Research reportEvidence for the interaction of glutamate and NK1 receptors in the periphery
Introduction
Several lines of evidence indicate that glutamate (GLU) plays a role in nociception in the periphery. For example, N-methyl-d-aspartate (NMDA), α-amino-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and kainate receptors have been demonstrated on unmyelinated axons at the dermal–epidermal junction of the rat glabrous skin [5]. Local peripheral injection of GLU or its agonists into the glabrous skin of the rat induces mechanical allodynia and hyperalgesia 5, 39, which is blocked by co-injection of appropriate antagonists [39]. Application of either GLU or kainate to peripheral skin initiates nociceptive reflex activity in an isolated spinal cord-tail preparation 1, 2. Finally, intraplantar injection of NMDA antagonists attenuates nociceptive behaviors resulting from intraplantar injection of formalin [8] or capsaicin [29].
In addition to GLU receptors, neurokinin 1 (NK1) receptors, for which substance P (SP) is the main natural ligand, have been localized on unmyelinated axons in the rat glabrous skin [7]. Activation of NK1 receptors following local injection of SP in glabrous skin results in mechanical allodynia and hyperalgesia which is blocked following co-injection of an NK1 antagonist [7]. Furthermore, peripheral application of SP has been reported to sensitize primary afferents in both normal and inflamed states 16, 20.
The presence of GLU and SP receptors on populations of unmyelinated cutaneous axons in glabrous skin, and the fact that GLU and SP or their ligands have physiological effects when injected locally raises the issue as to whether GLU and SP could interact in the periphery. This is an especially intriguing possibility since SP clearly enhances GLU- and NMDA-induced activity in spinal cord dorsal horn neurons 11, 12, 30, this enhancement being important in the generation of central sensitization 11, 23. Thus, a major goal of this study was to provide evidence to support the hypothesis that primary afferent neurons might well be subject to similar mechanisms that result in wind-up or central sensitization of spinal cord neurons. In pursuit of this goal, the objectives of the present study were: 1) to demonstrate GLU and SP receptors in cutaneous axons in the hairy skin of the rat tail, 2) to demonstrate that injection of GLU alone causes nociceptive behaviors that are ameliorated by co-injection with a GLU antagonist and 3) to show that activation of peripheral SP receptors enhances GLU-induced nociceptive behaviors.
Section snippets
Anatomical studies
Male Sprague–Dawley rats (125–200 g, n=3) were deeply anesthetized with nembutal (70 mg/kg, i.p.) and then perfused through the aorta with heparinized saline followed by a mixture of 1% glutaraldehyde and 1.25% paraformaldehyde in 0.1% picric acid in 0.1 M phosphate buffer at 4°C. Hairy skin from the tail was removed and cut into small blocks. Each block was immunostained with the ABC method (Vector Labs) using a previously described protocol [5]. Antibodies directed against either GluR1 (1 μ
Anatomical studies
Electron microscopic analyses demonstrated immunoreaction product for the NMDAR1 receptor subunit of the NMDA receptor (Fig. 1A), the GluR1 subunit of the AMPA receptor (Fig. 1B), and the NK1 receptor (Fig. 2) in unmyelinated axons at the dermal–epidermal junction in the tail skin. In all cases the reaction product was found along the axonal membrane as well as in the axoplasm, particularly associated with microtubules. Unlabeled axons were also observed. (Fig. 1, Fig. 2).
Dose–response relationships
Injection of 0.1, 1.0,
Discussion
A major goal of this study was to provide evidence to support the hypothesis that primary afferent neurons might be subject to similar mechanisms that result in wind-up or central sensitization of spinal cord neurons. In pursuit of this goal, we have shown that: 1) NMDA, non-NMDA or NK1 receptors are localized on cutaneous unmyelinated sensory axons innervating the hairy skin of the tail, 2) GLU injection into the tail skin results in nociceptive behaviors interpreted as mechanical allodynia
Acknowledgements
The authors would like to thank Brenda Kenworthy for her excellent secretarial assistance and Zhixia Ding for her skill and expertise in immunostaining and electron microscopy. This work was funded by NIH grants NS11255 (SMC, REC), NS27910 (SMC) and NS10161 (REC).
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