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Objective: Our aim was to survey astrocyte and microglial activation across four brain regions in a mouse model of chronic migraine., Background: Chronic migraine is a leading cause of disability, with higher rates in females. The role of central nervous system neurons and glia in migraine pathophysiology is not fully elucidated. Preclinical studies have shown abnormal glial activation in the trigeminal nucleus caudalis of male rodents. No current reports have investigated glial activation in both sexes in other important brain regions involved with the nociceptive and emotional processing of pain., Methods: The mouse nitroglycerin model of migraine was used, and nitroglycerin (10 mg/kg) or vehicle was administered every other day for 9 days. Prior to injections on days 1, 5, and 9, cephalic allodynia was determined by periorbital von Frey hair testing. Immunofluorescent staining of astrocyte marker, glial fibrillary protein (GFAP), and microglial marker, ionized calcium binding adaptor molecule 1 (Iba1), in male and female trigeminal nucleus caudalis, periaqueductal gray, somatosensory cortex, and nucleus accumbens was completed., Results: Behavioral testing demonstrated increased cephalic allodynia in nitroglycerin- versus vehicle-treated mice. An increase in the percent area covered by GFAP+ cells in the trigeminal nucleus caudalis and nucleus accumbens, but not the periaqueductal gray or somatosensory cortex, was observed in response to nitroglycerin. No significant differences were observed for Iba1 staining across brain regions. We did not detect significant sex differences in GFAP or Iba1 quantification., Conclusions: Immunohistochemical analysis suggests that, at the time point tested, immunoreactivity of GFAP+ astrocytes, but not Iba1+ microglia, changes in response to chronic migraine-associated pain. Additionally, there do not appear to be significant differences between males and females in GFAP+ or Iba1+ cells across the four brain regions analyzed., (© 2024 American Headache Society.)

Hyperalgesia occurs in the orofacial region of rats when estrogen levels are low, although the specific mechanism needs to be investigated further. Furthermore, oxidative stress plays an important role in the transmission of pain signals. This study aimed to explore the role of oxidative stress in orofacial hyperalgesia under low estrogen conditions. We firstly found an imbalance between oxidative and antioxidant capacity within the spinal trigeminal subnucleus caudalis (SP5C) of rats after ovariectomy (OVX), resulting in oxidative stress and then a decrease in the orofacial pain threshold. To investigate the mechanism by which oxidative stress occurs, we used virus as a tool to silence or overexpress the excitatory amino acid transporter 3 (EAAT3) gene. Further investigation revealed that the regulation of glutathione (GSH) and reactive oxygen species (ROS) can be achieved by regulating EAAT3, which in turn impacts the occurrence of oxidative stress. In summary, our findings suggest that reduced expression of EAAT3 within the SP5C of rats in the low estrogen state may decrease GSH content and increase ROS levels, resulting in oxidative stress and ultimately lead to orofacial hyperalgesia. This suggests that antioxidants could be a potential therapeutic direction for orofacial hyperalgesia under low estrogen conditions, though more research is needed to understand its mechanism., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The trigeminal spinal subnucleus caudalis (Sp5C), also known as the medullary dorsal horn, receives orofacial somatosensory inputs, particularly nociceptive inputs, from the trigeminal nerve. In the Sp5C, excitatory and inhibitory neurons, glutamatergic and GABAergic/glycinergic neurons, respectively, form the local circuits. The axons of the glutamatergic neurons in lamina I ascend toward the thalamic and parabrachial nuclei, and this projection is the main pathway of orofacial nociception. Additionally, the axons of the higher brain regions, including the locus coeruleus, dorsal raphe, and cerebral cortex, are sent to the Sp5C., Highlight: Among these descending projections, this review focuses on the functional profiles of the corticotrigeminal projections to the Sp5C, along with their anatomical aspects. The primary and secondary somatosensory and insular cortices are of particular interest., Conclusion: Corticotrigeminal projections from the somatosensory cortex to the Sp5C play a suppressive role in nociceptive information processing, whereas recent studies have demonstrated a facilitative role of the insular cortex in nociceptive information processing at the Sp5C level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The present study examined contribution of the transient receptor potential vanilloid 1 channel (TRPV1) to the chronic orofacial pain. Bilateral partial nerve ligation (PNL) of the mental nerve, a branch of trigeminal nerve, was performed to induce neuropathic pain. The withdrawal threshold in response to mechanical stimulation of the lower lip skin was substantially reduced after the surgery in the PNL rats while it remained unchanged in the sham rats. This reduction in the PNL rats was alleviated by pregabalin injected intraperitoneally (10 mg/kg) and intracisternally (10, 30, 100 μg). Furthermore, an intracisternal injection of AMG9810, an antagonist of TRPV1, (1.5, 5.0 μg) attenuated the reduction of withdrawal threshold. Spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) were recorded from the spinal trigeminal subnucleus caudalis (Vc) neurons in the brainstem slice, which receive the orofacial nociceptive signals. In the PNL rats, superfusion of capsaicin (0.03, 0.1 μM) enhanced their frequency without effect on the amplitude and the highest concentration (0.3 μM) increased both the frequency and amplitude. In the sham rats, only 0.3 μM capsaicin increased their frequency. Thus, capsaicin-induced facilitation of sEPSCs and mEPSCs in the PNL rats was significantly stronger than that in the sham rats. AMG9810 (0.1 μM) attenuated the capsaicin's effect. Capsaicin was ineffective on the trigeminal tract-evoked EPSCs in the PNL and sham rats. These results suggest that the chronic orofacial pain in the PNL model results from facilitation of the spontaneous excitatory synaptic transmission in the Vc region through TRPV1 at least partly., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Depression symptoms are often found in patients suffering from chronic pain, a phenomenon that is yet to be understood mechanistically. Here, we systematically investigate the cellular mechanisms and circuits underlying the chronic-pain-induced depression behavior. We show that the development of chronic pain is accompanied by depressive-like behaviors in a mouse model of trigeminal neuralgia. In parallel, we observe increased activity of the dopaminergic (DA) neuron in the midbrain ventral tegmental area (VTA), and inhibition of this elevated VTA DA neuron activity reverses the behavioral manifestations of depression. Further studies establish a pathway of glutamatergic projections from the spinal trigeminal subnucleus caudalis (Sp5C) to the lateral parabrachial nucleus (LPBN) and then to the VTA. These glutamatergic projections form a direct circuit that controls the development of the depression-like behavior under the state of the chronic neuropathic pain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of 'patches' of higher expression, interspersed within a less immunoreactive 'matrix', which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse area of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.

