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Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain
Abstract
To clarify the involvement of GluR2 and GluR3 subunits of AMPA receptor in orofacial neuropathic pain, we studied changes in nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation followed by infraorbital nerve (ION)-partial transection model applied to GluR2 or GluR3 delta7 knock-in (KI) mice. In these animals, last seven amino acids of GluR2 or GluR3 subunit, the binding sites of interacting protein, are deleted in vivo. Head-withdrawal threshold to mechanical stimulation of the whisker pad skin ipsilateral to ION-partial transection was significantly reduced at 1, 3, 5, 7, 11 and 14 days after transection compared with that before transection in wild-type mice. In the GluR2 and GluR3 delta7 KI mice, the head-withdrawal threshold did not change following ION-partial transection. The number of pERK-LI cells examined in Vc and C1-C2 in wild-type mice after the non-noxious stimulation was larger than that of GluR2 and GluR3 delta7 KI mice. The present findings suggest that GluR2 and GluR3 subunits of AMPA receptor play roles in the trigeminal nerve injury-mediated enhancement of Vc and C1-C2 neuronal excitability, and hyperalgesia.
... An investigation of glial cell activity in the trigeminal nucleus following ION ligature in rats showed that microglia and astrocytes are activated under the influence of the chemokine CCL2 at different time points, suggesting that glial cell activation is associated with the development and maintenance of PTNP [62]. Furthermore, neuroplastic changes and hyperexcitability of the central nervous system were suggested to result from substantial alterations in the expression of certain growth factors (e.g., NGF [40] and BDNF [45]) and markers of neuronal activity (e.g., p-p38 [46,51] and p-ERK [34,46,51,52]) in the brain stem and the higher central nervous system such as the mPFC. A study has shown that ERK, p38, and JNK MAPKs are accelerated in the TG of the CCI-ION rat model and that their inhibitors significantly reversed the effect of facial mechanical allodynia, suggesting that MAPKs are therapeutic targets for the treatment of chronic pain in PTNP [46]. ...
... <1> GDNF [38], Patched-1 (Hedgehog pathway readout) mRNA [36] <6> CGRP [30] Trigeminal ganglion Upregulation: [68] <7> IL-18 [31], p-IκB kinase [31], p-NF-κB p65 [31], p-p38 [31] <13> EphA4 [69], IL-1β [70], JAK2 [71], p-NF-κB [72], p-p38 [72], PTEN [71], VEGF [73] <11> ATF-3 [33], BrdU (mitotic marker) [33], CD11b [33], GFAP [33], NK1 receptor [33] <12> p-ERK [34] <13> OX-42 (microglial activation) [35,74], PPARG [75] Downregulation: ...
... Diacerein (IL-1β inhibitor)/intra-TG/mech. <12> Knockout of GluR2 and GluR3 subunits of AMPA receptor/mech.[34] <13> Pioglitazone (PPARγ agonist)/ip/mech.[75], ...
This review highlights potential molecular targets for treating neuropathic orofacial pain based on current findings in animal models. Preclinical research is currently elucidating the pathophysiology of the disease and identifying the molecular targets for better therapies using animal models that mimic this category of orofacial pain, especially post-traumatic trigeminal neuropathic pain (PTNP) and primary trigeminal neuralgia (PTN). Animal models of PTNP and PTN simulate their etiologies, that is, trauma to the trigeminal nerve branch and compression of the trigeminal root entry zone, respectively. Investigations in these animal models have suggested that biological processes, including inflammation, enhanced neuropeptide-mediated pain signal transmission, axonal ectopic discharges, and enhancement of interactions between neurons and glial cells in the trigeminal pathway, are underlying orofacial pain phenotypes. The molecules associated with biological processes, whose expressions are substantially altered following trigeminal nerve damage or compression of the trigeminal nerve root, are potentially involved in the generation and/or exacerbation of neuropathic orofacial pain and can be potential molecular targets for the discovery of better therapies. Application of therapeutic candidates, which act on the molecular targets and modulate biological processes, attenuates pain-associated behaviors in animal models. Such therapeutic candidates including calcitonin gene-related peptide receptor antagonists that have a reasonable mechanism for ameliorating neuropathic orofacial pain and meet the requirements for safe administration to humans seem worth to be evaluated in clinical trials. Such prospective translation of the efficacy of therapeutic candidates from animal models to human patients would help develop better therapies for neuropathic orofacial pain.
... The infraorbital nerve was exposed and dissected free from the surrounding tissue. As previously described by Miyamoto et al 31 and Xu et al, 56 the lateral half of the nerve was lifted from the maxillary bone and cut with scissors in some rats to produce a partial transection of the nerve (p-IONX); care was taken not to damage facial nerve branches. In other rats, a sham operation was performed but without any nerve injury. ...
... Several trigeminal neuropathic pain models have been developed to study the neural mechanisms underlying craniofacial neuropathic pain conditions. 1,[22][23][24]31,39,44,48,52,56 Following a chronic constriction injury of the infraorbital nerve, rats and mice exhibit a prolonged heat or mechanical hyperalgesia and mechanical allodynia in the vibrissal pad. 22,52,56 Iwata et al have reported that the mechanical thresholds in the ipsilateral vibrissal pad area are significantly lower than those of sham-operated rats for 4 weeks following inferior alveolar nerve transection, and that the contralateral side also shows transient decreases in mechanical thresholds. ...
... 23,24,36 In a mouse pIONX model, mechanical allodynia is also observed bilaterally. 31 The p-IONX rats in the present study also The baseline thresholds of p-IONX/pregabalin group and pIONX/saline group showed significant decreases in comparison to sham/pregabalin group. (1-way ANOVA followed by the Bonferroni post hoc test, p-IONX/pregabalin group versus shamoperated/pregabalin: P = .008; ...
Unlabelled:
The aim of this study was to determine whether pregabalin affects nociceptive behavior and central sensitization in a trigeminal neuropathic pain model. A partial infraorbital nerve transection (p-IONX) or sham operation was performed in adult male rats. Nociceptive withdrawal thresholds were tested with von Frey filaments applied to the bilateral vibrissal pads pre- and postoperatively. On postoperative day 7, the behavioral assessment was conducted before and at 30, 60, 120, and 180 minutes after and 24 hours after pregabalin (.1, 1, 10, 100 mg/kg intraperitoneally) or saline injection. The effects of pregabalin or saline were also examined on the mechanoreceptive field and response properties of nociceptive neurons recorded in the medullary dorsal horn at postoperative days 7 to 10. Reduced withdrawal thresholds reflecting bilateral mechanical allodynia were observed in p-IONX rats until postoperative day 28, but not in sham-operated rats. At postoperative day 7, pregabalin significantly and dose-dependently reversed the reduced mechanical withdrawal thresholds in p-IONX rats. Pregabalin also attenuated central sensitization of the neurons, as reflected in reversal of their reduced activation threshold, increased responses to pinch/pressure, and enhanced stimulus-response function. This study provides the first documentation that pregabalin attenuates the mechanical allodynia and central sensitization that characterize this trigeminal neuropathic pain model, and supports its clinical use for treating craniofacial neuropathic pain.
Perspective:
Trigeminal nerve injury in rats produced facial mechanical hypersensitivity and trigeminal central sensitization of medullary dorsal horn neurons that were markedly attenuated by systemically administered pregabalin, suggesting its potential clinical utility for orofacial neuropathic pain.
... Reports of sensation from the pulp that are nonnoxious arise from electrical stimulation, which is nonphysiological (32). As far as has been determined, the nociceptors in the pulp are non-mylelinated nerve terminals that could be the terminals of both Aδ and C fibers (33). There is no morphological differentiation between the terminals. ...
... The cell bodies of all of these neurons are in the trigeminal ganglion and the central processes end in the spinal nucleus of the trigeminal, predominantly in its caudal end (nucleus caudalis) by synapsing on second-order neurons, interneurons, and terminals of descending fibers. These terminals may contain calcitonin gene-related peptide (CGRP), substance P, or glutamate (33,34), which act as the principal synaptic messengers to the second-order neuron. The glutamate receptors in the post-synaptic membrane [N-methyl-D-aspartate (NMDA)] are a very promising target for new analgesic drugs, some of which are currently being developed. ...
The dental pulp, while exquisitely small, is extremely interesting. Its study has shed much light on basic biological processes such as protein production, mineralization, microvascular physiology, inflammation, pain, and most recently stem cell biology. The accumulation of knowledge on the dental pulp has been rapid over the past 30 years. With such efforts, one might think that our knowledge of the dental pulp is nearly complete, but this is not so. With the dental pulp, as with all other tissues, research always finds more questions than answers. For the specialty of Endodontics, knowledge of the tissue has reached such a point that newer, biological therapies are or shortly will be available whose effective application requires a good understanding of the biology involved. Clearly any review must be selective and will inevitably be incomplete, biased, and confused.
... Partial (one third to one half ) infraorbital nerve tight ligation with silk suture provides mechanical allodynia that does not recover in 25 days while face grooming diminishes within 7 days [5]. Similar functional outcome is reported with partial transection of the infraorbital nerve in another study [6]. In another previous report, sensation impairment persists more than 10 weeks after tight ligation of the mental nerve in mice, however, the behavioral test describes grabbing the mice from the back to take pain threshold measurements in this unnatural posture [7]. ...
