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1.
J Neurosci ; 21(21): 8690-6, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11606657

ABSTRACT

The present study investigated the effect of inhibiting the expression of Na(v)1.8 (PN3/SNS) sodium channels by an antisense oligodeoxynucleotide (ODN) on bladder nociceptive responses induced by intravesical acetic acid infusion in rats. Animals were injected intrathecally with either Na(v)1.8 antisense or mismatch ODN. Control cystometrograms under urethane anesthesia during intravesical saline infusion exhibited intercontraction intervals (ICIs) that were significantly longer in antisense-treated rats than in mismatch ODN-treated rats. Intravesical infusion of 0.1% acetic acid induced bladder hyperactivity as reflected by a 68% reduction in ICIs in mismatch ODN-treated rats but did not significantly reduce ICIs in antisense-treated rats. The number of Fos-positive cells after acetic acid administration were significantly reduced in the L6 spinal cord from antisense-treated animals, compared with mismatch ODN-treated animals. In addition, Na(v)1.8 immunoreactivity was reduced in L6 dorsal root ganglion neurons in the antisense-treated rat. In patch-clamp recordings, the conductance density of TTX-resistant sodium currents in dissociated bladder afferent neurons that were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall was also smaller in antisense-treated rats than in mismatch ODN-treated rats, whereas no changes were observed in TTX-sensitive currents. These results indicate that the Na(v)1.8 TTX-resistant sodium channels are involved in the activation of afferent nerves after chemical irritation of the bladder. These channels represent a new target for the treatment of inflammatory pain from visceral organs such as the urinary bladder.


Subject(s)
Neuropeptides/metabolism , Pain/physiopathology , Sodium Channels/metabolism , Acetic Acid , Administration, Intravesical , Animals , Disease Models, Animal , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiopathology , Injections, Spinal , NAV1.8 Voltage-Gated Sodium Channel , Neurons, Afferent/drug effects , Neurons, Afferent/metabolism , Neuropeptides/antagonists & inhibitors , Neuropeptides/drug effects , Oligonucleotides, Antisense/administration & dosage , Pain/chemically induced , Pain Measurement , Patch-Clamp Techniques , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, Sprague-Dawley , Sodium/metabolism , Sodium Channel Blockers , Sodium Channels/drug effects , Spinal Cord/drug effects , Spinal Cord/physiopathology , Tetrodotoxin/pharmacology , Urinary Bladder/drug effects , Urinary Bladder/innervation , Urinary Bladder/physiopathology , Visceral Afferents/drug effects , Visceral Afferents/metabolism
2.
Trends Neurosci ; 24(8): 473-8, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11476887

ABSTRACT

An important aspect of Na+ channel regulation is their distribution on neuronal membranes within the nervous system. The complexity of this process is brought by the molecular diversity of Na+ channels and differential regulation of their distribution. In addition, Na+ channel localization is a highly dynamic process depending on the status of the cell in vitro, and (patho)physiological condition of the organism in vivo. Nonetheless, the pharmacological manipulation of Na+ channel distribution should be possible and will hopefully bring safer and more-potent medicines in the future.


Subject(s)
Brain Chemistry/physiology , Nervous System/metabolism , Sodium Channels/analysis , Sodium Channels/metabolism , Animals
3.
J Neurosci Res ; 60(1): 37-44, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10723066

ABSTRACT

Voltage-gated sodium channels underlie the generation of action potentials in excitable cells. Various sodium channel isoforms have been cloned, functionally expressed and distinguished on the basis of their biophysical properties or differential sensitivity to tetrodotoxin (TTX). In the present study, we have investigated the immunolocalization of the TTX-sensitive sodium channel, rPN4/NaCh6/Scn8a, in discrete areas of the rat nervous system. Thus, in naïve animals, PN4 was abundantly expressed in brain, spinal cord, dorsal root ganglia (DRG) and peripheral nerve. The presence of PN4 at the nodes of Ranvier in the sciatic nerve suggests the importance of this sodium channel in peripheral nerve conduction. In addition, the pattern of PN4 immunolabeling was determined in DRG, spinal cord and sciatic nerve in rats subjected to chronic constriction nerve injury (CCI).


Subject(s)
Brain/metabolism , Ganglia, Spinal/metabolism , Nerve Tissue Proteins/metabolism , Sciatic Nerve/metabolism , Sodium Channels/metabolism , Spinal Cord/metabolism , Animals , Constriction, Pathologic , Immunohistochemistry , In Situ Hybridization , Ion Channel Gating , Male , Organ Specificity , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Sciatic Nerve/pathology
4.
Pain ; 80(1-2): 273-82, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10204740

ABSTRACT

P2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.