Background: The topical inflammatory soup can model the inflammation of the dura mater causing hypersensitivity and activation of the trigeminal system, a phenomenon present in migraineurs. Calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase are important in the sensitization process there. 5-HT 1B/1D receptor agonists, triptans are used as a treatment of migraine. Kynurenic acid an NMDA antagonist can act on structures involved in trigeminal activation., Aim: We investigated the effect of inflammatory soup induced dural inflammation on the calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase levels in the caudal trigeminal nucleus. We also tested whether pretreatment with a well-known antimigraine drug, such as sumatriptan and kynurenic acid, a compound with a different mechanism of action, can affect these changes and if their modulatory effects are comparable., Material and Methods: After subcutaneous sumatriptan or intraperitoneal kynurenic acid the dura mater of adult male Sprague-Dawley rats (n = 72) was treated with inflammatory soup or its vehicle (synthetic interstitial fluid). Two and a half or four hours later perfusion was performed and the caudal trigeminal nucleus was removed for immunohistochemistry., Results and Conclusion: Inflammatory soup increased calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase in the caudal trigeminal nucleus compared to placebo, which was attenuated by sumatriptan and kynurenic acid. This suggests the involvement of 5-HT 1B/1D and NMDA receptors in neurogenic inflammation development of the dura and thus in migraine attacks.

Activation of the trigeminal system causes the release of various neuropeptides, cytokines, and other immune mediators. Calcitonin gene-related peptide (CGRP), which is a potent algogenic mediator, is expressed in the peripheral sensory neurons of trigeminal ganglion (TG). It affects the inflammatory responses and pain sensitivity by modulating the activity of glial cells. The primary aim of this study was to use array analysis to investigate the effect of CGRP on the glial cells of TG in regulating nuclear factor kappa B (NF-κB) signaling genes and to further check if CGRP in the TG can affect neuron-glia activation in the spinal trigeminal nucleus caudalis. The glial cells of TG were stimulated with CGRP or Minocycline (Min) + CGRP. The effect on various genes involved in NF-κB signaling pathway was analyzed compared to no treatment control condition using a PCR array analysis. CGRP, Min + CGRP or saline was directly injected inside the TG and the effect on gene expression of Egr1 , Myd88 and Akt1 and protein expression of cleaved Caspase3 (cleav Casp3) in the TG, and c-Fos and glial fibrillary acidic protein (GFAP) in the spinal section containing trigeminal nucleus caudalis was analyzed. Results showed that CGRP stimulation resulted in the modulation of several genes involved in the interleukin 1 signaling pathway and some genes of the tumor necrosis factor pathway. Minocycline pre-treatment resulted in the modulation of several genes in the glial cells, including anti-inflammatory genes, and neuronal activation markers. A mild increase in cleav Casp3 expression in TG and c-Fos and GFAP in the spinal trigeminal nucleus of CGRP injected animals was observed. These data provide evidence that glial cells can participate in neuroimmune interaction due to CGRP in the TG via NF-κB signaling pathway.

Background: Purine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN)., Methods: Nerve endings isolated from the rat TCN were loaded with [ 3 H]D-aspartic acid ([ 3 H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [ 3 H]D-ASP release under different experimental conditions., Results: Synaptosomal [ 3 H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [ 3 H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [ 3 H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this "excess" release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [ 3 H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist., Conclusions: P2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.

Background: Vestibular migraine has recently been recognized as a novel subtype of migraine. However, the mechanism that relate vestibular symptoms to migraine had not been well elucidated. Thus, the present study investigated vestibular dysfunction in a rat model of chronic migraine (CM), and to dissect potential mechanisms between migraine and vertigo., Methods: Rats subjected to recurrent intermittent administration of nitroglycerin (NTG) were used as the CM model. Migraine- and vestibular-related behaviors were analyzed. Immunofluorescent analyses and quantitative real-time polymerase chain reaction were employed to detect expressions of c-fos and calcitonin gene-related peptide (CGRP) in the trigeminal nucleus caudalis (TNC) and vestibular nucleus (VN). Morphological changes of vestibular afferent terminals was determined under transmission electron microscopy. FluoroGold (FG) and CTB-555 were selected as retrograde tracers and injected into the VN and TNC, respectively. Lentiviral vectors comprising CGRP short hairpin RNA (LV-CGRP) was injected into the trigeminal ganglion., Results: CM led to persistent thermal hyperalgesia, spontaneous facial pain, and prominent vestibular dysfunction, accompanied by the upregulation of c-fos labeling neurons and CGRP immunoreactivity in the TNC (c-fos: vehicle vs. CM = 2.9 ± 0.6 vs. 45.5 ± 3.4; CGRP OD: vehicle vs. CM = 0.1 ± 0.0 vs. 0.2 ± 0.0) and VN (c-fos: vehicle vs. CM = 2.3 ± 0.8 vs. 54.0 ± 2.1; CGRP mRNA: vehicle vs. CM = 1.0 ± 0.1 vs. 2.4 ± 0.1). Furthermore, FG-positive neurons was accumulated in the superficial layer of the TNC, and the number of c-fos+/FG+ neurons were significantly increased in rats with CM compared to the vehicle group (vehicle vs. CM = 25.3 ± 2.2 vs. 83.9 ± 3.0). Meanwhile, CTB-555+ neurons dispersed throughout the VN. The structure of vestibular afferent terminals was less pronounced after CM compared with the peripheral vestibular dysfunction model. In vivo knockdown of CGRP in the trigeminal ganglion significantly reduced the number of c-fos labeling neurons (LV-CGRP vs. LV-NC = 9.9 ± 3.0 vs. 60.0 ± 4.5) and CGRP mRNA (LV-CGRP vs. LV-NC = 1.0 ± 0.1 vs. 2.1 ± 0.2) in the VN, further attenuating vestibular dysfunction after CM., Conclusions: These data demonstrates the possibility of sensitization of vestibular nucleus neurons to impair vestibular function after CM, and anti-CGRP treatment to restore vestibular dysfunction in patients with CM.

Background: The neurochemical background of the evolution of headache disorders, still remains partially undiscovered. Accordingly, our aim was to further explore the neurochemical profile of Complete Freund's adjuvant (CFA)-induced orofacial pain, involving finding the shift point regarding small molecule neurotransmitter concentrations changes vs. that of the previously characterized headache-related neuropeptides. The investigated neurotransmitters consisted of glutamate, γ-aminobutyric acid, noradrenalin and serotonin. Furthermore, in light of its influence on glutamatergic neurotransmission, we measured the level of kynurenic acid (KYNA) and its precursors in the kynurenine (KYN) pathway (KP) of tryptophan metabolism., Methods: The effect of CFA was evaluated in male Sprague Dawley rats. Animals were injected with CFA (1 mg/ml, 50 μl/animal) into the right whisker pad. We applied high-performance liquid chromatography to determine the concentrations of the above-mentioned compounds from the trigeminal nucleus caudalis (TNC) and somatosensory cortex (ssCX) of rats. Furthermore, we measured some of these metabolites from the cerebrospinal fluid and plasma as well. Afterwards, we carried out permutation t-tests as post hoc analysis for pairwise comparison., Results: Our results demonstrated that 24 h after CFA treatment, the level of glutamate, KYNA and that of its precursor, KYN was still elevated in the TNC, all diminishing by 48 h. In the ssCX, significant concentration increases of KYNA and serotonin were found., Conclusion: This is the first study assessing neurotransmitter changes in the TNC and ssCX following CFA treatment, confirming the dominant role of glutamate in early pain processing and a compensatory elevation of KYNA with anti-glutamatergic properties. Furthermore, the current findings draw attention to the limited time interval where medications can target the glutamatergic pathways.