... allodynia lasting as long as 4 weeks [5]. While the partial ligation and partial transection of the infraorbital nerve introduces increased facial grooming and mechanical allodynia , loose ligation of the infraorbital nerve with two 4-0 silk ligatures also induces thermal hyperalgesia in mice [4,6,12]. We report here a novel chronic orofacial neuropathic pain model inducing continuous pain related mechanical allodynia responses in mice. ...
Background
Trigeminal neuropathic pain attacks can be excruciating for patients, even after being lightly touched. Although there are rodent trigeminal nerve research models to study orofacial pain, few models have been applied to studies in mice. A mouse trigeminal inflammatory compression (TIC) model is introduced here which successfully and reliably promotes vibrissal whisker pad hypersensitivity.
Results
The chronic orofacial neuropathic pain model is induced after surgical placement of chromic gut suture in the infraorbital nerve fissure in the maxillary bone. Slight compression and chemical effects of the chromic gut suture on the portion of the infraorbital nerve contacted cause mild nerve trauma. Nerve edema is observed in the contacting infraorbital nerve bundle as well as macrophage infiltration in the trigeminal ganglia. Centrally in the spinal trigeminal nucleus, increased immunoreactivity for an activated microglial marker is evident (OX42, postoperative day 70). Mechanical thresholds of the affected whisker pad are significantly decreased on day 3 after chromic gut suture placement, persisting at least 10 weeks. The mechanical allodynia is reversed by suppression of microglial activation. Cold allodynia was detected at 4 weeks.
Conclusions
A simple, effective, and reproducible chronic mouse model mimicking clinical orofacial neuropathic pain (Type 2) is induced by placing chromic gut suture between the infraorbital nerve and the maxillary bone. The method produces mild inflammatory compression with significant continuous mechanical allodynia persisting at least 10 weeks and cold allodynia measureable at 4 weeks.
... While assays of nerve transection to branches of the trigeminal nerve also exist (209,210), they are likely more suitable to the study of mechanisms underlying pain in post-traumatic neuropathies due to, for instance, motor vehicle accident, sporting injury, or dental procedures. Transection or lesions to the TN are not putative contributors to primary TN and these assays will not be describe here. ...
Chronic primary orofacial pain (OFP) conditions such as painful temporomandibular disorders (pTMDs; i.e., myofascial pain and arthralgia), idiopathic trigeminal neuralgia (TN), and burning mouth syndrome (BMS) are seemingly idiopathic, but evidence support complex and multifactorial etiology and pathophysiology. Important fragments of this complex array of factors have been identified over the years largely with the help of preclinical studies. However, findings have yet to translate into better pain care for chronic OFP patients. The need to develop preclinical assays that better simulate the etiology, pathophysiology, and clinical symptoms of OFP patients and to assess OFP measures consistent with their clinical symptoms is a challenge that needs to be overcome to support this translation process. In this review, we describe rodent assays and OFP pain measures that can be used in support of chronic primary OFP research, in specific pTMDs, TN, and BMS. We discuss their suitability and limitations considering the current knowledge of the etiology and pathophysiology of these conditions and suggest possible future directions. Our goal is to foster the development of innovative animal models with greater translatability and potential to lead to better care for patients living with chronic primary OFP.
... Silencing of Kir4.1 led to pain-like behavior. The AMPA receptor, which is both a glutamate receptor and cation channel, was investigated in a study by Miyamoto et al. 52 Knockout mice of the Glur2 and Glur3 subunits of the AMPA receptor attenuated trigeminal neuropathic pain, as well as Kir4.1 was reduced in TG after CCI-ION. Potassium channel genes Kcnip3, Kcnj6, Kcnq2 and Kcnq3 were downregulated after CCI-ION in rats in a study by Korczeniewska et al. 45 Kcnk18 gene, coding for the TWIK-related spinal cord K þ channel (TRESK), was investigated in two separate studies. ...
Trigeminal neuralgia (TN) is a severe facial pain disease of unknown cause and unclear genetic background. To examine the existing knowledge about genetics in TN, we performed a systematic study asking about the prevalence of familial trigeminal neuralgia, and which genes that have been identified in human TN studies and in animal models of trigeminal pain. MedLine, Embase, Cochrane Library and Web of Science were searched from inception to January 2021. 71 studies were included in the systematic review. Currently, few studies provide information about the prevalence of familial TN; the available evidence indicates that about 1–2% of TN cases have the familial form. The available human studies propose the following genes to be possible contributors to development of TN: CACNA1A, CACNA1H, CACNA1F, KCNK1, TRAK1, SCN9A, SCN8A, SCN3A, SCN10A, SCN5A, NTRK1, GABRG1, MPZ gene, MAOA gene and SLC6A4. Their role in familial TN still needs to be addressed. The experimental animal studies suggest an emerging role of genetics in trigeminal pain, though the animal models may be more relevant for trigeminal neuropathic pain than TN per se. In summary, this systematic review suggests a more important role of genetic factors in TN pathogenesis than previously assumed.
... Algunos de los cambios más importantes en la producción de dolor neuropático se dan a nivel del subnúcleo caudalis, en el área espinal (Worsley, Allen, Billinton, King y Boissonade, 2014). Una vez activado el sistema trigeminal, es posible que se mantenga un estado de descargas ectópicas, produciendo una retroalimentación a nivel del núcleo caudalis (Oshinsky, 2014), en el cual se producen cambios en las cadenas de aminoácidos que contribuyen a la persistencia del dolor (Miyamoto et al., 2011). ...
Resumen: El dolor como condición patológica se comporta de forma distinta al dolor agudo o secundario, con la acti-vación de una serie de eventos fisiológicos y conductuales que lo convierten en un reto para el clínico. En el caso del dolor neuropático, existe evidencia de la presencia de alteraciones estructurales o funcionales en el sistema nervioso, que involucra una gran variabilidad de mecanismos y características. Este artículo busca ser un material de referencia de utilidad para la práctica clínica en el abordaje de las condiciones neuropáticas en la región orofacial, utilizando como base una revisión bibliográfica para contestar algunas preguntas comunes, como la frecuencia de la condición, patofisiología, patogénesis, estrategias de diagnóstico y tratamiento. Esta revisión no pretende ser exhaustiva, sino ofrecer un vistazo de algunos de los elementos que deben considerar-se, y motivar al clínico a mantenerse actualizado y comprender la complejidad del dolor crónico. El impacto en la vida del paciente al entender su condición y tener opciones para mejorar su funcionalidad y bienestar debe ser la prioridad para quienes deciden hacer suya esta tarea, es decir, ofrecer alivio a quienes padecen de dolor neuropático. Palabras clave Dolor neuropático, dolor crónico, neuropatías.
Abstract: Chronic Pain is a pathological condition which behaves differently from acute or secondary pain, with the activation of several mechanisms that might pose a challenge to the clinician. Neuropathic pain is an example of this type of conditions, and presents with structural or functional alterations in the nervous system, involving a great variability of mechanisms and characteristics. This article aims to be a reference material for clinical practice in the approach of neuropathic conditions in the orofacial region, using evidence from the literature to answer frequent questions, such as the frequency of the condition, pathophysiology, pathogenesis, strategies of diagnosis and treatment. This review aims to offer a glimpse of some of the elements that should be considered
... Toujours au niveau du caudalis, mais après régénérescence du nerf alvéolaire inférieur, les neurones WDR et LTMR présentent une augmentation de leur excitabilité. Cela suggère l'implication des neurones WDR et LTMR dans l'apparition et la persistance de la douleur neuropathique oro-faciale.Au niveau du caudalis, les récepteurs NMDA, AMPA, kainate et mGluR (G-protein coupled metabotropic) sont naturellement impliqués dans la transmission de l'information douloureuse(Miyamoto et al., 2011.La voie des mitogen-activated protein kinases (MAPK), voie de transduction cellulaire impliquant ERK au niveau des neurones, joue aussi un rôle dans la sensibilisation centrale trigéminale.Suzuki et al., (2013) ont mis en évidence une hyperalgésie aux stimuli mécaniques et thermiques sur la lèvre supérieure des rats après constriction chronique du nerf infra-orbitaire. Ce comportement s'associe à une augmentation ipsilatérale de la phosphorylation de ERK (phospho-ERK) au niveau du sous-noyau caudal. ...