Subject(s)
Neurons, Afferent/metabolism , Neurons, Afferent/ultrastructure , Peripheral Nervous System Diseases/metabolism , Receptors, Purinergic P2/metabolism , Animals , Blotting, Western , Cells, Cultured , Constriction, Pathologic/pathology , Ganglia, Spinal/pathology , Immunohistochemistry , Male , Neurons, Afferent/pathology , Peripheral Nervous System Diseases/pathology , Rabbits , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2X3 , Sciatic Nerve/pathology , Spinal Cord/pathology , Spinal Cord/ultrastructure
5.
Muscle Nerve ; 21(8): 1019-32, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9655120

ABSTRACT

The axonal distribution of voltage-dependent Na+ channels was determined during inflammatory demyelinating disease of the peripheral nervous system. Experimental allergic neuritis was induced in Lewis rats by active immunization. In diseased spinal roots Na+ channel immunofluorescence at many nodes of Ranvier changed from a highly focal ring to a more diffuse pattern and, as the disease progressed, eventually became undetectable. The loss of nodal channels corresponded closely with the development of clinical signs. Electrophysiological measurements and computations showed that a lateral spread of nodal Na+ channels could contribute significantly to temperature sensitivity and conduction block. During recovery new clusters of Na+ channels were seen. In fibers with large-scale demyelination, the new aggregates formed at the edges of adhering Schwann cells and appeared to fuse to form new nodes. At nodes with demyelination limited to paranodal retraction, Na+ channels were often found divided into two symmetric highly focal clusters. These results suggest that reorganization of Na+ channels plays an important role in the pathogenesis of demyelinating neuropathies.


Subject(s)
Axons/chemistry , Demyelinating Diseases/physiopathology , Neuritis, Autoimmune, Experimental/physiopathology , Sodium Channels/physiology , Animals , Axons/physiology , Electrophysiology , Female , Ion Channel Gating/physiology , Neural Conduction/physiology , Ranvier's Nodes/chemistry , Ranvier's Nodes/physiology , Rats , Rats, Inbred Lew , Schwann Cells/physiology , Spinal Nerve Roots/chemistry , Spinal Nerve Roots/cytology , Spinal Nerve Roots/physiopathology
6.
J Neurosci ; 18(6): 2174-87, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9482802

ABSTRACT

The novel sodium channel PN3/alpha-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXR sodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.


Subject(s)
Nervous System Diseases/metabolism , Neurons, Afferent/metabolism , Sodium Channels/drug effects , Sodium Channels/metabolism , Tetrodotoxin/pharmacology , Animals , Biological Transport/physiology , Drug Resistance , Immunohistochemistry , Male , Nerve Compression Syndromes/metabolism , Nervous System Diseases/pathology , Neuroma/metabolism , Neuroma/pathology , Patch-Clamp Techniques , Peripheral Nervous System Neoplasms/metabolism , Peripheral Nervous System Neoplasms/pathology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reference Values , Sciatic Nerve/injuries , Sciatic Nerve/metabolism , Sciatic Nerve/pathology , Sodium Channels/genetics , Tissue Distribution
8.
J Neurosci ; 16(16): 4914-22, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8756423

ABSTRACT

The distribution of Na+ channels in rat peripheral nerve was measured during development by using immunofluorescence. Small segments of sciatic nerve from postnatal day 0-13 (P0-P13) pups were labeled with an antibody raised against a well conserved region of the vertebrate Na+ channel. At day P0 axons contained almost no Na+ channel aggregates. The number of clusters increased dramatically throughout the first week. In almost all cases Na+ channels clustered in the vicinity of Schwann cell processes. At least four classes of aggregates were noted. Clusters formed singly at Schwann cell edges, in pairs or in broad regions between neighboring Schwann cells, and in more focal zones at presumptive nodes. Almost all Na+ channel aggregates had reached the latter stage by the end of the first week. Histograms plotting the frequency of occurrence of each cluster type suggested a sequence of events in node formation involving the initiation of channel aggregation by Schwann cell processes. The requirement for Schwann cells during sodium channel clustering was tested by blocking proliferation of these cells with the antimitotic agent mitomycin C. Na+ channel clustering was sharply reduced, whereas node formation was normal at a distal site along the same nerve. Immunocytochemical detection of myelin-associated glycoprotein (MAG) indicated that Schwann cells must begin to ensheathe axons before inducing Na+ channel clustering.


Subject(s)
Aging/physiology , Axons/metabolism , Peripheral Nerves/growth & development , Peripheral Nerves/metabolism , Sodium Channels/physiology , Animals , Animals, Newborn/metabolism , Cell Division/drug effects , Mitomycin/pharmacology , Myelin-Associated Glycoprotein/metabolism , Rats , Schwann Cells/cytology
9.
J Neurocytol ; 25(6): 403-12, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8835788

ABSTRACT

Rat sciatic nerve fibres were demyelinated by injection of lysolecithin and examined at several stages as Schwann cells proliferated, adhered, and initiated remyelination. Immunoperoxidase EM has been used to follow the clustering of Na+ channels that represents an early step in the formation of new nodes of Ranvier. At the peak of demyelination, 1 week post-injection, only isolated sites, suggestive of the original nodes, were labelled. As Schwann cells adhered and extended processes along the axons, regions of axonal Na+ channel immunoreactivity were often found just beyond their leading edges. These channel aggregates were associated only with the axolemma and Na+ channels were not detected on glial membranes. Sites with more than one cluster in close proximity and broadly labelled aggregates between Schwann cells suggested that new nodes of Ranvier formed as neighbouring Na+ channel groups merged. Schwann cells thus seem to play a major role in ion channel distributions in the axolemma. In all of these stages Na+ channel label was found primarily just outside the region of close contact between axon and Schwann cell. This suggests that Schwann cell adherence acts in part to exclude Na+ channels, or that diffusible substances are involved and can act some distance from regions of direct contact.


