Gregory L. Hargett

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.

Peripheral and central sensitization in remote spinal cord regions contribute to central neuropathic pain after spinal cord injury

ABSTRACT Central neuropathic pain (CNP) developing after spinal cord injury (SCI) is described by the region affected: above‐level, at‐level and below‐level pain occurs in dermatomes rostral, at/near, or below the SCI level, respectively. People with SCI and rodent models of SCI develop above‐level pain characterized by mechanical allodynia and thermal hyperalgesia. Mechanisms underlying this pain are unknown and the goals of this study were to elucidate components contributing to the generation of above‐level CNP. Following a thoracic (T10) contusion, forelimb nociceptors had enhanced spontaneous activity and were sensitized to mechanical and thermal stimulation of the forepaws 35 days post‐injury. Cervical dorsal horn neurons showed enhanced responses to non‐noxious and noxious mechanical stimulation as well as thermal stimulation of receptive fields. Immunostaining dorsal root ganglion (DRG) cells and cord segments with activating transcription factor 3 (ATF3, a marker for neuronal injury) ruled out neuronal damage as a cause for above‐level sensitization since few C8 DRG cells expressed AFT3 and cervical cord segments had few to no ATF3‐labeled cells. Finally, activated microglia and astrocytes were present in thoracic and cervical cord at 35 days post‐SCI, indicating a rostral spread of glial activation from the injury site. Based on these data, we conclude that peripheral and central sensitization as well as reactive glia in the uninjured cervical cord contribute to CNP. We hypothesize that reactive glia in the cervical cord release pro‐inflammatory substances which drive chronic CNP. Thus a complex cascade of events spanning many cord segments underlies above‐level CNP.

Localization of metabotropic glutamate receptors 2/3 on primary afferent axons in the rat

The goal of the present study is to determine the relationship of metabotropic glutamate receptors 2/3 (mGluR2/3) to dorsal root ganglion cells, peripheral primary afferent fibers in digital nerves and central primary afferent fibers in the spinal cord. We demonstrate that approximately 40% of L4 and L5 dorsal root ganglion cells contain mGluR2/3-like immunoreactivity. These mGluR2/3-positive cells are small in diameter (23 microm) and 76% stain for the isolectin Griffonia simplicifolia (I-B4), while 67% of I-B4 cells have mGluR2/3-like immunoreactivity. Electron microscopic analyses of mGluR2/3-like immunoreactivity in axons in digital nerves indicate that 32% of unmyelinated and 28% of myelinated axons are labeled. In the lumbar dorsal horn, mGluR2/3-like immunoreactivity is localized preferentially in lamina IIi with lighter staining in laminae III and IV. The dense mGluR2/3-like immunoreactivity in lamina IIi is consistent with the localization of these receptors in I-B4-labeled dorsal root ganglion cells. Elimination of primary afferent input following unilateral dorsal rhizotomies significantly decreases the mGluR2/3-like immunoreactivity density in the dorsal horn although some residual staining does remain, suggesting that many but not all of these receptors are located on primary afferent processes. The finding that mGluR2/3s are located on peripheral sensory axons suggests that they are involved in peripheral sensory transduction and can modulate transmission of sensory input before it reaches the spinal cord. This offers the possibility of altering sensory input, particularly noxious input, at a site that would avoid CNS side effects. Since many but not all of these receptors are located on primary afferent terminals, these receptors may also influence primary afferent transmission in the dorsal horn through presynaptic mechanisms and glutamatergic transmission in general through both presynaptic and postsynaptic mechanisms. Since these receptors are concentrated in lamina IIi and also largely co-localized with I-B4, they may have considerable influence on nociceptive processing by what are considered to be non-peptidergic primary afferent neurons.

Treatment with the NMDA antagonist memantine attenuates nociceptive responses to mechanical stimulation in neuropathic rats

Systemic treatment with the N-methyl-D-aspartate (NMDA) antagonist memantine (MEM) resulted in a decrease in mechanical hyperalgesia and mechanical allodynia in neuropathic rats. Bolus injections of 5, 10 and 20 mg/kg MEM (i.p.) attenuated withdrawal responses following mechanical stimulation for up to 6 h post-injection while saline had no effect. A more remarkable effect was observed following chronic treatment with MEM via mini-osmotic pumps (8 mg/kg per h for 7 days, i.p.), in which nociceptive responses were decreased during treatment and remained depressed for 3 days post-treatment. These antinociceptive effects can be obtained at doses which do not produce motor dysfunction. The results of this study demonstrate that MEM has a therapeutic effect on mechanical hyperalgesia and allodynia and may be considered as an alternative treatment for pathological pain in the clinical setting.