Les syndromes douloureux chroniques, inflammatoires ou neuropathiques, se caractérisent par une hypersensiblitité douloureuse, sous forme de douleurs spontanées et d’allodynie et d’hyperalgésie. L’isoforme γ de la protein kinase C (PKCγ), concentrée dans un type spécifique d’interneurones de la couche II interne (IIi) de la corne dorsale de la moelle ou du sous-noyau caudal du trijumeau (Sp5C) est impliqué dans mécanismes centraux de l’allodynie mécanique, une condition dans laquelle le toucher provoque une douleur. Nous avons utilisé des techniques comportementales et immunohistochimiques dans le système trigéminal.Le rôle de la PKCγ dans le développement de l’allodynie mécanique est bien établi après lésion nerveuse périphérique. Par contre, il l’est beaucoup moins dans l’allodynie d’origine inflammatoire. Nous avons testé l’hypothèse que l’allodynie mécanique persistante à la suite d’une inflammation périphérique provoquée par l’adjuvent complet de Freund (‘complete Freund’s adjuvant’ ou CFA) est bien due à une activation de la PKCγ. L’injection sous-cutanée de CFA au niveau de la zone d’insertion des vibrisses induit une allodynie persistante spécifiquement statique. L’immunomarquage phopho-ERK1/2 montre que l’expression de cette allodynie s’accompagne d’une activation d’interneurones des couches I-IIe et IIi-IIIe, dont des interneurones PKCγ de la couche IIi. Cette allodynie statique est supprimée par l’application intracisternale de l’antagoniste PKCγ, KIG31-1, avant l’injection de CFA, mais pas 3 jours après l’injection de CFA. Ainsi, comme pour l’allodynie mécanique neuropathique, l’activation de la PKCγ est nécessaire au développement de l’allodynie mécanique inflammatoire.Nous avons aussi examiné si l’activation de la PKCγ est suffisante pour le développement de l’allodynie mécanique. L’injection intracisternale de phorbol ester, 12,13-dibutyrate (PDBu), un activateur de la PKCγ, induit simultanément une allodynie mécanique statique et dynamique de la face. L’immunoréactivité phospho-ERK1/2 révèle que l’expression de ces deux allodynies mécaniques s’accompagne de la même activation d’interneurones des couches I-IIe et IIi-IIIe, dont des interneurones PKCγ de la couche IIi . Les effets de l’application de PDBu sont bloqués par l’application simultanée de KIG31-1.L’activation de la PKCγ seule est suffisante pour que se développe une allodynie mécanique, à la fois statique et dynamique. On sait que les interneurones PKCγ de la couche IIi sont directement activés par des afférences myélinisées mécaniques non nociceptives. Le niveau d’activation de la PKCγ contrôlerait la transmission de cette information vers les neurones de projection de la couche I, et donc la transformation du toucher en douleur.
... In line with this hypothesis, numerous studies indicate an increase in ERK and Akt phosphorylations by AMPAR activation in neural and non neural cells. [67][68][69][70] This study thus sheds a light on the potential effects of EAAs/AMPAR signaling in the spermatogenesis in vitro and provides a reference for a better understanding of spermatogenesis. ...
Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d‐aspartate ( d‐Asp) and N‐methyl‐ d‐aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC‐1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC‐1 cells. Subsequently, GC‐1 cells were incubated with medium containing l‐Glu, d‐Glu, d‐Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA‐treated GC‐1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p‐extracellular signal‐regulated kinase (ERK), p‐Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l‐Glu‐, d‐Glu‐, and NMDA‐treated GC‐1 cells. At 30 minutes and 2 hours of incubation, treated GC‐1 cells showed significantly higher expression levels of p‐ERK and p‐Akt. A consequent increase of PCNA and Aurora B expressions was induced by l‐Glu and NMDA, but not by d‐Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA‐treated GC‐1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.
... the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) neurons, as well as in trigeminal ganglion (TG) neurons [10,24]. Some recent studies reported that MAPK pathways in Vc and C1-C2 neurons were important in the development of orofacial neuropathic pain in a rat model of trigeminal nerve injury [12,19]. ION-CCI, inferior alveolar nerve transection, and upper cervical spinal nerve transection were reported to enhance phosphorylation of extracellular signal-regulated kinase (ERK) in Vc and C1-C2 neurons [15,22,25]. ...
We investigated the effect of botulinum neurotoxin type A (BoNT-A) on mechanical allodynia and hyperalgesia associated with infraorbital nerve constriction (ION-CCI) in rats. ION-CCI rats received a subcutaneous BoNT-A injection into the whisker pad area on day 7 postoperatively and underwent pain assessment on days 14 and 21 postoperatively. Rats were assigned to one of four treatment groups (n = 5 each): ION-CCI + BoNT-A 20 pg (low-dose group), ION-CCI + BoNT-A 200 pg (high-dose group), ION-CCI + saline, and Sham. Mechanical allodynia and hyperalgesia were evaluated preoperatively (baseline) and on days 7, 14, and 21 postoperatively. After noxious mechanical stimulation of whisker pad skin, the number and distribution pattern of the phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons were analyzed in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2). On day 21, nocifensive behavior was attenuated by high-dose but not low-dose BoNT-A administration. In addition, after noxious mechanical stimulation of whisker pad skin, the numbers of pERK-IR cells in the superficial laminae of Vc and C1-C2 were significantly lower in the high-dose BoNT-A group than in the ION-CCI + saline group. The present findings suggest that, by suppressing Vc neuronal activity, high-dose intradermal injection of BoNT-A at the site of ION innervation alleviates mechanical facial allodynia and hyperalgesia associated with ION-CCI.
... The partial transection of the right infraorbital nerve (ION) was performed by an intraoral access. 26,28 In detail, by a pair of sharp honed forceps with hooked tip, the right ION was exposed through a 2 to 2.5-mm long incision in the gingivo-buccal mucosa beginning just cranial to the first molar (supplementary Fig. 1, available online as Supplemental Digital Content at http://links. lww.com/PAIN/A186). ...
Clinical studies show that chronic pain can spread to adjacent or even distant body regions in some patients. However, little is known about how this happens. In the present study, we found that partial infraorbital nerve transection (p-IONX) in MRL/MPJ mice induced not only marked and long-lasting orofacial thermal hyperalgesia, but also thermal hyperalgesia from day 3 postoperatively (PO) and tactile allodynia from day 7 PO in bilateral hindpaws. Pain sensitization in the hindpaw was negatively correlated with facial thermal hyperalgesia at early but not late stage after p-IONX. Following a rapid activation of c-Fos, excitability and excitatory synaptic neurotransmission in lumbar dorsal horn neurons were elevated from day 3 and day 7 PO, respectively. In addition, microglial activation following p-IONX transmitted caudally from the Vc in the medulla to lumber dorsal horn in a time-dependent manner. Inhibition of microglial activation by minocycline at early but not late stage after p-IONX postponed and attenuated pain sensitization in the hindpaw. These results indicate that neuropathic pain following p-IONX in MRL/MPJ mice spreads from the orofacial region to distant somatic regions and that a rostral-caudal transmission of central sensitization in the spinal cord is involved in the spreading process of pain hypersensitivity.
... In contrast, neither GluR1 nor pGluR1 is changed in the rat spinal dorsal horn after L5 spinal nerve ligation [55]. The GluR2 and GluR3 subunits of AMPA receptor are implicated in the trigeminal nerve injury-mediated hyperalgesia [56]. One possibility for these differences in study findings could be due to the use of different models and/or different locations studied. ...
Neuropathic pain evoked by nerve injury is frequently accompanied by deterioration of emotional behaviors, but the underlying signaling mechanisms remain elusive. Glutamate (Glu) is the major mediator of excitatory synaptic transmission throughout the brain, and abnormal activity of the glutamatergic system has been implicated in the pathophysiology of pain and associated emotional comorbidities. In this study we used the partial sciatic nerve ligation (PSNL) model of neuropathic pain in rats to characterize the development of anxiety-like behavior, the expression of glutamatergic receptors, and the phosphorylation of extracellular signal-regulated kinase (ERK) in the hippocampus, the region that encodes memories related to emotions.
We found that the mechanical withdrawal threshold was significantly reduced and an anxiety-like behavior was increased as determined via open field tests and elevated plus-maze tests at 28 days after injury. No significant differences were found in the ratio of sucrose preference and immobility time detected by sucrose preference tests and forced swimming tests respectively, possibly due to the timing factor. The expression of N-methyl-D-aspartate (NMDA) receptor subtypes NR1 and NR2B, but not NR2A, GluR1, or GluR2 (the main subtype of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] receptor) in the hippocampus of injured rats was significantly reduced. Moreover, PSNL resulted in decreased phosphorylation of ERK1/2 in the hippocampus. Intriguingly, treatment with D-serine (a co-agonist of NMDA receptor, 1 g/kg intraperitoneally) reduced the anxiety-like behavior but not the mechanical hypersensitivity induced by PSNL.
PSNL can induce significant anxiety-like but not depression-like behavior, and trigger down-regulation of NMDA but not AMPA receptors in the hippocampus at 28 days after injury.
... Excitation of primary trigeminal afferents occur through ionotropic calcium and sodium ion-gated channels within cell membranes, and become depolarized when excitatory neurotransmitters such as glutamate bind to NMDA receptor subtypes NR2A/B [68]. AMPA receptors, specifically the GluR2 and GluR3 subtypes have also been shown toincrease neural excitation within the nucleus caudalis and upper cervical spinal neuronsupon injury to the trigeminal nerve [69]. Purinergic receptor subtypes P2X3/7 have also been shown to facilitate both peripheral sensitization among trigeminal nerve afferents, and also central sensitization among neurons within the caudalis region of the VBSNC [56,[70][71][72]. ...
