Sanja D. Novakovic

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Distribution of the Tetrodotoxin-Resistant Sodium Channel PN3 in Rat Sensory Neurons in Normal and Neuropathic Conditions

The novel sodium channel PN3/α-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 TTXRsodium 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.

Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury

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.

Differential distribution of the tetrodotoxin‐sensitive rPN4/NaCh6/Scn8a sodium channel in the nervous system

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). J. Neurosci. Res. 60:37–44, 2000

Regulation of Na+ channel distribution in the nervous system

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.

Sodium channel distribution in axons of hypomyelinated and MAG null mutant mice

Na+ channel organization was studied with immunofluorescence in the peripheral nervous system of mice genetically altered to produce abnormal myelin. In two of these strains, transcription of inserted transgenes was targeted to myelinating Schwann cells through linkage to a promoter for the myelin protein P0. Adults of both of these strains had hindlimb paralysis and a tremor on lifting by the tail. In one case, Schwann cells were eliminated via expression of the diphtheria toxin A chain (DT‐A). During postnatal days 3–7, Na+ channel clustering at forming nodes was dramatically reduced compared with that of normal animals. At 1–3 months of age, Na+ channel immunofluorescence was often found spread over long stretches of the axolemma, instead of being confined to nodal gaps. In the second P0‐linked transgenic model, Schwann cell expression of the large T antigen tsA‐1609 resulted in cell cycle dysfunction. Adult axons had regions of diffuse Na+ channel labeling. Focal clusters were rare within these zones, which were characterized by a series of cells of myelinating phenotype tightly apposed to the axon. Previous studies suggested that Schwann cells had to reach the stage of ensheathment characterized by periaxonal myelin associated glycoprotein (MAG) expression in order to induce Na+ channel clustering. However, in MAG‐deficient mice, Na+ channel labeling patterns within sciatic nerves were normal. J. Neurosci. Res. 50:321–336, 1997.

Disruption and reorganization of sodium channels in experimental allergic neuritis.

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.

Clusters of axonal Na+ channels adjacent to remyelinating Schwann cells

SummaryRat 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 postinjection, 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.

CONTROL OF MYELINATION, AXONAL GROWTH, AND SYNAPSE FORMATION IN SPINAL CORD EXPLANTS BY ION CHANNELS AND ELECTRICAL ACTIVITY

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.