Colocalization of metabotropic glutamate receptors in rat dorsal root ganglion cells

Glutamate is the main excitatory transmitter in both central and peripheral nervous systems. Discovery of metabotropic glutamate receptors (mGluRs) made it clear that glutamate can have excitatory or inhibitory effects on neuronal function, with group I mGluRs enhancing cell excitability but group II and III mGluRs decreasing excitability. The present study investigated the colocalization of mGluR subtypes representing groups I, II, or III in rat L5 dorsal root ganglion (DRG) cells. The analyses show that group III has the highest expression, with 75.0% of DRG cells expressing mGluR8, followed by group II, with 51.6% expressing mGluR2/3, followed by group I, with only 6.8% expressing mGluR1α. mGluR8 is expressed by small, medium, and large diameter cells. In contrast, mGluR1α and mGluR2/3 are expressed by mainly small and medium cells. Approximately half of cells expressing group I mGluR1α also express either group II mGluR2/3 or group III mGluR8. These mGluR1α double‐labeled populations are not likely to overlap since >1.0% of mGluR1α are triple‐labeled. As expected from the high percentage of single‐labeled mGluR2/3 and mGluR8 cells, there is a considerable population of double‐labeled cells with ≈30% of each population expressing both receptors. Due to the fact that the number of mGluR1α‐expressing cells in the DRG is low, the percentage of triple‐labeled cells is also low (≈1–2%). The prevalence of groups II and III indicate that glutamate could have a substantial inhibitory effect of primary afferent function, reducing and/or fine‐tuning sensory input before transmission to the spinal cord. These anatomical data highlight the potential inhibitory role glutamate may play in peripheral sensory transmission. J. Comp. Neurol. 501:780–789, 2007.

Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel

&NA; Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that ∼60% of SSTR2a‐labeled cells are positive for TRPV1. Conversely, ∼33% of TRPV1‐labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 &mgr;M octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) ‐induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co‐application of the SSTR antagonist cyclo‐somatostatin (c‐SOM). In vitro studies using a skin‐nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 &mgr;M OCT significantly reduces CAP‐induced activity and can prevent CAP‐induced desensitization. Furthermore, blockade of peripheral SSTRs with c‐SOM dramatically enhances CAP‐induced behaviors and nociceptor activity, demonstrating SSTR‐induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP‐induced excitation and does not effect OCT‐induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors.

Stereological analysis of Ca2+/calmodulin-dependent protein kinase IIα-containing dorsal root ganglion neurons in the rat: Colocalization with isolectin Griffonia simplicifolia, calcitonin gene-related peptide, or vanilloid receptor 1

The enzyme Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is widely distributed in the nervous system. A previous report describes immunostaining for CaMKIIα in dorsal root ganglion (DRG) neurons. In this study, CaMKIIα is colocalized in the rat with three putative markers of nociceptive DRG neurons, isolectin Griffonia simplicifolia (I‐B4), identifying small‐diameter, “peptide‐poor” neurons; calcitonin gene‐related peptide (CGRP), identifying “ peptide‐rich” neurons; or the vanilloid receptor 1 (VR1), identifying neurons activated by heat, acid, and capsaicin. Lumbar 4 and 5 DRG sections were labeled using immunofluorescence or lectin binding histochemistry, and percentages of single and double‐labeled CaMKIIα neurons were determined. Stereological estimates of total neuron number in the L4 DRG were 13,815 ± 2,798 and in the L5 DRG were 14,111 ± 4,043. Percentages of single‐labeled L4 DRG neurons were 41% ± 2% CaMKIIα, 38% ± 3% I‐B4, 44% ± 3% CGRP, and 32% ± 6% VR1. Percentages of single‐labeled L5 DRG neurons were 44% ± 5% CaMKIIα, 48% ± 2% I‐B4, 41% ± 7% CGRP, and 39% ± 14% VR1. For L4 and L5, respectively, estimates of double‐labeled CaMKIIα neurons showed 34% ± 2% and 38% ± 17% labeled for I‐B4, 25% ± 14% and 19% ± 10% labeled for CGRP, and 37% ± 7% and 38% ± 5% labeled for VR1. Conversely, for L4 and L5, respectively, 39% ± 14% and 38% ± 7% I‐B4 binding neurons, 24% ± 12% and 23% ± 10% CGRP neurons, and 42% ± 7% and 35% ± 7% VR1 neurons labeled for CaMKIIα. The mean diameter of CaMKIIα‐ labeled neurons was approximately 27 μm, confirming that this enzyme was preferentially localized in small DRG neurons. The results indicate that subpopulations of DRG neurons containing CaMKIIα are likely to be involved in the processing of nociceptive information. Thus, this enzyme may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory neurons. J. Comp. Neurol. 448:102–110, 2002.

