Elsevier

Brain Research

Volume 1041, Issue 1, 11 April 2005, Pages 38-47
Brain Research

Research report
Glutamate uptake is attenuated in spinal deep dorsal and ventral horn in the rat spinal nerve ligation model

https://doi.org/10.1016/j.brainres.2005.01.088Get rights and content

Abstract

Alteration of glutamatergic (GLU) neurotransmission within the spinal cord contributes to hyperalgesic and allodynic responses following nerve injury. In particular, changes in expression and efficacy of glutamate transporters have been reported. Excitatory, pain transmitting primary afferent neurons utilizing glutamate as an excitatory neurotransmitter project to both superficial (I–II) and deep (III–V) laminae of the dorsal horn. These experiments were designed to examine changes in glutamate uptake occurring concomitantly within the spinal deep dorsal and ventral horn in situ after experimentally induced neuropathic pain. In vivo voltammetry, using microelectrode arrays configured for enzyme-based detection of GLU were employed. Sprague–Dawley rats had either sham surgery or tight ligation of L5 and L6 spinal nerves (SNL). Four to six weeks later, the L4–L6 spinal cord of chloral hydrate-anesthetized animals was exposed, and ceramic-based glutamate microelectrodes equipped with glass micropipettes 50 μm from the recording surfaces were placed stereotaxically at sites within the spinal cord. Pressure ejection of GLU into the ipsilateral L5–L6 spinal cord resulted in a 72% reduction of GLU uptake in SNL rats compared to sham controls in the ipsilateral L5–L6 deep dorsal horn and a 96% reduction in the ventral horn. In contrast, in the same animals, the contralateral L5–L6 or the ipsilateral L4 spinal cord showed no change in glutamate uptake. The data suggest that spinal nerve ligation produced attenuated glutamate uptake activity extending into the deep dorsal and ventral horn. The study suggests that plasticity related to spinal nerve injury produces widespread alteration in glutamate transporter function that may contribute to the pathophysiology of neuropathic pain.

Introduction

Neuropathic pain, a particularly intractable condition, is thought to be mediated by plasticity of multiple systems within the central nervous system [11]. Allodynia, mechanical, and thermal hyperalgesia are commonly experienced by patients diagnosed with neuropathic pain. Central sensitization of the CNS that can lead to the expression of allodynia and hyperalgesia may be due to abnormal signal amplification within the CNS [25], [38]. Neuronal input from thinly myelinated Aδ and unmyelinated C fiber types, as well as myelinated Aβ fibers, can result in central sensitization [20]. In neuropathic pain models, spontaneous electrical impulses are generated along the path of injured peripheral afferent fibers [14], [20]. Evidence implicates this generation of ectopic firing as a causative factor in the development of central sensitization, resulting in allodynia and hyperalgesia [8], [12], [20]. In particular, a series of seminal studies suggest that enhanced Aβ fiber activity is sufficient to induce the enhanced tactile responses seen after nerve injury [14], [20], [28], [39].

Because of involvement of ectopic or enhanced spontaneous activity of primary afferent neurons, investigation of the pathogenesis of neuropathic pain is focused towards neurotransmission activity at the first central synapse within the spinal cord dorsal horn. At this location, primary afferent fibers synapse onto second-order neurons with glutamate being a major excitatory neurotransmitter. Glutamate is toxic when present in interstitial concentrations exceeding 1–3 μM, and is removed from extracellular fluid primarily by cellular uptake [7], [19]. Uptake is accomplished by sodium-dependent high-affinity glutamate transporters in the plasma membranes of both neurons and glia. Three glutamate transport protein subtypes isolated in the spinal cord (GLAST, GLT1, and EAAC1) are characterized as essential to maintain low resting levels of glutamate (<1 μM), and prevent over-stimulation of cells possessing excitatory amino acid receptors. A recent study suggests that following chronic constriction injury (CCI) of the sciatic nerve in the rat, glutamate transport proteins show bi-phasic changes in expression pattern in the superficial dorsal horn [30]. An initial up-regulation (1 and 4 days post CCI) was followed by a significant down-regulation (7 and 14 days post CCI) in glutamate transporter expression in superficial dorsal horn. In the same study, glutamate uptake activity in the dorsal horn was decreased 5 days after CCI. The glutamate transport protein down-regulation is proposed to lead to increased extracellular glutamate levels, which is thought to contribute to central sensitization seen after nerve injury [37].

Uptake activity of glutamate transporters is generally determined by in vitro synaptosomal preparations. These preparations, although informative, mix the chemistry of organelles, cells, and extracellular fluid. In situ determination of glutamate uptake activity potentially provides more meaningful assessment of glutamate transport protein activity within various laminae of the spinal cord. In situ glutamate uptake activity can be determined utilizing the superior temporal and spatial resolution of glutamate microelectrode arrays [3][24]. The present study extends previous work by demonstrating marked decrease in glutamate transport protein activity within deep dorsal horn and the ventral horn in L5–L6 spinal cord, 4–6 weeks after L5–L6 spinal nerve ligation.

Section snippets

Animal model

Experiments were performed on male Sprague–Dawley rats (Harlan, Indianapolis, IN), weighing 250–350 g. These animals were received 6 weeks post-birth and housed in pairs under a 12:12-h light/dark cycle (0600–1800 h light, 1800–0600 h dark), with free access to food and water. They were acclimated for 1 week before sham or L5 and L6 spinal nerve ligation (SNL) surgery [10], [16] was performed. Briefly, using sterile technique, the paraspinal muscles over the L5–S1 spinal processes were removed,

Self-referencing electrodes

To validate the selectivity of the ceramic electrodes for glutamate, site 2 was coated with glutamate oxidase enzyme while site 3 was prepared with the BSA/glutaraldehyde mixture without the enzyme present. Fig. 2B shows that removal of the glutamate oxidase step results in a dramatic reduction of the signal generated after microinjection of 78 μl of glutamate 200 mM from the adjoining micropipette, validating its selectivity for glutamate.

Glutamate dose response

Microejections of 50 mM glutamate into the rat spinal

Principal finding

The principal findings of this study were that spinal nerve ligation produced marked decrease of glutamate uptake in the L5–L6 region ipsilateral of the rat deep dorsal horn and ventral horn 4 to 6 weeks post-surgery. This suggests that events occurring after injuring primary afferent neurons lead to decreased glutamate transporter activity. The present study shows for the first time that attenuated glutamate transporter activity exists in both the dorsal and ventral horn after spinal nerve

Acknowledgments

The authors acknowledge the assistance of Dr. Greg Gerhardt, Peter Huettl, Francois Pomerene, and the staff of the Center for Sensor Technology at the University of Kentucky for manuscript critique and technical assistance.

Supported by USPHS grant AR046056.

References (40)

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    However, the role of GT under painful pathological conditions is less clear. As indicated some pain models seem to be associated with an upregulation of spinal GT (Sung et al., 2003; Yaster et al., 2011) some others show a down-regulation of spinal GT (Binns et al., 2005; Tawfik et al., 2008; Xin et al., 2009) while again others show decrease of glutamate uptake activity without modulation of spinal GT expression (Niederberger et al., 2006; Tao et al., 2005). However, the effect of injury induced changes in GT expression on pain behavior have not been investigated in most of these studies.

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