Parkinson's disease (PD) related pain can be assigned to either nociceptive pain or neuropathic pain, in which Transient receptor potential vanilloid 1 (TRPV1) has been demonstrated to play a pivotal role. Yet little research has examined possible involvement of TRPV1 in pain in PD. Here, we show that TRPV1 is highly expressed in PD and blocking TRPV1 can alleviate pain in PD. The level of TRPV1 in 6-OHDA induced semi mice model of PD was evaluated. The effect of TRPV1 and involved serotonin (5-HT) was also examined in the model. Unilateral injection of 6-OHDA in striatum significantly decreased thermal pain threshold and induced mechanical allodynia without changes in conditioned place preference. Immunostaining revealed that great increased expression in TRPV1 in the Vc of 6-OHDA lesioned mice compared with sham mice. TRPV1 sensitization was maintained by 5-HT/5-HT3A. In 6-OHDA-lesioned mice model of PD, TRPV1 sensitization might be implicated in the maintenance of behavioral hypersensitivity by enhanced descending 5-HT pain facilitation and dorsal horn 5-HT3AR mechanism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: According to our previous study, microglia P2X4 receptors (P2X4Rs) play a pivotal role in the central sensitization of chronic migraine (CM). However, the molecular mechanism that underlies the crosstalk between microglia P2X4Rs and neurons of the trigeminal nucleus caudalis (TNC) is not fully understood. Therefore, the aim of this study is to examine the exact P2X4Rs signalling pathway in the development of central sensitization in a CM animal model., Methods: We used an animal model with recurrent intermittent administration of nitroglycerin (NTG), which closely mimics CM. NTG-induced basal mechanical and thermal hypersensitivity were evaluated using a von Frey filament test and an increasing-temperature hot plate apparatus (IITC). We detected P2X4Rs, brain-derived neurotrophic factor (BDNF) and phosphorylated p38 mitogen-activated protein kinase (p-p38-MAPK) expression profiles in the TNC. We investigated the effects of a P2X4R inhibitor (5-BDBD) and an agonist (IVM) on NTG-induced hyperalgesia and neurochemical changes as well as on the expression of p-p38-MAPK and BDNF. We also detected the effects of a tropomyosin-related kinase B (TrkB) inhibitor (ANA-12) on the CM animal model in vivo. Then, we evaluated the effect of 5-BDBD and SB203580 (a p38-MAPK inhibitors) on the release and synthesis of BDNF in BV2 microglia cells treated with 50 μM adenosine triphosphate (ATP)., Results: Chronic intermittent administration of NTG resulted in chronic mechanical and thermal hyperalgesia, accompanied by the upregulation of P2X4Rs and BDNF expression. 5-BDBD or ANA-12 prevented hyperalgesia induced by NTG, which was associated with a significant inhibition of the NTG-induced increase in phosphorylated extracellular regulated protein kinases (p-ERK) and calcitonin gene related peptide (CGRP) release in the TNC. Repeated administration of IVM produced sustained hyperalgesia and significantly increased the levels of p-ERK and CGRP release in the TNC. Activating P2X4Rs with ATP triggered BDNF release and increased BDNF synthesis in BV2 microglia, and these results were then reduced by 5-BDBD or SB203580., Conclusions: Our results indicated that the P2X4R contributes to the central sensitization of CM by releasing BDNF and promoting TNC neuronal hyper-excitability. Blocking microglia P2X4R-BDNF signalling may have an effect on the prevention of migraine chronification.

Migraine is one of the most disabling neurological diseases worldwide; however, the mechanisms underlying migraine headache are still not fully understood and current therapies for such pain are inadequate. It has been suggested that inflammation and neuroimmune modulation in the gastrointestinal tract could play an important role in the pathogenesis of migraine headache, but how gut microbiomes contribute to migraine headache is unclear. In the present study, we investigated the effect of gut microbiota dysbiosis on migraine-like pain using broad-spectrum antibiotics and germ-free (GF) mice. We observed that antibiotics treatment-prolonged nitroglycerin (NTG)-induced acute migraine-like pain in wild-type (WT) mice and the pain prolongation was completely blocked by genetic deletion of tumor necrosis factor-alpha (TNFα) or intra-spinal trigeminal nucleus caudalis (Sp5C) injection of TNFα receptor antagonist. The antibiotics treatment extended NTG-induced TNFα upregulation in the Sp5C. Probiotics administration significantly inhibited the antibiotics-produced migraine-like pain prolongation. Furthermore, NTG-induced migraine-like pain in GF mice was markedly enhanced compared to that in WT mice and gut colonization with fecal microbiota from WT mice robustly reversed microbiota deprivation-caused pain enhancement. Together, our results suggest that gut microbiota dysbiosis contributes to chronicity of migraine-like pain by upregulating TNFα level in the trigeminal nociceptive system.

Background: Migraine is a neurovascular primary headache disorder, which causes significant socioeconomic problems worldwide. The pathomechanism of disease is enigmatic, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Migraine research of recent years has focused on neuropeptides, such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP1-38) as potential pathogenic factors and possible therapeutic offensives. The goal of present study was to investigate the simultaneous expression of CGRP and precursor of PACAP1-38 (preproPACAP) in the central region of the TS in a time-dependent manner following TS activation in rats., Methods: The right whisker pad of rats was injected with 50 μl Complete Freund's Adjuvant (CFA) or saline. A mechanical allodynia test was performed with von Frey filaments before and after treatment. Transcardial perfusion of the animals was initiated 24, 48, 72 and 120 h after injection, followed by the dissection of the nucleus trigeminus caudalis (TNC). After preparation, the samples were stored at - 80 °C until further use. The relative optical density of CGRP and preproPACAP was analyzed by Western blot. One-way ANOVA and Kruskal-Wallis followed by Tukey post hoc test were used to evaluate the data. Regression analysis was applied to explore the correlation between neuropeptides expression and hyperalgesia., Results: Orofacial CFA injection resulted in significant CGRP and preproPACAP release in the TNC 24, 48, 72 and 120 h after the treatment. The level of neuropeptides reached its maximum at 72 h after CFA injection, corresponding to the peak of facial allodynia. Negative, linear correlation was detected between the expression level of neuropeptides and value of mechanonociceptive threshold., Conclusion: This is the first study which suggests that the expression of CGRP and preproPACAP simultaneously increases in the central region of activated TS and it influences the formation of mechanical hyperalgesia. Our results contribute to a better understanding of migraine pathogenesis and thereby to the development of more effective therapeutic approaches.

