spinal cord (L1) of 2-d-old rats; controls received beta-galactosidase-secreting fibroblasts. After 6 hr to 12 d of survival, synaptic potentials (EPSP) elicited by two synaptic inputs, L5 dorsal root and ventrolateral funiculus (VLF), were recorded intracellularly in L5 motoneurons in vitro. Preparations treated with NT-3 implants exhibited enhanced monosynaptic synaptic transmission from both inputs, which persisted over the entire testing period. Unlike acute enhancement of transmission by NT-3 (Arvanian and Mendell, 2001a), the chronic effect could occur at connections not normally eliciting an NMDA receptor-mediated response at the time of NT-3 exposure. Using susceptibility to blockade of the NMDA receptor by Mg2+ and APV, we confirmed that chronic treatment with NT-3 did not enhance NMDA receptor activity at these connections. Cords treated with chronic NT-3 also transiently displayed polysynaptic components activated by VLF that were blocked by the NMDA receptor antagonist APV. These novel NMDA receptor-mediated potentials may reflect changes in interneurons near the site of fibroblast implantation. We conclude that chronic NT-3 enhances the potency of segmental and descending projections via mechanisms different from those underlying acute changes.
Topics: Animals; Animals, Newborn; Cells, Cultured; Collagen; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fibroblasts; In Vitro Techniques; Lumbosacral Region; Magnesium; Motor Neurons; Neurotrophin 3; Rats; Rats, Sprague-Dawley; Spinal Cord; Synapses; Synaptic Transmission; Time; beta-Galactosidase
PubMed: 14507970
DOI: 10.1523/JNEUROSCI.23-25-08706.2003 -
The Kaohsiung Journal of Medical... Apr 2003Increasing evidence implies the existence of a visceral pain pathway in the dorsal column of the spinal cord. Limited midline myelotomy has been used to treat...
Increasing evidence implies the existence of a visceral pain pathway in the dorsal column of the spinal cord. Limited midline myelotomy has been used to treat intractable pelvic cancer pain. However, no obvious evidence has been provided that high cervical punctate midline myelotomy (CPMM) relieves visceral pain originating from the abdomen. This study was designed to examine the pain relief effect of CPMM in a mouse model of visceral pain. Thirty-six Institute of Cancer Research (ICR) mice were divided into three groups: Group 1, healthy controls; Group 2, treated with CPMM at C1 and C2; and Group 3, a sham group that underwent laminectomy at C1 and C2 without CPMM. All animals were tested for antinociception in the writhing test 24 hours after surgery. Visceral pain-related behaviors were counted from 5-20 minutes after intraperitoneal injection of 0.6% acetic acid. Writhing test scores were not significantly different between Groups 1 (56.7 +/- 10.7) and 3 (50.7 +/- 17.4). However, Group 2 (30.0 +/- 14.3) showed more than 40% antinociception after treatment, and writhing test scores were significantly different from those of Groups 1 and 3 (p < 0.001). Our results confirm that midline punctate myelotomy can relieve visceral pain and imply that there is a pathway in the posterior funiculus that signals visceral pain. Punctate midline myelotomy at the cervical or high thoracic level may be an alternative strategy in the management of intractable visceral pain due to abdominal or pelvic cancers.
Topics: Animals; Cervical Vertebrae; Cordotomy; Male; Mice; Mice, Inbred ICR; Pain, Intractable; Pelvic Pain; Spinal Cord; Viscera
PubMed: 12795344
DOI: 10.1016/S1607-551X(09)70465-6