Elsevier

Brain Research

Volume 895, Issues 1–2, 23 March 2001, Pages 178-185
Brain Research

Research report
Characterization of the glutamatergic system for induction and maintenance of allodynia

https://doi.org/10.1016/S0006-8993(01)02069-8Get rights and content

Abstract

Glutamate is the main excitatory neurotransmitter in the central nervous system and has been shown to be involved in spinal nociceptive processing. We previously demonstrated that intrathecal (i.t.) administration of prostaglandin (PG) E2 and PGF induced touch-evoked pain (allodynia) through the glutamatergic system by different mechanisms. In the present study, we characterized glutamate receptor subtypes and glutamate transporters involved in induction and maintenance of PGE2- and PGF-evoked allodynia. In addition to PGE2 and PGF, N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not kainate, induced allodynia. PGE2- and NMDA-induced allodynia were observed in NMDA receptor ε4 (NR2D) subunit knockout (GluRε4(−/−)) mice, but not in ε1 (NR2A) subunit knockout (GluRε1(−/−)) mice. Conversely, PGF- and AMPA-induced allodynia were observed in GluRε1(−/−) mice, but not in GluRε4(−/−) mice. The induction of allodynia by PGE2 and NMDA was abolished by the NMDA receptor ε2 (NR2B) antagonist CP-101,606 and neonatal capsaicin treatment. PGF- and AMPA-induced allodynia were not affected by CP-101,606 and by neonatal capsaicin treatment. On the other hand, the glutamate transporter blocker dl-threo-β-benzyloxyaspartate (dl-TBOA) blocked all the allodynia induced by PGE2, PGF, NMDA, and AMPA. These results demonstrate that there are two pathways for induction of allodynia mediated by the glutamatergic system and suggest that the glutamate transporter is essential for the induction and maintenance of allodynia.

Introduction

The dorsal horn of the spinal cord is an important site for pain transmission and many substances are involved in the modulation of incoming pain information [47]. Glutamate is the main excitatory neurotransmitter in the central nervous system and mediates fast neurotransmission at the vast majority of excitatory synapses via N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptors [11], [30], [31], [32]. The NMDA receptor channel is formed by GluRε (NR2) and GluRζ (NR1) subunits, and highly active NMDA receptor channels are produced only when the GluRζ subunit is expressed together with any one of four GluRε subunits (GluRε1–GluRε4). The non-NMDA receptor group is further divided pharmacologically into α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate subtypes. Activation of NMDA receptors on dorsal horn neurons by this amino acid is frequently thought to be an essential step in the development of spinal sensitization and hyperalgesia [24]. Central sensitization produces a prolonged and significant enhancement of responses to both noxious and innocuous stimuli. Recent studies from our laboratory and others have demonstrated that prostaglandins (PGs), the products of the cyclooxygenase pathway of arachidonic acid cascade, are critical for the processing of pain not only by sensitizing peripheral terminals of primary afferent fibers but also by sensitizing the processing of pain information at the spinal level. For example, prostaglandin E2 (PGE2) was released from the spinal cord in vivo upon various noxious stimuli and inflammatory insults [23]. Intrathecal (i.t.) delivery of cyclooxygenase inhibitors blocked pain responses induced by subcutaneous formalin and NMDA-induced hyperalgesia [21], [22], and abolished the enhanced excitability of dorsal horn convergent neurons to both noxious and innocuous mechanical stimuli after ischemia [10]. Conversely, i.t. administration of PGE2 into conscious mice induced hyperalgesia to noxious stimuli [41], [43] and allodynia to tactile innocuous stimuli [27], [29]. Intrathecal administration of PGF also induced mechanical allodynia [28]. Glutamate has been assumed to play a significant role in induction of allodynia as well as hyperalgesia. Present knowledge of involvement of the glutamatergic system in PGE2- and PGF-induced allodynia derives from two types of approaches: genetic manipulations, in which the gene for a selected GluRε subtype is disrupted [27], and pharmacological blockade, in which the induction of allodynia by PGE2 and PGF was inhibited with NMDA and non-NMDA antagonists [26]. This was also supported by the findings that glutamate was released from the spinal cord in response to PGE2[33] and that glutamate content increased in the dorsal horn following nerve injury [18]. The i.t. injection of the glycine receptor antagonist strychnine or the GABAA receptor antagonist bicuculline induced allodynia, which was reversed by NMDA and non-NMDA receptor antagonists [34], [46], suggesting that the imbalance between excitatory and inhibitory amino acids is a cause of induction of allodynia.

Released glutamate is rapidly eliminated from the extracellular space by a high-affinity transport system that is present in both nerve endings and surrounding glial cells [37]. The transport process is considered to be primarily responsible for the termination of neurotransmitter action of glutamate and the prevention of neuronal damage from excessive activation of glutamate receptors. Five distinct high-affinity, sodium-dependent glutamate transporters have been cloned from rodent and human tissues: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4 and EAAT5, and these proteins have distinct structures, functions, and distributions [37]. Immunohistochemical studies have suggested that GLAST and GLT-1 are both in astrocytes, while EAAC1 and EAAT4 are primarily neuronal glutamate transporters. In addition, the re-uptake process is also essential for the recycling of the amino acid pool that replenishes the supply of neurotransmitter in glutamatergic terminals.

We previously demonstrated that i.t. glutamate induced allodynia in conscious mice [26]. In the present study, we characterized glutamate receptor subtypes involved in the glutamate-induced allodynia and compared it with PGE2- and PGF-induced allodynia. In order to investigate the role of glutamate transporters in the allodynia, we used dl-threo-β-benzoyloxyaspartate (dl-TBOA) [38], a newly synthesized, nontransportable inhibitor of EAAT1 and EAAT2, and EAAT3, the three main transporters present in the spinal cord.

Section snippets

Drugs

PGE2 and PGF were generous gifts from Ono Central Research Institute (Osaka, Japan). They were stored in ethanol solution at −20°C. For injection, an aliquot of the stock solution was put into a borosilicate tube, and the ethanol was removed by evaporation to dryness under nitrogen gas. Sterile saline was then added to dissolve the PGE2 or PGF. NMDA was obtained from Sigma (St. Louis, MO). AMPA and kainate were purchased from Research Biochemicals Internationals (Natick, MA). d

Effect of i.t. glutamate receptor agonists on mechanical allodynia

We previously showed that i.t. glutamate produced allodynia over a wide range of dosages from 0.1 pg to 100 ng/mouse and that allodynia evoked by i.t. administration of PGE2 and PGF were mediated by the glutamatergic system [26]. In an initial approach to characterize glutamate receptors involved in the PGE2- and PGF-induced allodynia, we examined the ability of agonists for ionotropic glutamate receptors to induce allodynia in conscious mice. The i.t. administration of NMDA, AMPA, but not

Discussion

Activation of primary afferent fibers can induce states of facilitated spinal sensory processing, resulting in spontaneous agitation, hyperalgesia, and allodynia. Whereas i.t. application of glutamate receptor agonists elicits hyperalgesia and spontaneous agitation of behavioral responses such as licking, biting and scratching [1], [8], [21], [36], glutamate receptor antagonists produce analgesia. Despite extensive evidence to implicate spinal NMDA receptors and subsequent nitric oxide

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports, and Culture of Japan and Scientific Research (B) and (C) from Japan Society of the Promotion of Science, and by grants from the Science Research Promotion Fund of the Japan Private School Promotion Foundation and Jinsenkai Foundation of Osaka Medical College.

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