Background: Although the mechanism of chronic migraine (CM) is unclear, it might be related to central sensitization and neuronal persistent hyperexcitability. The tyrosine phosphorylation of NR2B (NR2B-pTyr) reportedly contributes to the development of central sensitization and persistent pain in the spinal cord. Central sensitization is thought to be associated with an increase in synaptic efficiency, but the mechanism through which NR2B-pTyr regulates synaptic participation in CM-related central sensitization is unknown. In this study, we aim to investigate the role of NR2B-pTyr in regulating synaptic plasticity in CM-related central sensitization., Methods: Male Sprague-Dawley rats were subjected to seven inflammatory soup (IS) injections to model recurrent trigeminovascular or dural nociceptor activation, which is assumed to occur in patients with CM. We used the von Frey test to detect changes in mechanical withdrawal thresholds, and western blotting and immunofluorescence staining assays were performed to detect the expression of NR2B-pTyr in the trigeminal nucleus caudalis (TNC). NR2B-pTyr was blocked with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo [3,4-d] pyrimidine (PP2) and the protein tyrosine kinase inhibitor genistein to detected the changes in calcitonin gene-related peptide (CGRP), substance P (SP), and the synaptic proteins postsynaptic density 95 (PSD95), synaptophysin (Syp), synaptotagmin1 (Syt-1). The synaptic ultrastructures were observed by transmission electron microscopy (TEM), and the dendritic architecture of TNC neurons was observed by Golgi-Cox staining., Results: Statistical analyses revealed that repeated infusions of IS induced mechanical allodynia and significantly increased the expression of NR2B Tyr-1472 phosphorylation (pNR2B-Y1472) and NR2B Tyr-1252 phosphorylation (pNR2B-Y1252) in the TNC. Furthermore, the inhibition of NR2B-pTyr by PP2 and genistein relieved allodynia and reduced the expression of CGRP, SP, PSD95, Syp and Syt-1 and synaptic transmission., Conclusions: These data indicate that NR2B-pTyr might regulate synaptic plasticity in central sensitization in a CM rat model. The inhibition of NR2B tyrosine phosphorylation has a protective effect on threshold dysfunction and migraine attacks through the regulation of synaptic plasticity in central sensitization.

Age-related changes in striatal function are potentially important for predicting declining memory performance over the adult life span. Here, we used fMRI to measure functional connectivity of caudate subfields with large-scale association networks and positron emission tomography to measure striatal dopamine transporter (DAT) density in 51 older adults (age 65-86 years) who received annual cognitive testing for up to 7 years (mean = 5.59, range 2-7 years). Analyses showed that cortical-caudate functional connectivity was less differentiated in older compared with younger adults ( n = 63, age 18-32 years). Unlike in younger adults, the central lateral caudate was less strongly coupled with the frontal parietal control network in older adults. Older adults also showed less "decoupling" of the caudate from other networks, including areas of the default network (DN) and the hippocampal complex. Contrary to expectations, less decoupling between caudate and the DN was not associated with an age-related reduction of striatal DAT, suggesting that neurobiological changes in the cortex may drive dedifferentiation of cortical-caudate connectivity. Reduction of specificity in functional coupling between caudate and regions of the DN predicted memory decline over subsequent years at older ages. The age-related reduction in striatal DAT density also predicted memory decline, suggesting that a relation between striatal functions and memory decline in aging is multifaceted. Collectively, the study provides evidence highlighting the association of age-related differences in striatal function to memory decline in normal aging., Competing Interests: The authors declare no conflict of interest.

Acid-sensing ion channel 3 (ASIC3) is abundant in the trigeminal nervous system and is most sensitive to a slight pH decrease. Recent studies have indicated that ASIC3 in the peripheral trigeminal ganglia is likely involved in the pathogenesis of migraine pain. However, it is unclear whether this receptor plays a role in recurrent migraine, namely, migraine chronicity. Here, we aimed to investigate the role of ASIC3 in an animal model of recurrent migraine (RM). In this study, we established a rat model of RM through repeated administration of inflammatory soup (IS) onto the dura. Then, we tested the mechanical pain thresholds of the face and hindpaws by von Frey filaments. qRT-PCR, Western blot and immunofluorescence labelling were used to detect the expression and localization of ASIC3 in the trigeminal nucleus caudalis (TNC). The protein levels of calcitonin gene-related peptide (CGRP), its receptor component receptor activity modifying protein 1 (RAMP1) and c-Fos were analysed following treatment with the ASIC3 inhibitor APETx2 and activator 2-guanidine-4-methylquinazoline (GMQ). We found decreased pain thresholds after repeated dural inflammatory stimulation, which suggested the establishment of an RM model. Based on this model, we observed elevated expression of ASIC3 in the TNC group compared to that in the Sham group. ASIC3 was primarily expressed in neurons but not in astrocytes of the TNC. Moreover, APETx2 attenuated tactile allodynia and significantly decreased the expression of c-Fos, CGRP and RAMP1, while GMQ aggravated these effects compared to those observed in the IS + vehicle group. These findings indicate a critical role of ASIC3 channels in the pathophysiology of RM, and ASIC3 might represent a potential therapeutic target to prevent the progression of migraine.

The pain sensation system is highly conserved among species, thus animal models have been used to investigate relevant tissues. The focus for head-specific pain has been on the primary nociceptive neurons in the trigeminal pathway, i.e. trigeminal ganglia. The secondary nociceptive neurons of the trigeminal pathway, trigeminal nucleus caudalis (TNC), have not been assessed. We expect different gene expression profiles compared to the homologous spinal cord dorsal horn (SDH), as several signalling substances provoke head-specific pain but not peripheral pain. We aim to provide expression profiles of TNC and SDH, tissues highly relevant for pain- and migraine-studies. We extracted RNA from laser capture microdissected laminae I-V from TNC and SDH from six Wistar rats for RNA-Sequencing. We showed the expression profiles of genes involved in neural signal transmission and found that among all G protein-coupled receptors Gabbr1 was highest expressed in both tissues. Among the migraine-associated genes we showed that Cacna1a, where non-synonyms mutations can cause familial hemiplegic migraine, was highly expressed with a slightly lower expression in TNC than in SDH. To show the genetic differences between the two homologous systems we performed a differential expression analysis, revealing 1696 genes higher and 1895 genes lower expressed genes in TNC than in SDH, of which many were neuronal-related. The high number of differentially expressed genes shows the large genetic difference between the trigeminal and spinothalamic system. Our results contribute to the characterization of nociceptive pathways, which may help us understanding why several signalling molecules cause headache and no peripheral pain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background A common characteristic of migraine-inducing substances is that they cause headache and no pain in other areas of the body. Few studies have compared pain mechanisms in the trigeminal and spinal systems and, so far, no major differences have been noted. We compared signalling molecules in the trigeminal and spinothalamic system after infusion of the migraine-provoking substance glyceryltrinitrate. Method A catheter was placed in the femoral vein of rats and one week later glyceryltrinitrate 4 µg/kg/min was infused for 20 min. Protein expression in the dura mater, trigeminal ganglion, nucleus caudalis, dorsal root ganglion and the dorsal horn of the thoracic spinal cord was analysed at different time points using western blotting and immunohistochemistry. Results Glyceryltrinitrate caused a threefold increase in expression of phosphorylated extracellular signal-regulated kinases at 30 min in the dura mater and nucleus caudalis ( P < 0.05) and at 2 h in the trigeminal ganglion with very few expressions in the dorsal root ganglion. In the nucleus caudalis, expression of phosphorylated extracellular signal-regulated kinases and Cam KII increased 2.6-fold and 3.2-fold, respectively, at 2 h after glycerytrinitrate infusion ( P < 0.01). p-CREB/ATF-1 upregulation was observed only at 30 min ( P < 0.05) in the nucleus caudalis. None of these markers showed increased expression in the regions of thoracic spinal cord dorsal horn. Conclusion The dura, trigeminal ganglion and nucleus caudalis are activated shortly after glycerytrinitrate infusion with long-lasting expression of phosphorylated extracellular signal-regulated kinases observed in the nucleus caudalis. These activations were not observed at the spinal level.