Chronic orofacial pain is a multifaceted health problem that like many other forms ofchronic pain bears deleterious effects upon quality of life as well as psychological andphysiological well-being. Due to a poorly understood etiology, effective treatment strategies are lacking and tend to lack a guiding integrative conceptual framework toform the basis and development of intervention. This review seeks to provide an updatedreview of the comorbid psychological disorders and characteristics that are common among chronic orofacial pain patients, while also examining the pathophysiological mechanisms underlying orofacial pain. Rather than consider the emotional, cognitive, andneuroendocrine influences upon pain perception and severity individually, these factorsshould be viewed as working in concert with one another. It is this interplay amongst distinct psychological characteristics governing the patient along with physiologicalmechanisms that exacerbate the pain. Together, the goal is to identify unique characteristics surrounding orofacial pain and offer some plausible insights for effectivetreatment outcomes.
... Prior to any treatment, a baseline mechanical threshold (at t = d0) for eliciting the head withdrawal responses was determined in all test groups over 3training sessions on 2 successive days. Mice were tested for their responses to mechanical stimulation in the masseter muscle region according to modified methods of Imbe et al [30] and Miyamoto et al [31]. The test results were recorded with a calibrated Electronic von Frey TM Anesthesiometer (IITC Inc., Woodland Hills, CA). ...
The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.
... Like originally reported by Vos et al. (1994) in this model, we also observed contralateral behavioral hypersensitivity, even though less severe compared with that from the injury side, in a small percentage of CCI-ION rats. This is consistent with the bilateral effects in ION or inferior alveolar nerve injury models observed in other studies Miyamoto et al., 2011;Cao et al., 2013). The bilateral effects could derive from dysregulation of circulating factors such as pro-inflammatory cytokines (Anderson & Rao, 2001), nerve growth factors (Anderson et al., 1998;Anderson & Rao, 2001), or descending facilitatory mechanisms (Shimizu et al., 2009;Chai et al., 2012) by unilateral nerve injury. ...
Injury to the trigeminal nerve often results in the development of chronic pain states including tactile allodynia, or hypersensitivity to light touch, in orofacial area, but its underlying mechanisms are poorly understood. Peripheral nerve injury has been shown to cause up-regulation of thrombospondin-4 (TSP4) in dorsal spinal cord that correlates with neuropathic pain development. In this study, we examined whether injury-induced TSP4 is critical in mediating orofacial pain development in a rat model of chronic constriction injury to the infraorbital nerve.
Orofacial sensitivity to mechanical stimulation was examined in a unilateral infraorbital nerve ligation rat model. The levels of TSP4 in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 spinal cord (Vc/C2) from injured rats were examined at time points correlating with the initiation and peak orofacial hypersensitivity. TSP4 antisense and mismatch oligodeoxynucleotides were intrathecally injected into injured rats to see if antisense oligodeoxynucleotide treatment could reverse injury-induced TSP4 up-regulation and orofacial behavioural hypersensitivity.
Our data indicated that trigeminal nerve injury induced TSP4 up-regulation in Vc/C2 at a time point correlated with orofacial tactile allodynia. In addition, intrathecal treatment with TSP4 antisense, but not mismatch, oligodeoxynucleotides blocked both injury-induced TSP4 up-regulation in Vc/C2 and behavioural hypersensitivity.
Our data support that infraorbital nerve injury leads to TSP4 up-regulation in trigeminal spinal complex that contributes to orofacial neuropathic pain states. Blocking this pathway may provide an alternative approach in management of orofacial neuropathic pain states.
... Very little research has focused on the possible circuitry and mechanisms involved in the development of contralateral orofacial hyperalgesia after deep tissue orofacial injury. Traditionally, orofacial pain research has focused on the activation of the subnucleus caudalis (Vc) of the spinal trigeminal nucleus (STN)34567 . Despite the emphasis of orofacial pain research in the Vc, the subnucleus interpolaris/subnucleus caudalis transition zone (Vi/Vc) of the STN has been more recently shown to be involved in mechanisms of deep tissue trigeminal pain891011. ...
Background
Our previous studies have shown that complete Freund’s adjuvant (CFA)-induced masseter inflammation and microinjection of the pro-inflammatory cytokine interleukin-1β (IL-1β) into the subnucleus interpolaris/subnucleus caudalis transition zone of the spinal trigeminal nucleus (Vi/Vc) can induce contralateral orofacial hyperalgesia in rat models. We have also shown that contralateral hyperalgesia is attenuated with a lesion of the rostral ventromedial medulla (RVM), a critical site of descending pain modulation. Here we investigated the involvement of the RVM-Vi/Vc circuitry in mediating contralateral orofacial hyperalgesia after an injection of CFA into the masseter muscle.
Results
Microinjection of the IL-1 receptor antagonist (5 nmol, n=6) into the ipsilateral Vi/Vc attenuated the CFA-induced contralateral hyperalgesia but not the ipsilateral hyperalgesia. Intra-RVM post-treatment injection of the NK1 receptor antagonists, RP67580 (0.5-11.4 nmol) and L-733,060 (0.5-11.4 nmol), attenuated CFA-induced bilateral hyperalgesia and IL-1β induced bilateral hyperalgesia. Serotonin depletion in RVM neurons prior to intra-masseter CFA injection prevented the development of contralateral hyperalgesia 1–3 days after CFA injection. Inhibition of 5-HT3 receptors in the contralateral Vi/Vc with direct microinjection of the select 5-HT3 receptor antagonist, Y-25130 (2.6-12.9 nmol), attenuated CFA-induced contralateral hyperalgesia. Lesions to the ipsilateral Vc prevented the development of ipsilateral hyperalgesia but did not prevent the development of contralateral hyperalgesia.
Conclusions
These results suggest that the development of CFA-induced contralateral orofacial hyperalgesia is mediated through descending facilitatory mechanisms of the RVM-Vi/Vc circuitry.
The spinal N-methyl-D-aspartate receptor (NMDAR), particularly their subtypes NR2A and NR2B, plays pivotal roles in neuropathic and inflammatory pain. However, the roles of NR2A and NR2B in orofacial pain and the exact molecular and cellular mechanisms mediating nervous system sensitization are still poorly understood. Here, we exhaustively assessed the regulatory effect of NMDAR in mediating peripheral and central sensitization in orofacial neuropathic pain. Von-Frey filament tests showed that the inferior alveolar nerve (IANX) induced ectopic allodynia behavior in the whisker pad of mice. Interestingly, mechanical allodynia was reversed in mice lacking NR2A and NR2B. IANX also promoted the production of peripheral sensitization-related molecules, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, brain-derived neurotrophic factor (BDNF), and chemokine upregulation (C-C motif) ligand 2 (CCL2), and decreased the inward potassium channel (Kir) 4.1 on glial cells in the trigeminal ganglion, but NR2A conditional knockout (CKO) mice prevented these alterations. In contrast, NR2B CKO only blocked the changes in Kir4.1, IL-1β, and TNF-α and further promoted the production of CCL2. Central sensitization-related c-fos, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) were promoted and Kir4.1 was reduced in the spinal trigeminal caudate nucleus by IANX. Differential actions of NR2A and NR2B in mediating central sensitization were also observed. Silencing of NR2B was effective in reducing c-fos, GFAP, and Iba-1 but did not affect Kir4.1. In contrast, NR2A CKO only altered Iba-1 and Kir4.1 and further increased c-fos and GFAP. Gain-of-function and loss-of-function approaches provided insight into the differential roles of NR2A and NR2B in mediating peripheral and central nociceptive sensitization induced by IANX, which may be a fundamental basis for advancing knowledge of the neural mechanisms’ reaction to nerve injury.
We investigated the effects of social defeat stress (SDS) and treadmill running on masseter muscle nociception, which was quantified by the orofacial formalin test and c‐Fos and FosB immunoreactivities in the upper cervical spinal cord (C1/C2) region in male mice. After daily SDS or non‐SDS conditioning for 10 days, SDS‐conditioned mice were categorized into SDS‐susceptible versus resilient mice. Several mice, including non‐SDS‐conditioned, SDS‐susceptible, and resilient mice, were selected to assess masseter muscle nociception on Day 11. SDS conditioning for 10 days increased masseter muscle‐evoked nocifensive behaviors and c‐Fos and FosB expression in SDS‐susceptible compared to non‐SDS and resilient mice. The remaining SDS‐susceptible and non‐SDS mice were subjected to an additional 10 days of SDS plus treadmill running or sedentary sessions before assessing masseter muscle nociception on Day 21. Daily treadmill running sessions reduced enhanced masseter muscle nociception in SDS‐susceptible mice but not in non‐SDS mice. The preventive effects of daily treadmill running immediately after each SDS conditioning for 10 days on orofacial nocifensive behaviors were assessed on Day 11. Treadmill running conducted immediately after daily SDS inhibited enhanced orofacial nocifensive behaviors. These findings indicate that repeated SDS increases masseter muscle nociception, which could be prevented by daily treadmill running exercise.
Various studies proved spinal AMPA receptors were involved in the formation of neuropathic pain. In this study, we investigated the effect of methyl cinnamate (MC), a flavoring agent widely used in food and commodity industry, on CCI-induced upregulation of spinal AMPARs and pain hypersensitive behaviors. Results indicated that MC treatment dosage-dependently inhibited CCI-induced mechanical and thermal hypersensitivity. To further investigate the effect of MC after the formation of neuropathic pain, MC at the dosage of 100 mg/kg was administrated on day 7-14 on CCI rats. Results showed that MC treatment for seven days alleviated CCI-induced pain hypersensitivity after the formation of neuropathic pain. MC treatment reversed CCI-induced upregulation of GluR2, GluR3 and phosphorylation of GluR1. Further, MC dosage-dependently alleviated CCI-induced activation of mTOR and the downstream p70s6k. MC dosage-dependently induced activation of AMPK. All the MC-induced effects in CCI rats were completely reversed by Compound C, a AMPK inhibitor. These results meant MC treatment mitigated CCI-induced upregualtion of spinal AMPA receptors and pain hypersensitive behaviors through actviation of AMPK.