Subject(s)
Axons/metabolism , Demyelinating Diseases/pathology , Schwann Cells/physiology , Sodium Channels/metabolism , Amino Acid Sequence , Animals , Cell Adhesion , Demyelinating Diseases/chemically induced , Female , Immunohistochemistry , Lysophosphatidylcholines/pharmacology , Microscopy, Electron , Microscopy, Fluorescence , Molecular Sequence Data , Rats , Rats, Inbred Lew , Schwann Cells/cytology , Sciatic Nerve/drug effects , Sciatic Nerve/pathology
10.
Glia ; 15(2): 188-94, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8567070

ABSTRACT

Mouse sciatic nerves from the degeneration-resistant strain C57BL/6/Wld (Ola) were surgically injected with lysolecithin to induce focal demyelination. Three days later they were transected adjacent to the spinal cord to eliminate contact of the axons with their cell bodies. The Na+ channel distribution was assessed by immunocytochemistry and followed at several stages of remyelination. Control experiments were performed on nerves that were injected but not cut. At (3 + 4) days, namely, nerves cut 3 days post-injection and examined 4 days after cutting, axons contained fully demyelinated regions. Na+ channel clusters appeared only at heminodes and at isolated sites that are likely to represent original nodes of Ranvier. During the next few days proliferating Schwann cells adhered to the axons and extended their processes. Clusters of Na+ channels appeared at their edges, and as the Schwann cells elongated the distance between these aggregates increased. A few clusters associated with neighboring Schwann cells approached each other and appeared to coalesce at sites where presumably new nodes of Ranvier would be formed. Beyond (3 + 6) days excessive degeneration of the transected axons precluded further observations. In the uncut controls, the spatio-temporal sequence of Schwann cell proliferation and channel patch formation and movement was similar to that described above, although myelin formation was somewhat faster than in the cut axons. It is concluded that Na+ channel aggregation associated with the early stages of remyelination is not dependent upon continuous communication of the axon with its cell body and is under local control.


Subject(s)
Axons/physiology , Myelin Sheath/physiology , Sciatic Nerve/metabolism , Sodium Channels/physiology , Amino Acid Sequence , Animals , Cell Survival/physiology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Microscopy, Electron , Molecular Sequence Data , Myelin Sheath/ultrastructure , Ranvier's Nodes/metabolism , Ranvier's Nodes/physiology , Sciatic Nerve/ultrastructure , Wallerian Degeneration/physiology
11.
Brain Res Dev Brain Res ; 88(1): 68-78, 1995 Aug 28.
Article in English | MEDLINE | ID: mdl-7493408

ABSTRACT

The involvement of axonal electrical activity and ion channels as mediators of neuron-glial communication during myelin formation has been tested in explant culture. Transverse slices of embryonic mouse spinal cord were maintained under conditions normally leading to extensive myelination. Axonal conduction was measured optically through the use of a voltage-sensitive dye. Glial development was at a very early stage at the time of plating, and oligodendrocyte precursor cells had not yet appeared. Spontaneous electrical activity was blocked either by tetrodotoxin or by elevation of external K+ concentrations. Myelin development was unaffected by tetrodotoxin and was also present, though quantitatively reduced, in elevated K+. Tetraethylammonium ion (TEA+), a blocker of many K+ channels, almost entirely eliminated myelination at a concentration of 1 mM, but axonal growth and conduction were unaffected. Synapse formation was followed both morphologically and functionally, and was altered neither by conduction block nor by 1 mM TEA+. It is concluded that in the spinal cord oligodendrocyte development and myelination can proceed in the absence of axonal action potentials, but ion channels, possibly in glial membranes, play an important role in these events.


Subject(s)
Axons/physiology , Ion Channels/metabolism , Myelin Sheath/physiology , Spinal Cord/growth & development , Synapses/physiology , Action Potentials/physiology , Animals , Axons/drug effects , Axons/ultrastructure , Electrophysiology , Ion Channels/drug effects , Mice , Myelin Sheath/ultrastructure , Oligodendroglia/metabolism , Oligodendroglia/physiology , Organ Culture Techniques , Potassium Channels/drug effects , Potassium Channels/metabolism , Potassium Channels/physiology , Spinal Cord/drug effects , Spinal Cord/ultrastructure , Synapses/drug effects , Synapses/ultrastructure , Tetraethylammonium Compounds/pharmacology , Tetrodotoxin/pharmacology
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