Plasticity in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits in the rat dorsal horn following deafferentation

Immunostaining for GluR1 and GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is prominent in laminae I and II of the normal dorsal horn, with much less staining in deeper laminae. Three days after dorsal rhizotomy, image analysis demonstrates a significant decrease in staining density for both antibodies in the superficial dorsal horn. By contrast, staining density is significantly increased in laminae III, IV and the reticulated region of lamina V for GluR1 only. This increase suggests that AMPA receptors contribute to deafferentation or radicular pain by at least two mechanisms: (1) up-regulation of GluR1 subunits of the AMPA receptor in deeper laminae, and (2) enhanced Ca2+ permeability of dorsal horn neurons because AMPA receptors lacking the GluR2 subunit are Ca2+ permeable. Glutamate activation of these receptors could initiate or potentiate second messenger cascades that could well contribute to neuronal changes documented in the dorsal horn of deafferented animals.

Pain after Discontinuation of Morphine Treatment Is Associated with Synaptic Increase of GluA4-Containing AMPAR in the Dorsal Horn of the Spinal Cord

Withdrawal from prescribed opioids results in increased pain sensitivity, which prolongs the treatment. This pain sensitivity is attributed to neuroplastic changes that converge at the spinal cord dorsal horn. We have recently reported that repeated morphine administration triggers an insertion of GluA2-lacking (Ca2+-permeable) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) in the hippocampus. This finding together with the reported involvement of AMPAR in the mechanisms underlying inflammatory pain led us to hypothesize a role for spinal AMPAR in opioid-induced pain behavior. Mice treated with escalating doses of morphine showed hypersensitivity to mechanical stimulation. Intrathecal administration of a Ca2+-permeable AMPAR selective blocker disrupted morphine-induced mechanical sensitivity. Analysis of the expression and phosphorylation levels of AMPAR subunits (GluA1/2/3/4) in homogenates and in postsynaptic density fractions from spinal cord dorsal horns showed an increase in GluA4 expression and phosphorylation in the postsynaptic density after morphine. Co-immunoprecipitation analyses suggested an increase in GluA4 homomers (Ca2+-permeable AMPAR) and immunohistochemical staining localized the increase in GluA4 levels in laminae III–V. The excitatory postsynaptic currents (EPSCs) recorded in laminae III–V showed enhanced sensitivity to Ca2+-permeable AMPAR blockers in morphine-treated mice. Furthermore, current–voltage relationships of AMPAR-mediated EPSCs showed that rectification index (an indicator of Ca2+-permeable AMPAR contribution) is increased in morphine-treated but not in saline-treated mice. These effects could be reversed by infusion of GluA4 antibody through patch pipette. This is the first direct evidence for a role of GluA4-containing AMPAR in morphine-induced pain and highlights spinal GluA4-containing AMPAR as targets to prevent the morphine-induced pain sensitivity.

Distribution of glycine-immunoreactive profiles in the monkey spinal cord: A light microscopic and ultrastructural study

The present study analyzed the relationships of glycine (GLY)‐immunoreactive (‐IR) and unlabeled profiles in the primate spinal cord. Light microscopic analysis demonstrated GLY‐IR profiles in laminae III‐VII, with fewer labeled profiles in laminae I, II, VIII, IX and X. The dorsal part of the lateral funiculus and the dorsal funiculus contained few labeled axons, in contrast to all other areas of white matter, which were heavily labeled. At the electron microscopic level, GLY‐IR terminals in monkeys contained mainly round, with occasional pleomorphic, clear vesicles; however, F‐type GLY‐IR terminals synapsing on motoneurons contained pleomorphic vesicles. This seems to be an important species difference because vesicles in GLY‐IR terminals in rat and cat are predominantly oval or elliptical.