The aim of this study was to determine whether pregabalin affects nociceptive behavior and central sensitization in a trigeminal neuropathic pain model. A partial infraorbital nerve transec-tion (p-IONX) or sham operation was performed in adult male rats. Nociceptive withdrawal thresholds were tested with von Frey filaments applied to the bilateral vibrissal pads pre-and postoperatively. On postoperative day 7, the behavioral assessment was conducted before and at 30, 60, 120, and 180 minutes after and 24 hours after pregabalin (.1, 1, 10, 100 mg/kg intraperitone-ally) or saline injection. The effects of pregabalin or saline were also examined on the mechanorecep-tive field and response properties of nociceptive neurons recorded in the medullary dorsal horn at postoperative days 7 to 10. Reduced withdrawal thresholds reflecting bilateral mechanical allodynia were observed in p-IONX rats until postoperative day 28, but not in sham-operated rats. At postop-erative day 7, pregabalin significantly and dose-dependently reversed the reduced mechanical withdrawal thresholds in p-IONX rats. Pregabalin also attenuated central sensitization of the neurons, as reflected in reversal of their reduced activation threshold, increased responses to pinch/pressure, and enhanced stimulus-response function. This study provides the first documentation that pregabalin attenuates the mechanical allodynia and central sensitization that characterize this trigeminal neuro-pathic pain model, and supports its clinical use for treating craniofacial neuropathic pain. Perspective: Trigeminal nerve injury in rats produced facial mechanical hypersensitivity and trigeminal central sensitization of medullary dorsal horn neurons that were markedly attenuated by systemically administered pregabalin, suggesting its potential clinical utility for orofacial neuro-pathic pain.
Dental pain, including toothache, is one of the most prevalent types of orofacial pain, causing severe, persistent pain that has a significant negative effect on quality of life, including eating disturbances, mood changes, and sleep disruption. As the primary cause of toothache pain is injury to the uniquely innervated dental pulp, rodent models of this injury provide the opportunity to study neurobiological mechanisms of tissue injury-induced persistent pain. Here we evaluated behavioral changes in mice with a dental pulp injury (DPI) produced by mechanically exposing the pulp to the oral environment. We monitored the daily life behaviors of mice with DPI, including measures of eating, drinking, and movement. During the first 48hours, the only parameter affected by DPI was locomotion, which was reduced. There was also a significant short-term decrease in the amount of weight gained by DPI animals that was not related to food consumption. As cold allodynia is frequently observed in individuals experiencing toothache pain, we tested whether mice with DPI demonstrate an aversion to drinking cold liquids using a cold-sucrose consumption test. Surprisingly, mice with DPI increased their consumption of sucrose solution, to over 150% of baseline, regardless of temperature. Both the weight loss and increased sucrose intake in the first 2days of injury were reversed by administration of indomethacin. These findings indicate that enhanced sucrose consumption may be a reliable measure of orofacial pain in rodents, and suggest that alterations in energy expenditure and motivational behaviors are under-recognized outcomes of tooth injury.
Overuse of medications used to treat migraine headache can produce a chronic daily headache, termed medication overuse headache (MOH). Although "overuse" of opioids, triptans, and over-the-counter analgesics can all produce MOH, the neuronal mechanisms remain unknown. Headache pain is likely to be produced by stimulation of primary afferent neurons that innervate the intracranial vasculature and the resulting activation of medullary dorsal horn (MDH) neurons. The present study compared the receptive field properties of MDH dura-sensitive neurons in rats treated with morphine to those given vehicle. Animals were implanted with osmotic minipumps or pellets for sustained subcutaneous administration of morphine or vehicle 6-7 d before recording from dura-sensitive neurons. Electrical and mechanical activation thresholds from the dura were significantly lower in chronic morphine-treated animals when compared to vehicle controls. In addition, sustained morphine increased the cutaneous receptive field sizes. The presence of diffuse noxious inhibitory controls (DNICs) was examined by placing the tail in 55 degrees C water during concomitant noxious thermal stimulation of the cutaneous receptive field, usually located in the ophthalmic region. The DNIC stimulus produced significant inhibition of heat-evoked activity in vehicle- but not chronic morphine-treated animals. Inactivation of the rostral ventromedial medulla with 4% lidocaine reinstated DNICs in chronic morphine-treated animals. These results are consistent with studies demonstrating a loss of DNICs in patients that suffer from chronic daily headache and may partially explain why overuse of medication used to treat migraine can induce headaches.
Spinal cord GluR2-lacking AMPA receptors (AMPARs) contribute to nociceptive hypersensitivity in persistent pain, but the molecular mechanisms underlying this event are not completely understood. We report that complete Freund's adjuvant (CFA)-induced peripheral inflammation induces synaptic GluR2 internalization in dorsal horn neurons during the maintenance of CFA-evoked nociceptive hypersensitivity. This internalization is initiated by GluR2 phosphorylation at Ser(880) and subsequent disruption of GluR2 binding to its synaptic anchoring protein (GRIP), resulting in a switch of GluR2-containing AMPARs to GluR2-lacking AMPARs and an increase of AMPAR Ca(2+) permeability at the synapses in dorsal horn neurons. Spinal cord NMDA receptor-mediated triggering of protein kinase C (PKC) activation is required for the induction and maintenance of CFA-induced dorsal horn GluR2 internalization. Moreover, preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site impairs CFA-evoked nociceptive hypersensitivity during the maintenance period. These results suggest that dorsal horn GluR2 internalization might participate in the maintenance of NMDA receptor/PKC-dependent nociceptive hypersensitivity in persistent inflammatory pain.
Although most projection neurons in lamina I express the neurokinin 1 receptor (NK1r), we have identified a population of large multipolar projection cells that lack the NK1r, are characterized by the high density of gephyrin puncta that coat their cell bodies and dendrites, and express the transcription factor Fos in response to noxious chemical stimulation. Here we show that these cells have a very high density of glutamatergic input from axons with strong immunoreactivity for vesicular glutamate transporter 2 that are likely to originate from excitatory interneurons. However, they receive few contacts from peptidergic primary afferents or transganglionically labeled Adelta nociceptors. Unlike most glutamatergic synapses in superficial laminas, those on the gephyrin-coated cells contain the GluR4 subunit of the AMPA receptor. A noxious heat stimulus caused Fos expression in 38% of the gephyrin-coated cells but in 85% of multipolar NK1r-immunoreactive cells. These findings are consistent with the suggestion that there is a correlation between function and morphology for lamina I neurons but indicate that there are at least two populations of multipolar neurons that differ in receptor expression, excitatory inputs, and responses to noxious stimulation. Although there are only approximately 10 gephyrin-coated cells on each side per segment in the lumbar enlargement, they constitute approximately 18% of the lamina I component of the spinothalamic tract at this level, which suggests that they play an important role in transmission of nociceptive information to the cerebral cortex. Our results also provide the first evidence that postsynaptic GluR4-containing AMPA receptors are involved in spinal nociceptive transmission.
In order to evaluate mechanisms that may underlie the sensitization of trigeminal spinal subnucleus caudalis (Vc; the medullary dorsal horn) and upper cervical spinal cord (C1-C2) nociceptive neurons to heat, cold and mechanical stimuli following topical capsaicin treatment of the facial skin, nocifensive behaviors as well as phosphorylation of extracellular regulated-kinase (pERK) in Vc and C1-C2 neurons were studied in rats.
Compared to vehicle application, capsaicin application to the lateral facial skin produced 1 hour later a flare in the skin, and also induced significantly greater nocifensive behaviors to heat, cold or mechanical stimulus of the lateral facial skin. The intrathecal (i.t.) injection of the MEK inhibitor PD98059 markedly attenuated the nocifensive behaviors to these stimuli in capsaicin-treated rats. Moreover, the number of pERK-like immunoreactive (pERK-LI) cells in Vc and C1-C2 was significantly larger following the heat, cold and mechanical stimuli in capsaicin-treated rats compared with vehicle-treated rats. The number of pERK-LI cells gradually increased following progressive increases in the heat or mechanical stimulus intensity and following progressive decrease in the cold stimulus. The ERK phosphorylation in Vc and C1-C2 neurons was strongly inhibited after subcutaneous injection of the capsaicin antagonist capsazepine in capsaicin-treated rats.
The present findings revealed that capsaicin treatment of the lateral facial skin causes an enhancement of ERK phosphorylation in Vc and C1-C2 neurons as well as induces nocifensive behavior to heat, cold and mechanical simulation of the capsaicin-treated skin. The findings suggest that TRPV1 receptor mechanisms in rat facial skin influence nociceptive responses to noxious cutaneous thermal and mechanical stimuli by inducing neuroplastic changes in Vc and C1-C2 neurons that involve in the MAP kinase cascade.
