Susan M. Carlton

Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin

Immunohistochemical staining for the glutamate receptor subtypes N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) results in a significant number of labeled unmyelinated axons in the glabrous skin of the rat hindpaw. Injection of glutamate into the rat hindpaw results in behavioral changes interpreted as mechanical allodynia and mechanical hyperalgesia. The anatomical findings provide a reasonable explanation for the action of the exogenous peripheral glutamate, namely that activation of these receptors leads to increased primary afferent activity in unmyelinated axons and thus to pain behaviors. AMPA receptors are frequently associated with small clear vesicles in the axoplasm of the unmyelinated axons, many of which have been previously shown to contain high concentrations of glutamate. This finding indicates that these might be autoreceptors and so glutamate itself might regulate certain types of peripheral impulse traffic. The presence of peripheral glutamate receptors associated with unmyelinated axons suggests the possibility that glutamate antagonists applied peripherally might prevent or attenuate some pain-related behaviors.

Staining of glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic nerve constriction injury.

The change in staining density of glial fibrillary acidic protein (GFAP) was analyzed in rats that sustained a chronic constriction injury produced by sutures tied loosely around one sciatic nerve. This injury model of peripheral neuropathy resulted in a behavioral hyperalgesia evidenced by a decrease in mean foot withdrawal latency to radiant heat. Increased GFAP immunostaining was observed in the gray matter of the spinal cord ipsilateral to the lesion and specific to spinal segments in which the sciatic nerve is distributed. Elevated GFAP staining density was attributed primarily to hypertrophy of astrocytes rather than their proliferation or migration since counts of astrocyte profiles demonstrated no significant difference when comparing the lesioned to the control side. The magnitude of the increase in GFAP staining correlated with the degree of hyperalgesia. Thus, these data suggest that astrocytes participate in the sequelae occurring in the dorsal horn following constriction injury of a peripheral nerve.

Peripheral group I metabotropic glutamate receptors modulate nociception in mice

The metabotropic glutamate receptors (mGluRs) are found throughout the central nervous system, where they modulate neuronal excitability and synaptic transmission. Here we report the presence of phospholipase C-coupled group I mGluRs (mGluR1 and mGluR5) outside the central nervous system on peripheral unmyelinated sensory afferents. Given their localization on predominantly nociceptive afferents, we investigated whether these receptors modulate nociceptive signaling, and found that agonist-induced activation of peripheral group I mGluRs leads to increased sensitivity to noxious heat, a phenomenon termed thermal hyperalgesia. Furthermore, group I mGluR antagonists not only prevent, but also attenuate established formalin-induced pain. Taken together, these results suggest that peripheral mGluRs mediate a component of hyperalgesia and may be therapeutically targeted to prevent and treat inflammatory pain.

Receptor localization in the mammalian dorsal horn and primary afferent neurons

The dorsal horn of the spinal cord is a primary receiving area for somatosensory input and contains high concentrations of a large variety of receptors. These receptors tend to congregate in lamina II, which is a major receiving center for fine, presumably nociceptive, somatosensory input. There are rapid reorganizations of many of these receptors in response to various stimuli or pathological situations. These receptor localizations in the normal and their changes after various pertubations modify present concepts about the wiring diagram of the nervous system. Accordingly, the present work reviews the receptor localizations and relates them to classic organizational patterns in the mammalian dorsal horn.

Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery

The present study determines the proportions of unmyelinated cutaneous axons at the dermal–epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal–epidermal junction immunostain for either N‐methyl‐D‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non‐NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Aδ and Aβ, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large‐diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive. J. Comp. Neurol. 391: 78–86, 1998.