Introduction Research in development of new migraine therapeutics is hindered by the lack of suitable, predictive animal models. Cilostazol provokes headache in healthy humans and migraineurs by increasing intracellular cAMP levels. We aimed to investigate whether cilostazol could provoke headache-like behaviours and c-fos expression in rats. In order to evaluate the predictive validity of the model, we examined the response to the migraine specific drug sumatriptan. Methods The effect of cilostazol (125 mg/kg p.o.) in female Sprague Dawley rats was evaluated on a range of spontaneous behavioural parameters, light sensitivity and mechanical sensitivity thresholds. We also measured c-fos expression in the trigeminal nucleus caudalis. Results Cilostazol increased light sensitivity and grooming behaviour. These manifestations were not inhibited by sumatriptan. Cilostazol also induced c-fos expression in the trigeminal nucleus caudalis. Furthermore, trigeminal - but not hind paw hyperalgesia was observed. Conclusion The altered behaviours are suggestive of cilostazol induced headache with migraine-like features, but not specific. The presence of head specific hyperalgesia and the c-fos response in the trigeminal nucleus caudalis imply that the model involves trigeminal nociception. The model will be useful for studying mechanisms related to the cAMP pathway in headache, but its predictive properties appear to be more limited due to the lack of response to sumatriptan.

Background: Frequent mild head injuries or concussion along with the presence of headache may contribute to the persistence of concussion symptoms., Methods: In this study, the acute effects of recovery between mild head injuries and the frequency of injuries on a headache behavior, trigeminal allodynia, was assessed using von Frey testing up to one week after injury, while histopathological changes in the trigeminal pain pathway were evaluated using western blot, ELISA and immunohistochemistry.  RESULTS: A decreased recovery time combined with an increased mild closed head injury (CHI) frequency results in reduced trigeminal allodynia thresholds compared to controls. The repetitive CHI group with the highest injury frequency showed the greatest reduction in trigeminal thresholds along with greatest increased levels of calcitonin gene-related peptide (CGRP) in the trigeminal nucleus caudalis. Repetitive CHI resulted in astrogliosis in the central trigeminal system, increased GFAP protein levels in the sensory barrel cortex, and an increased number of microglia cells in the trigeminal nucleus caudalis., Conclusions: Headache behavior in rats is dependent on the injury frequency and recovery interval between mild head injuries. A worsening of headache behavior after repetitive mild head injuries was concomitant with increases in CGRP levels, the presence of astrocytosis, and microglia proliferation in the central trigeminal pathway. Signaling between neurons and proliferating microglia in the trigeminal pain system may contribute to the initiation of acute headache after concussion or other traumatic brain injuries.

Background: Clinical and experimental studies have pointed to the possible involvement of the transient receptor potential ankyrin type-1 (TRPA1) channels in migraine pain. In this study, we aimed to further investigate the role of these channels in an animal model of migraine using a novel TRPA1 antagonist, ADM&#95;12, as a probe., Methods: The effects of ADM&#95;12 on nitroglycerin-induced hyperalgesia at the trigeminal level were investigated in male rats using the quantification of nocifensive behavior in the orofacial formalin test. The expression levels of the genes coding for c-Fos, TRPA1, calcitonin gene-related peptide (CGRP) and substance P (SP) in peripheral and central areas relevant for migraine pain were analyzed. CGRP and SP protein immunoreactivity was also evaluated in trigeminal nucleus caudalis (TNC)., Results: In rats bearing nitroglycerin-induced hyperalgesia, ADM&#95;12 showed an anti-hyperalgesic effect in the second phase of the orofacial formalin test. This effect was associated to a significant inhibition of nitroglycerin-induced increase in c-Fos, TRPA1 and neuropeptides mRNA levels in medulla-pons area, in the cervical spinal cord and in the trigeminal ganglion. No differences between groups were seen as regards CGRP and SP protein expression in the TNC., Conclusions: These findings support a critical involvement of TRPA1 channels in the pathophysiology of migraine, and show their active role in counteracting hyperalgesia at the trigeminal level.