Video recordings of free behavior and responses to mechanical facial stimulation were analyzed to assess whether chronic constriction injury (CCI) to the rat's infraorbital nerve (IoN) results in behavioral alterations indicative of neuropathic pain. A unilateral CCI was produced by placing loose chromic gut ligatures around the IoN. After CCI to the IoN, rats exhibited changes in both non-evoked and evoked behavior. Behavioral changes developed in two phases. Early after CCI (postoperative days 1-15), rats showed increased face-grooming activity with face-wash strokes directed to the injured nerve territory, while the responsiveness to stimulation of this area was decreased. Later after CCI (postoperative days 15-130), the prevalence of asymmetric face grooming was reduced but remained significantly increased compared to control rats. The early hyporesponsiveness was abruptly replaced by an extreme hyperresponsiveness: all stimulus intensities applied to the injured nerve territory evoked the "maximal" response (brisk head withdrawal, avoidance behavior plus directed face grooming). This response was never observed in control rats. Concurrently, IoN ligation rats showed a limited increase in the responsiveness to stimulation of the contralateral IoN territory, and around postoperative days 30-40 the responsiveness to stimulation of facial areas outside the IoN territories also increased. The hyperresponsiveness to stimulation of the ligated IoN territory slightly decreased from 60 d postoperative. Throughout the study, IoN ligation rats showed decreased exploratory behavior, displayed more freezing-like behavior, had a slower body weight gain, and a higher defecation rate, compared to control rats. The behavioral alterations observed after CCI to the IoN are indicative of severe sensory disturbances within the territory of the injured nerve: mechanical allodynia develops after a period of relative hypo-/anesthesia during which behavioral signs of recurrent spontaneous, aversive (possibly painful) sensations (paresthesias/dysesthesias) are maximal.
Postsynaptic Ca2+ elevation during synaptic transmission is an important trigger for short- and long-term changes in synaptic strength in the vertebrate central nervous system. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors, a subfamily of glutamate receptors, mediate much of the excitatory synaptic transmission in the brain and spinal cord. It has been shown that a subtype of the AMPA receptor is Ca2+-permeable and is present in the subpopulations of neurons. When synaptically localized, these receptors should mediate postsynaptic Ca2+ influx, providing a trigger for changes in synaptic strength. Here we show that Ca2+-permeable AMPA receptors are synaptically localized on a subpopulation of dorsal horn neurons, and that they provide a synaptically gated route of Ca2+ entry, and that activation of these receptors strengthens synaptic transmission mediated by AMPA receptors. This pathway for postsynaptic Ca2+ influx may provide a new form of activity-dependent modulation of synaptic strength.
Secondary trigeminal neuralgia (STN) follows an injury to the trigeminal nerve or one of its branches. Although rare, this condition results in great suffering and it is notoriously difficult to treat. The experimental analysis of painful neuropathy due to damage to the innervation of the limbs (e.g., the sciatic nerve) has progressed rapidly in recent years, but very few reports have appeared concerning experimental neuropathy in the trigemenial region. We report here an experimental rat model of trigeminal neuropathic pain produced by a chronic constriction injury to the infraorbital nerve (CCI-ION), and on a method that detects heat-evoked pain-related behavior. Rats with the CCI-ION have clear signs of heat-hyperalgesia when stimulated on the snout (the vibrissal pad). The hyperalgesia is seen both ipsi- and contralateral to the side of nerve injury, but is significantly more severe ipsilaterally, and lasts about 12 days.
In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.
Here, we show that N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission was also observed when an anti-NSF monoclonal antibody (mAb) was loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function.
We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.
Synaptic clustering of neurotransmitter receptors is crucial for efficient signal transduction and integration in neurons. PDZ domain-containing proteins such as PSD-95/SAP90 interact with the intracellular C termini of a variety of receptors and are thought to be important in the targeting and anchoring of receptors to specific synapses. Here, we show that PICK1 (protein interacting with C kinase), a PDZ domain-containing protein, interacts with the C termini of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in vitro and in vivo. In neurons, PICK1 specifically colocalizes with AMPA receptors at excitatory synapses. Furthermore, PICK1 induces clustering of AMPA receptors in heterologous expression systems. These results suggest that PICK1 may play an important role in the modulation of synaptic transmission by regulating the synaptic targeting of AMPA receptors.
The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors. NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture (Gu et al., 1996) and expressed by dorsal horn neurons in situ (Nagy et al., 1994; Engelman et al., 1997). We used kainate-induced cobalt uptake as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and combined this with markers of nociceptive primary afferents in the postnatal rat dorsal horn. We have shown that cobalt-positive neurons are located in lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1 receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the nociceptive pathway. On the basis of all of these observations, we propose that Ca2+-permeable AMPA receptors are localized to mediate transmission of nociceptive information.
To monitor changes in α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the
AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was
transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron
microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation
induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking
events required synapticN-methyl-d-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor–mediated transmission observed during
long-term potentiation and activity-dependent synaptic maturation.
The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane.
Activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase in dorsal horn neurons of the spinal cord by peripheral noxious stimulation contributes to short-term pain hypersensitivity. We investigated ERK activation by peripheral inflammation and its involvement in regulating gene expression in the spinal cord and in contributing to inflammatory pain hypersensitivity. Injection of complete Freund's adjuvant (CFA) into a hindpaw produced a persistent inflammation and a sustained ERK activation in neurons in the superficial layers (laminae I-IIo) of the dorsal horn. CFA also induced an upregulation of prodynorphin and neurokinin-1 (NK-1) in dorsal horn neurons, which was suppressed by intrathecal delivery of the MEK (MAP kinase kinase) inhibitor U0126. CFA-induced phospho-ERK primarily colocalized with prodynorphin and NK-1 in superficial dorsal horn neurons. Although intrathecal injection of U0126 did not affect basal pain sensitivity, it did attenuate both the establishment and maintenance of persistent inflammatory heat and mechanical hypersensitivity. Activation of the ERK pathway in a subset of nociceptive spinal neurons contributes, therefore, to persistent pain hypersensitivity, possibly via transcriptional regulation of genes, such as prodynorphin and NK-1.
We investigated electrophysiological changes in chronically axotomized and neighboring intact dorsal root ganglion (DRG) neurons in rats after either a peripheral axotomy consisting of an L5 spinal nerve ligation (SNL) or a central axotomy produced by an L5 partial rhizotomy (PR). SNL produced lasting hyperalgesia to punctate indentation and tactile allodynia to innocuous stroking of the foot ipsilateral to the injury. PR produced ipsilateral hyperalgesia without allodynia with recovery by day 10. Intracellular recordings were obtained in vivo from the cell bodies (somata) of axotomized and intact DRG neurons, some with functionally identified peripheral receptive fields. PR produced only minor electrophysiological changes in both axotomized and intact somata in L5 DRG. In contrast, extensive changes were observed after SNL in large- and medium-sized, but not small-sized, somata of intact (L4) as well as axotomized (L5) DRG neurons. These changes included (in relation to sham values) higher input resistance, lower current and voltage thresholds, and action potentials with longer durations and slower rising and falling rates. The incidence of spontaneous activity, recorded extracellularly from dorsal root fibers in vitro, was significantly higher (in relation to sham) after SNL but not after PR, and occurred in myelinated but not unmyelinated fibers from both L4 (9.1%) and L5 (16.7%) DRGs. We hypothesize that the changes in the electrophysiological properties of axotomized and intact DRG neurons after SNL are produced by a mechanism associated with Wallerian degeneration and that the hyperexcitability of intact neurons may contribute to SNL-induced hyperalgesia and allodynia.
Activity-dependent changes in synaptic function are believed to underlie the formation of memories. A prominent example is long-term potentiation (LTP), whose mechanisms have been the subject of considerable scrutiny over the past few decades. I review studies from our laboratory that support a critical role for AMPA receptor trafficking in LTP and experience-dependent plasticity.
The quantity of neurotransmitter released into the synaptic cleft, the reliability with which it is released, and the response of the postsynaptic cell to that transmit- ter all contribute to the strength of a synaptic connection. The presynaptic nerve terminal is a major regulatory site for activity-dependent changes in synaptic func- tion. Ionotropic receptors for the inhibitory amino acid GABA, expressed on the presynaptic terminals of crustacean motor axons and vertebrate sensory neurons, were the first well-defined mechanism for the heterosynaptic transmitter-mediated regulation of transmitter release. Recently, presynaptic ionotropic receptors for a large range of transmitters have been found to be widespread throughout the central and peripheral nervous systems. In this review, we first consider some general theoretical issues regarding whether and how presynaptic ionotropic re- ceptors are important regulators of presynaptic function. We consider the criteria that should be met to identify a presynaptic ionotropic receptor and its regulatory function and review several examples of presynaptic receptors that meet at least some of those criteria. We summarize the classic studies of presynaptic inhibition mediated by GABA-gated Cl channels and then focus on presynaptic nicotinic ACh receptors and presynaptic glutamate receptors. Finally, we briefly discuss evidence for other types of presynaptic ionotropic receptors.
Glutamate receptors of the AMPA type (AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1-4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I-III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining.
