Elsevier

Brain Research

Volume 900, Issue 1, 4 May 2001, Pages 119-127
Brain Research

Research report
Ion channels associated with the ectopic discharges generated after segmental spinal nerve injury in the rat

https://doi.org/10.1016/S0006-8993(01)02274-0Get rights and content

Abstract

In an attempt to identify important ion channels contributing to the generation of ectopic discharges, the present study examined the effects of ion channel blockers on ectopic discharges of injured sensory neurons after spinal nerve ligation. The main focus of the study was to examine the effect of the sodium channel blocker, tetrodotoxin (TTX), in order to identify important subtype(s) (i.e. TTX-sensitive and TTX-resistant) of sodium channels that are involved in ectopic discharge generation. In addition, the effects of potassium and calcium channel blockers were also tested for comparison with the results of previous studies. The dorsal root ganglion (DRG) of the injured segment was removed along with the dorsal root (DR) and the spinal nerve 7–14 days after spinal nerve ligation in the rat. The tissue was placed in an in-vitro recording chamber consisting of multiple compartments that were independently perfused with 35°C artificial cerebrospinal fluid (ACSF). Single unit recordings were made from teased DR fibers. Once a spontaneously active unit was found and characterized, ACSF containing a channel blocker was perfused to the DRG, the site where almost all ectopic discharges originate after spinal nerve ligation. All the recorded spontaneously active units were found to be Aβ and Aδ fibers (no C fibers were detected). Perfusion of the DRG with a sodium channel blocker (lidocaine) at a dose much less than that required to block conduction of action potentials, significantly inhibited ectopic discharges in all recorded fibers. In addition, ectopic discharges were inhibited by TTX perfused to the DRG at a dose much lower (average of 22.1 nM) than that required to block TTX-resistant subtypes of sodium channels. The data suggest that TTX-sensitive sodium channels are likely to be involved in the generation of ectopic discharges. The present study also confirmed the results of previous studies on the additional potential roles of potassium and calcium channels, thus suggesting that multiple ion channels are likely to be involved in the generation of ectopic discharges.

Introduction

Injured sensory neurons often produce spontaneous discharges at an abnormal site — ectopic discharges. The repetitive and prolonged firing of such abnormal discharges for days and weeks may have important consequences. These include plastic changes in the central nervous system and production of abnormal sensations such as pain [18], [22], [51], [52]. Therefore, studying the mechanism of ectopic discharge generation is important.

The mechanisms of ectopic discharge generation are not clear. One likely mechanism is the up-regulation or mobilization of one or more ion channels after a peripheral nerve injury. Supporting this hypothesis is the fact that applications of various channel blockers to the injury site have been shown to inhibit ectopic discharges. These include blockers for sodium channels [15], [36], [39], [40], [54], potassium channels [27], [36], [54], and various subtypes of calcium channels [53], [54]. The information on sodium channels is of particular interest not only because their blockers inhibit the ectopic discharges, but also because the density of the sodium channels has been shown to be increased [13], [35], [36] and because it has been demonstrated that a new type of sodium channel appears at the injury site [42], [43], [50].

Functionally, sodium channels have been classified by their sensitivity to tetrodotoxin (TTX). Most of the cloned subtypes of sodium channels are referred to as TTX-sensitive since they are blocked by a low dose of TTX (<100 nM). On the other hand, a few (e.g. SNS/PN3 and NaN) subtypes of sodium channels are referred to as TTX-resistant because they require a much higher dose (>1 μM) of TTX to be blocked [26]. Although there seems to be general agreement that changes in sodium channels play an important role in the generation of ectopic discharges after a peripheral nerve injury, it is not clear which specific subtype is critically important nor is it clear whether it belongs to a family of TTX-sensitive or TTX-resistant subtypes.

Using an in-vitro electrophysiological set-up, the present study examined ectopic discharges generated from injured sensory neurons after spinal nerve ligation in the rat. Various ion channel blockers were added to the perfusion solution of the DRG, commonly known as the site where practically all ectopic discharges originate after spinal nerve ligation [33]. The present study examined the sensitivity of ectopic discharges to TTX in an attempt to identify the critically important subtype(s) of sodium channels involved in ectopic discharge generation. In addition, the effects of potassium and calcium channel blockers were also tested to confirm the results of many previous studies.

Section snippets

Experimental animals and surgical operation

Male Sprague–Dawley rats weighing 125–150 g were used in this study. The experimental protocol was approved by the Animal Care and Use Committee of the University of Texas Medical Branch. Under gaseous anesthesia with a mixture of halothane (2% for induction and 0.8% for maintenance) and a 2:1 flow ratio of N2O and O2, spinal nerves were tightly ligated at a site distal to the DRG following a similar approach to the operation used in the spinal nerve ligation (SNL) model of neuropathic pain [28]

The origin of ectopic discharges

The originating site of ectopic discharges was verified on some units. Since several units were frequently recorded from a single preparation, a destructive lesion experiment could not be done in all units. At the end of the experiment, successive resections were made while recording the discharges from the last unit of some preparations. We concluded that the discharges recorded in the dorsal root were originated from the DRG when a unit discharged continuously after a resection at the

Discussion

The present study examined the effects of various ion channel blockers on spontaneous activity originating from an abnormal impulse generating site in injured sensory neurons (i.e. ectopic discharges). Using an in-vitro recording set-up, the ectopic discharges were recorded from single units isolated from the DR of the segments that had been injured by tightly ligating the spinal nerves 7–14 days prior to the recording session. We applied ion channel blockers to the DRG, and the outcome was

Summary

After segmental spinal nerve injury in the rat, ectopic discharges were recorded from the DR fascicles using an in-vitro recording set-up. Various ion channel blockers were applied to the DRG, where most ectopic discharges are known to originate in this preparation. The main focus of this study was to test the sensitivity of ectopic discharges to TTX in order to identify the types of sodium channels that are important for the generation of ectopic discharges. Ectopic discharges were inhibited

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