Objective: A new mammalian tachykinin peptide encoded in a TAC4 gene was identified and designated as hemokinin-1 (HK-1). A representative of the tachykinin peptide family is substance P (SP), and the function of SP has been well characterized as a pain transmitter or modulator, while it is possible that HK-1 is involved in pruriceptive processing, but, as yet, the distribution of HK-1 peptide in the trigeminal sensory system is still unknown. Thus, the aim of the present study was to elucidate the distribution of HK-1, while comparing the expression of SP, in the trigeminal ganglion and trigeminal sensory nuclear complex., Design: The trigeminal ganglion and the brain stem of male SD rats were used in the immunohistochemical study. Since the amino acid sequence in the carboxyl-terminal regions of HK-1 and SP is common, polyclonal antibodies of HK-1 and SP derived from 6 amino acids consisting of amino-terminal regions of these peptides were produced in guinea pig and rabbit, respectively. The immunohistochemical staining of HK-1 and SP was conducted using frozen sections of the trigeminal ganglion and brain stem in rats., Results: Immunohistochemical studies revealed the expression of HK-1 in small- and medium-sized trigeminal ganglion neurons, in the paratrigeminal nucleus, and in lamina I of the trigeminal nucleus caudalis, while there was no immunoreactivity of HK-1 in the trigeminal nucleus principalis, trigeminal nucleus oralis, and trigeminal nucleus interpolaris., Conclusion: These findings indicate that HK-1 is a target molecule for treatment of itch in the orofaicial regions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although it is well known that migraine pain is enhanced by photic stimulation of the eye, the mechanisms underlying this response are not yet understood. Noxious stimulation to the dura is known to activate trigeminal spinal subnucleus caudalis and upper cervical spinal cord (Vc/C1) neurons, causing migraine pain. Intense photic stimulation to the eye is also known to activate certain Vc/C1 neurons, thus increasing migraine pain. In this study, we hypothesized that Vc/C1 neurons receiving noxious dural input would be further activated by intense photic stimulation, resulting in the enhancement of migraine pain. However, mechanisms underlying the interactions between dural and photic sensory information in Vc/C1 neurons is unknown. To evaluate the above hypothesis, we studied phosphorylated extracellular signal-regulated kinase (pERK) -immunoreactive (IR) cells in Vc/C1 in dural mustard oil (DMO)-administrated rats. The change in neuronal excitability of Vc/C1 nociceptive neurons receiving input from the dura in DMO rats was examined and tested if those neurons were modulated by intense flush light stimulation. There were many pERK-IR cells in the lateral portion of Vc/C1 after MO administration to the dura. Flashlight presentation to the eye in DMO rats caused an enhancement of ERK phosphorylation in Vc/C1 neurons and pERK-IR cells were significantly suppressed after intracisternal administration of MEK1 inhibitor PD98059. Dura-light sensitive (DL) neurons were recorded in the lateral portion of Vc/C1 and photic responses of DL neurons were significantly enhanced following dural MO administration. These findings indicate that DL Vc/C1 neurons in DMO rats intensified their responses to intense photic stimulation and that ERK phosphorylation in Vc/C1 neurons receiving noxious dural input increased with intense photic stimulation, suggesting that Vc/C1 nociceptive neurons are involved in the enhancement of dural nociception associated with intense light stimulation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Antioxidants have been proven to weaken hyperalgesia in neuropathic pain. Endogenous antioxidant defense system may have a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) pathway in regulating the activation of the trigeminovascular system (TGVS) and hypersensitivity in nitroglycerin (NTG)-induced hyperalgesia rats., Methods: The expression levels of Nrf2, HO, HO1, and NQO1 in the trigeminal nucleus caudalis (TNC) were detected by western blot. Immunofluorescence was used to demonstrate the cell-specific localization of Nrf2 in TNC. Sulforaphane, a Nrf2 activator, was administered to NTG-induced rats. Then, the number of c-Fos- and nNOS-immunoreactive neurons in TNC was evaluated using immunofluorescence, and c-Fos and nNOS protein levels were quantified using western blot. Von Frey hair testing was used to evaluate the tactile thresholds of rats at different time points in different groups., Results: Total cellular and nuclear levels of the proteins Nrf2, HO1, and NQO1 were elevated in TNC after NTG injection, and Nrf2 was found to be located in the nucleus and cytoplasm of the neurons. Sulforaphane pretreatment significantly increased the nuclear Nrf2, HO1, and NQO1 levels in TNC. In addition, sulforaphane exposure effectively inhibited the expression of nNOS and c-Fos, reduced the number of nNOS and c-Fos immunoreactive neurons in TNC, and attenuated the tactile thresholds induced by NTG injection., Conclusion: Oxidative stress was involved in nitroglycerin-induced hyperalgesia. Activation of the Nrf2/ARE pathway inhibited the activation of TGVS and prevented the induction of hyperalgesia. Sulforaphane might therefore be an effective agent for hyperalgesia. Further studies are needed to discover the underlying mechanisms of the process.

ATP induces pain via activation of purinergic receptors in nociceptive sensory nerves. ATP signaling is terminated by ATP hydrolysis mediated by cell surface-localized ecto-nucleotidases. Using enzymatic histochemical staining, we show that ecto-ATPase activity is present in mouse trigeminal nerves. Using immunofluorescence staining, we found that ecto-NTPDase3 is expressed in trigeminal nociceptive neurons and their projections to the brainstem. In addition, ecto-ATPase activity and ecto-NTPDase3 are also detected in the nociceptive outermost layer of the trigeminal subnucleus caudalis. Furthermore, we demonstrate that incubation with anti-NTPDase3 serum reduces extracellular ATP degradation in the nociceptive lamina of both the trigeminal subnucleus caudalis and the spinal cord dorsal horn. These results are consistent with neuronal NTPDase3 activity modulating pain signal transduction and transmission by affecting extracellular ATP hydrolysis within the trigeminal nociceptive pathway. Thus, disruption of trigeminal neuronal NTPDase3 expression and localization to presynaptic terminals during chronic inflammation, local constriction and injury may contribute to the pathogenesis of orofacial neuropathic pain., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Background: Dry mouth is known to cause severe pain in the intraoral structures, and many dry mouth patients have been suffering from intraoral pain. In development of an appropriate treatment, it is crucial to study the mechanisms underlying intraoral pain associated with dry mouth, yet the detailed mechanisms are not fully understood. To evaluate the mechanisms underlying pain related to dry mouth, the dry-tongue rat model was developed. Hence, the mechanical or heat nocifensive reflex, the phosphorylated extracellular signal-regulated kinase and phosphorylated GluR1-IR immunohistochemistries, and the single neuronal activity were examined in the trigeminal spinal subnucleus caudalis of dry-tongue rats., Results: The head-withdrawal reflex threshold to mechanical, but not heat, stimulation of the tongue was significantly decreased on day 7 after tongue drying. The mechanical, but not heat, responses of trigeminal spinal subnucleus caudalis nociceptive neurons were significantly enhanced in dry-tongue rats compared to sham rats on day 7. The number of phosphorylated extracellular signal-regulated kinase-immunoreactive cells was also significantly increased in the trigeminal spinal subnucleus caudalis following noxious stimulation of the tongue in dry-tongue rats compared to sham rats on day 7. The decrement of the mechanical head-withdrawal reflex threshold (HWT) was reversed during intracisternal administration of the mitogen-activated protein kinase kinase 1 inhibitor, PD98059. The trigeminal spinal subnucleus caudalis neuronal activities and the number of phosphorylated extracellular signal-regulated kinase-immunoreactive cells following noxious mechanical stimulation of dried tongue were also significantly decreased following intracisternal administration of PD98059 compared to vehicle-administrated rats. Increased number of the phosphorylated GluR1-IR cells was observed in the trigeminal spinal subnucleus caudalis of dry-tongue rats, and the number of phosphorylated GluR1-IR cells was significantly reduced in PD98059-administrated rats compared to the vehicle-administrated tongue-dry rats., Conclusions: These findings suggest that the pERK-pGluR1 cascade is involved in central sensitization of trigeminal spinal subnucleus caudalis nociceptive neurons, thus resulting in tongue mechanical hyperalgesia associated with tongue drying., (© The Author(s) 2016.)