As we have shown previously, virtually all AMPAr-immunoreactive puncta were immunostained for GluR2. In laminae I-II, approximately 65% were GluR1-positive and approximately 60% were GluR3-positive, while in lamina III the corresponding values were 34% (GluR1) and 80% (GluR3). Puncta stained with antibody against the C-terminus of GluR4 (which only detects the long form of this subunit) made up 23% of the AMPAr-containing puncta in lamina I, approximately 8% of those in lamina II and 46% of those in lamina III. Some overlap between GluR1 and GluR3 was seen in each region, but in lamina I GluR1 and GluR4 were present in largely non-overlapping populations. The GluR4 puncta often appeared to outline dendrites of individual neurons in the superficial laminae. Virtually all of the AMPAr-positive puncta were immunostained for PSD-95, and 98% of PSD-95 puncta contained AMPAr-immunoreactivity. Staining for GluR1, GluR2 and GluR3 was absent in sections from mice in which these subunits had been knocked out, while the punctate staining for PSD-95 was absent in mice with a mutation that prevents accumulation of PSD-95 at synapses.
Our results suggest that virtually all glutamatergic synapses in laminae I-III of adult rat spinal cord contain AMPArs. They show that synapses in laminae I-II contain GluR2 together with GluR1 and/or GluR3, while the long form of GluR4 is restricted to specific neuronal populations, which may include some lamina I projection cells. They also provide further evidence that immunostaining for AMPAr subunits following antigen retrieval is a reliable method for detecting these receptors at glutamatergic synapses.
Glutamate receptor-interacting protein 1 (GRIP1) and GRIP2 are closely related proteins that bind GluR2-containing AMPA receptors and couple them to structural and signaling complexes in neurons. Cerebellar long-term synaptic depression (LTD) is a model system of synaptic plasticity that is expressed by persistent internalization of GluR2-containing AMPA receptors. Here, we show that genetic deletion of both GRIP1 and GRIP2 blocks LTD expression in primary cultures of mouse cerebellar neurons but that single deletion of either isoform allows LTD to occur. In GRIP1/2 double knock-out Purkinje cells, LTD can be fully rescued by a plasmid-driving expression of GRIP1 and partially rescued by a GRIP2 plasmid. These results indicate that the GRIP family comprises an essential molecular component for cerebellar LTD.
A rat model has been developed to characterize the responses of brainstem trigeminal neurons to orofacial deep and cutaneous tissue inflammation and hyperalgesia. Complete Freund’s adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. The TMJ and PO inflammation resulted in orofacial behavioral hyperalgesia and allodynia that peaked within 4–24 h and persisted for at least 2 weeks. Compared to cutaneous CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a selective upregulation of preprodynorphin mRNA in the trigeminal spinal complex, an enhanced medullary dorsal horn hyperexcitability, and a greater trigeminal Fos protein expression, a marker of neuronal activation. The Fos-LI induced by TMJ inflammation persisted longer, was more intense, particularly in the superficial laminae, and more widespread rostrocaudally. Thus, the inflammatory irritant produces a stronger effect in deep than in cutaneous orofacial tissue. As there is heavy innervation of the TMJ by unmyelinated nerve endings, a strong nociceptive primary afferent barrage is expected following inflammation. An increase in TMJ C-fiber input after inflammation and strong central neuronal activation may initiate central hyperexcitability and contribute to persistent pain associated with temporomandibular disorders. Since deep inputs may be more effective in inducing central neuronal excitation than cutaneous inputs, greater sensory disturbances may occur in pain conditions involving deep tissues than in those involving cutaneous tissues.
Ca(2+)-permeable AMPA receptors are densely expressed in the spinal dorsal horn, but their functional significance in pain processing is not understood. By disrupting the genes encoding GluR-A or GluR-B, we generated mice exhibiting increased or decreased numbers of Ca(2+)-permeable AMPA receptors, respectively. Here, we demonstrate that AMPA receptors are critical determinants of nociceptive plasticity and inflammatory pain. A reduction in the number of Ca(2+)-permeable AMPA receptors and density of AMPA channel currents in spinal neurons of GluR-A-deficient mice is accompanied by a loss of nociceptive plasticity in vitro and a reduction in acute inflammatory hyperalgesia in vivo. In contrast, an increase in spinal Ca(2+)-permeable AMPA receptors in GluR-B-deficient mice facilitated nociceptive plasticity and enhanced long-lasting inflammatory hyperalgesia. Thus, AMPA receptors are not mere determinants of fast synaptic transmission underlying basal pain sensitivity as previously thought, but are critically involved in activity-dependent changes in synaptic processing of nociceptive inputs.
We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2?1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, MycGluR2?1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.
We have examined and compared the effects of systemically applied MK-801, an NMDA receptor/channel blocker, on the wind-up and facilitation of the flexor reflex during and after conditioning stimulation (CS) of C-afferents in rats with intact sciatic nerves or 13-16 days after axotomy. In rats with intact sciatic nerves, intravenous MK-801 (0.5 mg/kg) partially reduced wind-up and totally blocked reflex facilitation following C-fiber CS to the sural nerve. In contrast, 13-16 days after unilateral section of the sciatic nerve, the same dose of MK-801 failed to reduce the wind-up and reflex facilitation following C-fiber CS to the axotomized sural nerve, although the duration of reflex facilitation was significantly shortened. These findings indicate that the involvement of NMDA receptors in mediating activity-dependent spinal hyperexcitability is substantially reduced after peripheral nerve section, possibly reflecting a reduced release of glutamate by primary sensory afferents.
AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.
AMPA glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system and play a role in the synaptic plasticity underlying learning and memory. AMPA receptors are heteromeric complexes of four homologous subunits (GluR1-4) that differentially combine to form a variety of AMPA receptor subtypes. These subunits are thought to have a large extracellular amino-terminal domain, three transmembrane domains and an intracellular carboxy-terminal domain. AMPA receptors are localized at excitatory synapses and are not found on adjacent inhibitory synapses enriched in GABA(A) receptors. The targeting of neurotransmitter receptors, such as AMPA receptors, and ion channels to synapses is essential for efficient transmission. A protein motif called a PDZ domain is important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses. Here we identify a synaptic PDZ domain-containing protein GRIP (glutamate receptor interacting protein) that specifically interacts with the C termini of AMPA receptors. GRIP is a new member of the PDZ domain-containing protein family which has seven PDZ domains and no catalytic domain. GRIP appears to serve as an adapter protein that links AMPA receptors to other proteins and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain.
Here we report an interaction between AMPA receptor subunits and a single PDZ domain-containing protein called PICK1 which is known to bind protein kinase C alpha (PKC alpha). The interaction occurs within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal alternative splice variants of AMPA receptor subunits. No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif. The PDZ domain of PICK1 is required for the interaction and the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1 and GluR2 in the yeast two-hybrid assay. A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha indicating that the same domain of PICK1 binds both PKC alpha and GluRs. Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2; short splice variant) but not by GST-ct-GluR1 (long splice variant). Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody. Transient expression of both proteins in COS cells reveals colocalization and an altered pattern of distribution for each protein from when they are expressed individually. This novel interaction provides a possible regulatory mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha.
We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.
AMPA-type glutamate receptors (AMPA-Rs) mediate a majority of excitatory synaptic transmission in the brain. In hippocampus, most AMPA-Rs are hetero-oligomers composed of GluR1/GluR2 or GluR2/GluR3 subunits. Here we show that these AMPA-R forms display different synaptic delivery mechanisms. GluR1/GluR2 receptors are added to synapses during plasticity; this requires interactions between GluR1 and group I PDZ domain proteins. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors continuously; this occurs only at synapses that already have AMPA-Rs and requires interactions by GluR2 with NSF and group II PDZ domain proteins. The combination of regulated addition and continuous replacement of synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained.
A rat model has been developed to characterize the responses of brainstem trigeminal neurons to orofacial deep and cutaneous tissue inflammation and hyperalgesia. Complete Freund's adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. The TMJ and PO inflammation resulted in orofacial behavioral hyperalgesia and allodynia that peaked within 4-24 h and persisted for at least 2 weeks. Compared to cutaneous CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a selective upregulation of preprodynorphin mRNA in the trigeminal spinal complex, an enhanced medullary dorsal horn hyperexcitability, and a greater trigeminal Fos protein expression, a marker of neuronal activation. The Fos-LI induced by TMJ inflammation persisted longer, was more intense, particularly in the superficial laminae, and more widespread rostrocaudally. Thus, the inflammatory irritant produces a stronger effect in deep than in cutaneous orofacial tissue. As there is heavy innervation of the TMJ by unmyelinated nerve endings, a strong nociceptive primary afferent barrage is expected following inflammation. An increase in TMJ C-fiber input after inflammation and strong central neuronal activation may initiate central hyperexcitability and contribute to persistent pain associated with temporomandibular disorders. Since deep inputs may be more effective in inducing central neuronal excitation than cutaneous inputs, greater sensory disturbances may occur in pain conditions involving deep tissues than in those involving cutaneous tissues.
Ca2+-permeable non-N-methyl-D-aspartate receptors are found in the spinal dorsal horn and represent a presumptive target for glutamatergic transmission in nociceptive processing. This study characterized the analgesic profile associated with the blockade of these spinal receptors by intrathecally delivered agents known to act at these receptors, the spider venom Joro toxin (JST) and philanthotoxin.
