The changes in expression of three subtypes of TTX sensitive sodium channels in sensory neurons after spinal nerve ligation.

Our previous studies showed that the ectopic discharges in injured sensory neurons and mechanical allodynia that developed after spinal nerve ligation were significantly reduced by application of a low concentration of tetrodotoxin (TTX) to the corresponding dorsal root ganglion (DRG) of the ligated spinal nerve. Based on these data, we hypothesized that expression of TTX-sensitive sodium channels is up-regulated in the injured sensory neurons and that such up-regulation plays an important role in the generation of ectopic discharges and thus pain behaviors in spinal nerve ligated neuropathic rats. To test this hypothesis, the present study examined the changes in three subtypes of TTX-sensitive sodium channels in the DRG after spinal nerve ligation. The changes in the total amount of mRNA for alpha-subunits of sodium channel brain type I (type I), brain type II (type II) and brain type III (type III) were determined by RNase protection assays (RPA). The population of DRG neurons expressing type III sodium channel protein was examined by an immunohistochemical method with antibodies to type III sodium channels. In the normal DRG, the level of mRNA for the type I sodium channel is high while that for type II and type III is very low. After spinal nerve ligation, the expression of type III mRNA was significantly increased at 16-h postoperatively (PO), doubled by 3 days PO and then was maintained at this high level until the end of the experiment (7 days PO). By contrast, the amount of mRNA for type I and type II sodium channels started to decrease at 1 day PO and were reduced to 25-50% of the normal control levels by 7 days after nerve ligation. Neurons showing positive immunostaining for type III sodium channels were rare ( approximately 3.2% of total population) in the normal DRG but increased after nerve ligation to 21% and 15% of the total neuronal population by 1 day and 7 days PO, respectively. Type III immunoreactivity was found preferentially in medium to large sized neurons. Thus the majority of neurons with cell bodies having diameters > or =40 microm became type III-positive after nerve ligation. The data indicate that the increased expression of type III sodium channels in axotomized sensory neurons may be the critical factor for the TTX sensitivity of ectopic discharges in injured sensory neurons and thus the generation of ectopic discharges and neuropathic pain behaviors in spinal nerve ligated rats.     

Low dose of tetrodotoxin reduces neuropathic pain behaviors in an animal model

We hypothesize that the accumulation of tetrodotoxin (TTX) sensitive sodium channels in injured dorsal root ganglion (DRG) neurons plays a critically important role in the generation of ectopic discharges and mechanical allodynia after peripheral nerve injury. Using the segmental spinal nerve (L5) ligation model of neuropathic pain, this hypothesis was tested by examining the effect of TTX on the mechanical sensitivity of the affected hind paw. Various concentrations of TTX were applied topically to the L5 DRG by using chronically implanted polyethylene tubing. The data showed that application of TTX at low doses (12.5–50 nM), which are far less than those needed for blocking action potential conduction, produced a significant elevation of mechanical threshold in the paw for foot withdrawals, a sign of reduced allodynic behaviors. The data suggest that TTX-sensitive subtypes of sodium channels play an important role in maintaining allodynic behaviors in an animal model of neuropathic pain.     

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 na?ve 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).     

Brain extract induces the tetrodotoxin-sensitive action potentials in a rat skeletal muscle cell line (L6)

Tetrodotoxin (TTX) usually blocks the action potential in adult vertebrate innervated skeletal muscle, i.e. the action potential is operated by a TTX-sensitive sodium channel. The L6 cultured myotube, however, has TTX-resistant action potential that is usually found in skeletal muscle following denervation or during fetal and neonatal stages in rat. The TTX-resistivity of the L6 myotubes might be due to a lack of a neurotrophic substance. The present study revealed that brain extract had a trophic substance that is capable of inducing the TTX-sensitive sodium channels in uninnervated cultured L6 myotubes. Seventeen to 18 days old cultures were used and the maximum rate of rise of action potential was measured as an index of the sodium channel density. In control cultures, the maximum rate of rise was about 120 V/s and did not change by TTX application. However, in cultures with the brain extract the value was 142 V/s and reduced to 86 V/s by TTX. The reduced maximum rate of rise by TTX in the latter cultures means that exposure of L6 myotubes to brain extract resulted in an increase in TTX-sensitive sodium channels and a decrease in TTX-resistant sodium channels. Therefore, exposure to a factor in brain extract can elicit development of TTX-sensitive sodium channels in isolated L6 muscle cells that have been grown as a cell line in the absence of neurons for over 10 years.     