The functional significance of non-peptidergic C-fibers in orofacial pain processing is largely unknown. The present study examined the effects of the selective elimination of isolectin B4 (IB4)-binding (IB4(+)) neurons on formalin-induced face rubbing behavior (FRB) in the upper lip of rats and c-Fos-immunoreactive (c-Fos-IR) cells in the trigeminal subnucleus caudalis (Vc). IB4 conjugated to neurotoxin, saporin (IB4-Sap), blank-saporin (Bl-Sap), or saline (Sal) was injected into the cisterna magna. IB4-Sap treatments significantly decreased IB4(+) terminals in lamina II of Vc and IB4(+) trigeminal ganglia neurons, whereas Sal- and BI-Sap treatments did not. The number of formalin-induced FRB 15-30 min after the formalin injection was significantly higher in IB4-Sap-treated rats than in Sal- or Bl-Sap-treated rats, and was associated with an increase in c-Fos-IR cells. The systemic preadministration of the GABAA antagonist, bicuculline, and agonist, muscimol, had stronger decreasing effects on FRB and c-Fos-IR cells in IB4-Sap-treated rats than the preadministration of Sal, whereas the opposite effects were observed in Sal- and Bl-Sap-treated rats. These results indicate that IB4(+) neurons in the trigeminal nerve play antinociceptive regulatory roles in formalin-induced orofacial pain processing and that GABAA receptor functions at segmental and supratrigeminal sites have complex modulatory influences on antinociceptive roles., (Copyright © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

In this study, we demonstrate the existence on trigeminal caudal nucleus (TCN) glutamatergic terminals of α4β2 nicotinic receptors (nAChRs) capable of enhancing the terminals' spontaneous release of [(3)H]d-aspartate ([(3)H]D-Asp). In rat TCN synaptosomes, spontaneous [(3)H]D-Asp release was increased by nicotine and the nicotinic receptor agonists (±)epibatidine and RJR2403. The increase was potentiated by the positive allosteric modulator of nAChRs LY2087101, inhibited by the nicotinic antagonists mecamylamine (MEC) and dihydro-β-erythroidine hydrobromide (DHβE), and unaffected by α-bungarotoxin (α-BgTx) and methyllycaconitine (MLA). Evidence of functional interaction was observed between the α4β2 nAChRs and cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors co-localized on the TCN synaptosomes. Brief pre-exposure of synaptosomes to 30 μM nicotine or 10 μM RJR2403 abolished the AMPA (100 μM) -induced potentiation of [K(+)]e-evoked [(3)H]D-Asp release, an effect that seems to be caused by nicotine-induced increases in the internalization of AMPA receptors. Indeed, the effects of nicotine-pretreatment were not seen in synaptosomes containing pre-entrapped pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA endocytosis, while entrapment of pep2-SVKE, an inactive peptide used as negative control, was inefficacious. These findings show that nicotine can negatively modulate the function of AMPA receptors present on glutamatergic nerve terminals in the rat TCN. Dynamic control of AMPA receptors by the nicotinic cholinergic system has been observed under other experimental conditions, and it can contribute to the control of synaptic plasticity such as long-term depression and potentiation. Nicotine's ability to reduce the functionality of presynaptic AMPA receptors could contribute to its analgesic effects by diminishing glutamatergic transmission from the primary afferent terminals that convey nociceptive input to TCN., (Published by Elsevier Ltd.)

Peripheral neuropathy is a frequent complication of diabetes mellitus and a common symptom of neuropathic pain, the mechanism of which is complex and involves both peripheral and central components of the sensory system. The lamina II of the medullary dorsal horn, called the substantia gelatinosa (SG), is well known to be a critical site for processing of orofacial nociceptive information. Although there have been a number of studies done on diabetic neuropathy related to the orofacial region, the action of neurotransmitter receptors on SG neurons in the diabetic state is not yet fully understood. Therefore, we used the whole-cell patch clamp technique to investigate this alteration on SG neurons in both streptozotocin (STZ)-induced diabetic mice and offspring from diabetic female mice. STZ (200 mg/kg)-injected mice showed a small decrease in body weight and a significant increase in blood glucose level when compared with their respective control group. However, application of different concentrations of glycine, gamma-aminobutyric acid (GABA) and glutamate on SG neurons from STZ-injected mice did not induce any significant differences in inward currents when compared to their control counterparts. On the other hand, the offspring of diabetic female mice (induced by multiple injections of STZ (40 mg/kg) for 5 consecutive days) led to a significant decrease in both body weight and blood glucose level compared to the control offspring. Glycine and glutamate responses in the SG neurons of the offspring from diabetic female mice were similar to those of control offspring. However, the GABA response in SG neurons of offspring from diabetic female mice was greater than that of control offspring. Furthermore, the GABA-mediated responses in offspring from diabetic and control mice were examined at different concentrations ranging from 3 to 1,000 μM. At each concentration, the GABA-induced mean inward currents in the SG neurons of offspring from diabetic female mice were larger than those of control mice. These results demonstrate that SG neurons in offspring from diabetic mice are more sensitive to GABA compared to control mice, suggesting that GABA sensitivity may alter orofacial pain processing in offspring from diabetic female mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Aims: Calcitonin gene-related peptide (CGRP) and glyceryl trinitrate (GTN) infusion in migraineurs provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective in the treatment of acute migraine. We hypothesized that CGRP infusion would increase molecular markers of neuronal activation in migraine-relevant tissues of the rat., Methods: CGRP was infused intravenously (i.v.) in freely moving rats to circumvent factors like anesthesia, acute surgery and severe hypotension, the three confounding factors for c-Fos expression. The trigeminal nucleus caudalis (TNC) was isolated at different time points after CGRP infusion. The level of c-Fos mRNA and protein expression in TNC were analyzed by qPCR and immunohistochemistry. c-Fos-stained nuclei were also counted in the nucleus tractus solitarius (NTS) and caudal ventrolateral medulla (CVLM), integrative sites in the brain stem for processing cardiovascular signals. We also investigated Zif268 protein expression (another immediate early gene) in TNC. The protein expression of p-ERK, p-CREB and c-Fos was analyzed in dura mater, trigeminal ganglion (TG) and TNC samples using Western blot., Results: CGRP infusion caused a significant dose-dependent fall in mean arterial blood pressure. No significant activation of c-Fos in the TNC at mRNA and protein levels was observed after CGRP infusion. A significant increase in c-Fos protein was observed in the NTS and CVLM in the brain stem. Zif268 expression in the TNC was also not changed after CGRP infusion. p-ERK was increased in the dura mater 30 minutes after CGRP infusion., Conclusion: CGRP infusion increased the early expression of p-ERK in the dura mater but did not increase c-Fos and Zif268 expression in the TNC. The rats may, thus, differ from migraine patients, in whom infusion of CGRP caused headache and a delayed migraine attack. The rat CGRP infusion model with c-Fos or Zif268 as neuronal pain markers in TNC is unsuitable for antimigraine drug testing., (© International Headache Society 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