Philanthotoxin (0.5, 2.5, or 5 microg) or JST (5 microg) was given spinally before thermal injury to the paw. JST (5 microg) was also given 10 min before subcutaneous formalin injection, after intraplantar administration of carrageenan, and to rats that were allodynic due to tight ligation of spinal nerves. Lower doses of JST (0.25 and 1.0 microg) were given before formalin injection and testing of thermal latencies. Thermal latencies were measured using a Hargreaves box, mechanical thresholds using von Frey hairs, and formalin response by means of counting flinches.
Both agents blocked thermal injury-induced mechanical allodynia. JST (5 microg) given 1 h after carrageenan blocked induction of thermal hyperalgesia and mechanical allodynia. JST (5 microg) had no effect in the formalin test, on allodynia after spinal nerve ligation, or when given 3 h after carrageenan. The lowest dose (0.25 microg JST) at pretreatment intervals of 60-120 min resulted in modest hypoalgesia during phase 1 formalin and thermal testing.
The behavioral effect of intrathecal Ca2+-permeable non-N-methyl-D-aspartate antagonists indicates an important role for this spinal receptor in regulating hyperalgesic states induced by tissue injury and inflammation and reveals an action that is distinct from those observed with other glutamate receptor antagonists.
The effects of inferior alveolar nerve (IAN) transection on escape behavior and MDH neuronal activity to noxious and nonnoxious stimulation of the face were precisely analyzed. Relative thresholds for escape from mechanical stimulation applied to the whisker pad area ipsilateral to the transection were significantly lower than that for the contralateral and sham-operated whisker pad until 28 days after the transection, then returned to the preoperative level at 40 days after transection. A total of 540 neurons were recorded from the medullary dorsal horn (MDH) of the nontreated naive rats [low-threshold mechanoreceptive (LTM), 27; wide dynamic range (WDR), 31; nociceptive specific (NS), 11] and sham-operated rats with skin incision (LTM, 34; WDR, 30; NS, 23) and from the ipsilateral (LTM, 82; WDR, 82; NS, 31) and contralateral MDH relative to the IAN transection (LTM, 77; WDR, 82; NS, 33). The electrophysiological properties of these neurons were precisely analyzed. Background activity of WDR neurons on the ipsilateral side relative to the transection was significantly increased at 2-14 days after the operation as compared with that of naive rats. Innocuous and noxious mechanical-evoked responses of LTM and WDR neurons were significantly enhanced at 2-14 days after IAN transection. The mean area of the receptive fields of WDR neurons was significantly larger on the ipsilateral MDH at 2-7 days after transection than that of naive rats. We could not observe any modulation of thermal responses of WDR and NS neurons following IAN transection. Also, no MDH neurons were significantly affected in the rats with sham operations. The present findings suggest that the increment of neuronal activity of WDR neurons in the MDH following IAN transection may play an important role in the development of the mechano-allodynia induced in the area adjacent to the area innervated by the injured nerve.
No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA(A) receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.
The aim of the present study was to demonstrate the convergence of inputs from masseter muscle (MM) and tooth pulp (TP) onto C1 spinal neurons and to determine whether the afferent fibers express the functional vanilloid receptor (VR1). Extracellular single-unit recordings were made from 61 C1 units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram signal in pentobarbital anesthetized rats. Eighty-four percent of C1 neurons responding to TP stimulation also responded to the ipsilateral MM stimulation. Of these neurons, 61% were considered to be afferent inputs from Adelta-fibers and the remaining units (39%) were C-fibers, based on calculation of the nerve conduction velocity. Intramuscular injection of capsaicin (0.05 and 0.1%) produced a reduction in a MM-induced C1 neuronal activity in a dose-dependent manner and this effect was antagonized by pretreatment with an antagonist of VR1, capsazepine. Some of these units were also excited by noxious heat stimulation (> 43 degrees C). The trigeminal root ganglion (TRG) neurons that innervated the MM were retrogradely labeled with Fluorogold (FG) and the small-diameter FG-labeled TRG neurons expressed the immunoreactivity for VR1. After intramuscular mustard oil injection (noxious chemical stimulation), the C1 neuronal activity induced by both touch and pinch stimuli was enhanced and their receptive field sizes were significantly expanded. These changes were reversed within 15-20 min. These results suggest that there may be the convergence of noxious afferents inputs from the MM and TP afferents on the same C1 neurons in rats, and that the afferent fibers expressing the functional VR1 may contribute to the hyperalgesia and/or referred pain associated with temporomandibular joint disorder.
A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.
The aim of this study was to characterize the properties of somatosensory neurons in the first 2 cervical spinal dorsal horns (C1 and C2 DHs) and compare them with those previously described for the rostral subnucleus caudalis (rVc). A total of 74 nociceptive neurons classified as wide-dynamic-range (WDR) or nociceptive-specific (NS), as well as 72 low-threshold mechanoreceptive (LTM) neurons, was studied in urethane/chloralose-anesthetized rats. The majority of LTM neurons were located in laminae III/IV and had a small mechanoreceptive field (RF) that included the posterior face and cervical tissues. In contrast, the nociceptive neurons were located in laminae I/II or V/VI, and the RF of each C1 and C2 DH nociceptive neuron included a part of the face and in 47% of them the RF included a region supplied by upper cervical afferents. There was a gradual caudal shift in the neuronal RF from nasal/intraoral tissues towards the neck as recording sites progressed from rVc to C1 and C2 DHs. In contrast to LTM neurons, many C1 and C2 DH nociceptive neurons received mechanosensitive convergent afferent inputs from cervical and craniofacial deep tissues (e.g., tongue muscles or temporomandibular joint), and over 50% could be activated by hypoglossal (XII) nerve electrical stimulation. We propose that C1 and C2 DHs represent part of the caudal extension of the Vc, and that Vc and C1 and C2 DHs may act together as one functional unit to process nociceptive information from craniofacial and cervical tissues, including that from deep craniofacial tissues.
Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.
Postnatal glutamatergic principal neuron synapses are typically presumed to express only calcium-impermeable (CI), GluR2-containing AMPARs under physiological conditions. Here, however, we demonstrate that long-term potentiation (LTP) in CA1 hippocampal pyramidal neurons causes rapid incorporation of GluR2-lacking calcium-permeable (CP)-AMPARs: CP-AMPARs are present transiently, being replaced by GluR2-containing AMPARs approximately 25 min after LTP induction. Thus, CP-AMPARs are physiologically expressed at CA1 pyramidal cell synapses during LTP, and may be required for LTP consolidation.
In order to deal effectively with danger, it is imperative to know about it. This is what nociceptors do--these primary sensory neurons are specialized to detect intense stimuli and represent, therefore, the first line of defense against any potentially threatening or damaging environmental inputs. By sensing noxious stimuli and contributing to the necessary reactions to avoid them--rapid withdrawal and the experience of an intensely unpleasant or painful sensation, nociceptors are essential for the maintenance of the body's integrity. Although nociceptive pain is clearly an adaptive alarm system, persistent pain is maladaptive, essentially an ongoing false alarm. Here, we highlight the genesis of nociceptors during development and the intrinsic properties of nociceptors that enable them to transduce, conduct, and transmit nociceptive information and also discuss how their phenotypic plasticity contributes to clinical pain.
Ca(2+)-permeable-AMPA receptors (AMPARs) are expressed in the superficial dorsal horn (SDH, laminae I/II) of the spinal cord, the area involved in transmission and modulation of sensory information, including nociception. A possible role of Ca(2+)-permeable-AMPARs in synaptic strengthening has been suggested in postnatal DH cultures, but their role in the long-lasting activity-dependent synaptic plasticity of primary afferent neurotransmission in the adult mouse SDH has not been investigated. In the present study the role of Ca(2+)-permeable-AMPARs in the regulation of long-lasting synaptic plasticity, specifically long-term potentiation (LTP) and long-term depression (LTD) in the SDH, was investigated using mice deficient in AMPAR GluR2 subunit. We show here that the GluR2 mutants exhibited no changes in passive membrane properties, but a significant increase in rectification of excitatory postsynaptic currents, the finding suggesting increased expression of Ca(2+)-permeable-AMPARs. In the absence of GluR2, high-frequency stimulation (HFS) of small-diameter primary afferent fibers induced LTP that is enhanced and non-saturating in the SDH at both primary afferent Adelta- and/or C-fibers monosynaptic and polysynaptic pathways, whereas neuronal excitability and paired-pulse depression were normal. The LTP could be induced in the presence of the NMDA receptor antagonist d-AP5, and L-type Ca(2+) channel blockers, suggesting that Ca(2+)-permeable-AMPARs are sufficient to induce LTP in the SDH neurons of adult mouse spinal cord. In contrast, the induction of HFS-LTD is reduced in the SDH of GluR2 mutants. These results suggest an important role for AMPAR GluR2 subunit in regulating synaptic plasticity with potential relevance for long-lasting hypersensitivity in pathological states.
Presynaptic ionotropic receptors and control of transmitter release
- Engelman
Engelman HS, MacDermott AB. Presynaptic ionotropic receptors and control of transmitter
release. Nat. Rev. Neurosci. 2004; 5:135–145. [PubMed: 14735116]
Role of capsaicin-sensitive primary afferent inputs from the masseter muscle in the C1 spinal neurons responding to tooth-pulp stimulation in rats
- Takeda