The effects of dorsal rhizotomy and spinal cord isolation on calcitonin gene-related peptide-labeled terminals in the rat lumbar dorsal horn☆

In the present study, the origin of calcitonin gene-related peptide (CGRP) to the dorsal horn in the rat lumbar spinal cord is investigated. CGRP immunoreactivity is examined following multiple unilateral and bilateral dorsal rhizotomies and isolated cord preparations (spinal cords are isolated by transecting the cord in two places and cutting all dorsal roots between the transections). Seven to 11 days after surgery, unilateral multiple dorsal rhizotomies result in a drastic decrease in CGRP-stained terminals on the operated side; following bilateral dorsal rhizotomies and isolated cord preparations, one or two CGRP varicosities remain in the dorsal horn in each section. The numbers of CGRP-immunostained varicosities observed in the latter two preparations are not significantly different, suggesting that few if any axons descending from the brain contribute to the CGRP terminal population in the spinal cord dorsal horn. Based on these data, we hypothesize that dorsal root ganglion cells are the only source of CGRP to the rat lumbar dorsal horn.

Peripheral administration of NMDA, AMPA or KA results in pain behaviors in rats

The present study investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor subtypes in peripheral pain transmission. Activation of NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-pro- pionic acid (AMPA) and kainate acid (KA) receptors in glabrous skin of the rat hindpaw resulted in mechanical allodynia and mechanical hyperalgesia. These agonist- induced pain behaviors were attenuated following peripheral injection of appropriate antagonists (MK-801 and CNQX). Thus, activation of NMDA, AMPA or KA receptors at the level of the peripheral nerve terminal can produce nociceptive behavior. These data suggest that topical application of glutamate receptor antagonists may be useful in treating pain disorders. Since all three receptor subtypes are involved in peripheral pain transmission, however, it will be necessary to antagonize multiple glutamate receptor subtypes to achieve effective pain relief.

Peripheral NMDA and non-NMDA glutamate receptors contribute to nociceptive behaviors in the rat formalin test

THE present study demonstrates that local cutaneous administration of either the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 or the non-NMDA glutamate receptor antagonist 6-cyano7-nitroquinoxaline-2,3-dione (CNQX) significantly attenuates formalin-induced nociceptive behaviors. Specifically, pretreatment with either drug reduced the magnitude and time course of lifting and licking behavior in the late phase of formalin pain; however, flinching behavior was not affected. In contrast, posttreatment of formalin pain with either antagonist did not affect lifting and licking behavior, although flinching behavior was mildly attenuated. We hypothesize that these actions result from blocking of peripheral glutamate receptors located on unmyelinated axons at the dermal–epidermal junction. These data suggest that peripheral glutamate receptors on cutaneous axons can be manipulated to reduce certain aspects of pain of peripheral origin. This route of administration offers the advantage of avoiding the side effects of systemic administration.

Opioid receptors on peripheral sensory axons

Opioid receptors have been demonstrated by light microscopic techniques in fine cutaneous nerves in naive animals. The present study extends these findings by showing that 29 and 38% of unmyelinated cutaneous sensory axons can be immunostained for mu- or delta-opioid receptors respectively. Local cutaneous injection of DAMGO, a mu-opioid ligand, ameliorates the nociceptive behaviors caused by local cutaneous injection of glutamate, a purely nociceptive chemical stimulus showing that the mu-receptors are functional. By contrast the delta-opioid ligand [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE) had no effect on these behaviors. These findings indicate a wider function for opioid receptors in naive animals than previously envisioned.

Inflammation-induced changes in peripheral glutamate receptor populations

The ionotropic glutamate receptors N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) have been localized on subpopulations of unmyelinated and myelinated sensory axons in normal skin. Behavioral studies indicate that activation of these receptors results in nociceptive behaviors and contributes to inflammatory pain. The goal of the present study was to determine if these glutamate receptors might contribute to the peripheral hypersensitivity observed in inflammation. The major findings were that 48 h following complete Freunds adjuvant (CFA)-induced inflammation, the proportions of unmyelinated axons labeled for NMDA, AMPA or KA receptors were 61%, 43% and 48%, respectively, in cutaneous nerves in the inflamed paw compared to 48%, 22% and 27%, respectively, in the non-inflamed paw. The proportions of myelinated axons labeled for NMDA, AMPA or KA receptors were 61%, 61% and 43%, respectively, compared to 43%, 42% and 28%, respectively, in the non-inflamed hindpaw. These increases were all significant. These data indicate that the number of sensory axons containing ionotropic glutamate receptors increases during inflammation, and this may be a contributing factor to peripheral sensitization in inflammation.