A cadmium-sensitive, tetrodotoxin-resistant sodium channel in bullfrog autonomic axons

The major component of the compound action potential of the bullfrog cardiac nerve was not blocked by TTX (10(-5) M) although the conduction velocity of these fibers was decreased by 50%. The TTX-resistant compound action potential was completely blocked by 100-300 microM CdCl2 but not by 5 mM CoCl2 or NiCl2. This cadmium-sensitive action potential was only partially reduced by removal of calcium from the medium but was abolished by replacement of sodium with sucrose, tetramethylammonium or choline. Therefore, in addition to the classical TTX-sensitive sodium channels, the parasympathetic axons of the bullfrog may also utilize a TTX-insensitive, cadmium-sensitive sodium channel.     

TNF-α enhances the currents of voltage gated sodium channels in uninjured dorsal root ganglion neurons following motor nerve injury

The ectopic discharges observed in uninjured dorsal root ganglion (DRG) neurons following various lesions of spinal nerves have been attributed to functional alterations of voltage-gated sodium channels (VGSCs). Such mechanisms may be important for the development of neuropathic pain. However, the pathophysiology underlying the functional modulation of VGSCs following nerve injury is largely unknown. Here, we studied this issue with use of a selective lumbar 5 ventral root transection (L5-VRT) model, in which dorsal root ganglion (DRG) neurons remain intact. We found that the L5-VRT increased the current densities of TTX-sensitive Na channels as well as currents in Nav1.8, but not Nav1.9 channels in uninjured DRG neurons. The thresholds of action potentials decreased and firing rates increased in DRG neurons following L5-VRT. As we found that levels of tumor necrosis factor-alpha (TNF-α) increased in cerebrospinal fluid (CSF) and in DRG tissue after L5-VRT, we tested whether the increased TNF-α might result in the changes in sodium channels. Indeed, recombinant rat TNF (rrTNF) enhanced the current densities of TTX-S and Nav1.8 in cultured DRG neurons dose-dependently. Furthermore, genetic deletion of TNF receptor 1 (TNFR-1) in mice attenuated the mechanical allodynia and prevented the increase in sodium currents in DRG neurons induced by L5-VRT. These data suggest that the increase in sodium currents in uninjured DRG neurons following nerve injury might be mediated by over-production of TNF-α.     

VR1 protein expression increases in undamaged DRG neurons after partial nerve injury

Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron-specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1-immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1-IR of the A-fibre somata increased approximately 3-fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C-fibres. Furthermore, we found that VR1-IR persisted in the transected sciatic nerve proximal to the lesion, despite its down-regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.     

Sodium channels, excitability of primary sensory neurons, and the molecular basis of pain.

Following nerve injury, primary sensory neurons (dorsal root ganglion [DRG] neurons, trigeminal neurons) exhibit a variety of electrophysiological abnormalities, including increased baseline sensitivity and/or hyperexcitability, which can lead to abnormal burst activity that underlies pain, but the molecular basis for these changes has not been fully understood. Over the past several years, it has become clear that nearly a dozen distinct sodium channels are encoded by different genes and that at least six of these (including at least three distinct DRG- and trigeminal neuron-specific sodium channels) are expressed in primary sensory neurons. The deployment of different types of sodium channels in different types of DRG neurons endows them with different physiological properties. Dramatic changes in sodium channel expression, including downregulation of the SNS/PN3 and NaN sodium channel genes and upregulation of previously silent type III sodium channel gene, occur in DRG neurons following axonal transection. These changes in sodium channel gene expression are accompanied by a reduction in tetrodotoxin (TTX)-resistant sodium currents and by the emergence of a TTX-sensitive sodium current which recovers from inactivation (reprimes) four times more rapidly than the channels in normal DRG neurons. These changes in sodium channel expression poise DRG neurons to fire spontaneously or at inappropriately high frequencies. Changes in sodium channel gene expression also occur in experimental models of inflammatory pain. These observations indicate that abnormal sodium channel expression can contribute to the molecular pathophysiology of pain. They further suggest that selective blockade of particular subtypes of sodium channels may provide new, pharmacological approaches to treatment of disease involving hyperexcitability of primary sensory neurons.     

Ectopic discharges and adrenergic sensitivity of sensory neurons after spinal nerve injury

At various times after spinal nerve injury, dorsal root ganglia (DRGs) from injured segments were removed with attached dorsal roots and spinal nerves. In an in vitro recording chamber, spontaneously active units were recorded from teased dorsal root fascicles. Sustained spontaneous activity could first be recorded at 13 h after the ligation, but adrenergic sensitivity did not develop until 24 h after the injury. Almost all recorded activity originated from the DRG. Thus, the DRG is the most common site for ectopic discharge generation after spinal nerve injury and separate mechanisms seem to be involved in the development of ectopic discharges and adrenergic sensitivity.