We examined the substrates for ocular nociception in adult male Sprague-Dawley rats. Capsaicin application to the ocular surface in awake rats evoked nocifensive responses and suppressed spontaneous grooming responses. Thus, peripheral capsaicin was able to activate the central pathways encoding ocular nociception. Our capsaicin stimulus evoked c-Fos expression in a select population of neurons within rostral trigeminal nucleus caudalis in anesthetized rats. These activated neurons also received direct contacts from corneal afferent fibers traced with cholera toxin B from the corneal surface. However, the central terminals of the corneal afferents that contacted capsaicin-activated trigeminal neurons did not contain TRPV1. To determine if TRPV1 expression had been altered by capsaicin stimulation, we examined TRPV1 content of corneal afferents in animals that did not receive capsaicin stimulation. These studies confirmed that while TRPV1 was present in 30% of CTb-labeled corneal afferent neurons within the trigeminal ganglion, TRPV1 was only detected in 2% of the central terminals of these corneal afferents within the trigeminal nucleus caudalis. Other TRP channels were also present in low proportions of central corneal afferent terminals in unstimulated animals (TRPM8, 2%; TRPA1, 10%). These findings indicate that a pathway from the cornea to rostral trigeminal nucleus caudalis is involved in corneal nociceptive transmission, but that central TRP channel expression is unrelated to the type of stimulus transduced by the peripheral nociceptive endings., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Large conductance, calcium-activated potassium channels [big potassium (BK) channel] consist of a tetramer of pore-forming α-subunit and distinct accessory β-subunits (β1-4) that modify the channel's properties. In this study, we analyzed the effects of BK channel activators and blockers on glutamate and γ-aminobutyric acid (GABA) release from synaptosomes isolated from the cerebral cortices or trigeminal caudal nuclei (TCN) of rats. Real-time polymerase chain reaction was used to characterize BK channel α and β(1-4) subunit expression in the cortex and in the trigeminal ganglia (TG), whose neurons project primary terminal afferents into the TCN. Immunocytochemistry was used to localize these subunits on cortical and TCN synaptosomes. The BK channels regulating [(3)H]D-aspartate release from primary afferent nerve terminals projecting into the TCN displayed limited sensitivity to iberiotoxin, whereas those expressed on cortical synaptosomes were highly sensitive to this toxin. BK channels did not appear to be present on GABAergic nerve terminals from the TCN since [(3)H]-γ-aminobutyric acid release in this model was unaffected by BK channel activators or blockers. Gene expression studies revealed expression levels of the α subunit in the TG that were only 31.2 ± 2.1% of those found in cortical tissues. The β4 subunit was the accessory subunit expressed most abundantly in both the cortex and TG. Levels of β1 and β2 were low in both these areas although β2 expression in the TG was higher than that found in the cortex. Immunocytochemistry experiments showed that co-localization of α and β4 subunits (the accessory subunit most abundantly expressed in both brain areas) was more common in TCN synaptosomes than in cortical synaptosomes. On the basis of these findings, it is reasonable to hypothesize that BK channels expressed on glutamatergic terminals in the TCN and cortex have distinct pharmacological profiles, which probably reflect different α and β subunit combinations. Channels in the cortex seem to be composed mainly of α subunits and to a lesser degree by α and β4 subunits, whereas in the TG the α + β4 combination seems to prevail (although α and/or α + β2 channels cannot be excluded). In light of the BK channels' selective control of excitatory transmission and their pharmacological diversity, their effects on primary glutamatergic afferents projecting to TCN represent a potential target for drug therapy of migraines and other types of orofacial pain.

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic subtype of migraine with aura caused by missense mutations in the CACNA1A gene, which encodes the pore-forming α1 subunit of voltage-gated neuronal CaV2.1 (P/Q-type) calcium channels. Transgenic knock-in mice expressing the CACNA1A R192Q mutation that causes FHM-1 in patients show a greater susceptibility to cortical spreading depression, the likely underlying mechanism of typical human migraine aura. The aim of this study was to compare neuronal activation within the trigeminal pain pathways in response to nociceptive trigeminovascular stimulation in wild-type and R192Q knock-in mice. After sham surgery or electrical stimulation of the superior sagittal sinus for 2h, or stimulation preceded by treatment with naratriptan, mice underwent intracardiac perfusion, and the brain, including the brainstem, was removed. Fos expression was measured in the trigeminocervical complex (TCC) and the lateral (ventroposteromedial, ventrolateral), medial (parafascicular, centromedian) and posterior thalamic nuclei. In the TCC of wild-type animals, the number of Fos-positive cells increased significantly following dural stimulation compared to the sham control group (P<0.001) and decreased after naratriptan treatment (P<0.05). In R192Q knock-in mice, there was no significant difference between the stimulated and sham (P=0.10) or naratriptan pre-treated groups (P=0.15). The number of Fos-positive cells in the R192Q stimulated group was significantly lower compared to the wild-type stimulated mice (P<0.05). In the thalamus, R192Q mice tended to be more sensitive to stimulation compared to the sham control in the medial and posterior nuclei, and between the two strains of stimulated animals there was a significant difference in the centromedian (P<0.005), and posterior nuclei (P<0.05). The present study suggests that the FHM-1 mutation affects more rostral brain structures in this experimental paradigm, which offers a novel perspective on possible differential effects of mutations causing migraine in terms of phenotype-genotype correlations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α2δ1 (Cavα2δ1) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Cavα2δ1 up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Cavα2δ1 with gabapentin, a ligand for the Cavα2δ1 proteins, or Cavα2δ1 antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Cavα2δ1 in orofacial pain processing. Importantly, increased Cavα2δ1 in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Cavα2δ1 up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.

Corneal pain is mediated by primary afferent fibers projecting to the dorsal horn of the medulla, specifically the trigeminal nucleus caudalis. In contrast to reflex responses, the conscious perception of pain requires transmission of neural activity to higher brain centers. Ascending pain transmission is mediated primarily by pathways to either the thalamus or parabrachial nuclei. We previously showed that some corneal afferent fibers preferentially contact parabrachial-projecting neurons in the rostral pole of the trigeminal nucleus caudalis, but the role of these projection neurons in transmitting noxious information from the cornea has not been established. In the present study, we show that noxious stimulation of the corneal surface activates neurons in the rostral pole of the nucleus caudalis, including parabrachially projecting neurons that receive direct input from corneal afferent fibers. We used immunocytochemical detection of c-Fos protein as an index of neuronal activation after noxious ocular stimulation. Animals had previously received injections of a retrograde tracer into either thalamic or parabrachial nuclei to identify projection neurons in the trigeminal dorsal horn. Noxious stimulation of the cornea induced c-Fos in neurons sending projections to parabrachial nuclei, but not thalamic nuclei. We also confirmed that corneal afferent fibers identified with cholera toxin B preferentially target trigeminal dorsal horn neurons projecting to the parabrachial nucleus. The parabrachial region sends ascending projections to brain regions involved in emotional and homeostatic responses. Activation of the ascending parabrachial system may explain the extraordinary salience of stimulation of corneal nociceptors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To test the hypothesis that the astrocytic chemokine (C-C motif) ligand 2 (CCL2) plays an important role in nocifensive behaviors after experimental tooth movement (ETM), the expression and cellular localization of CCL2 and astrocyte activation in the medullary dorsal horn (MDH) were determined by immunohistochemistry in rats. The dose-dependent effects of intrathecal C-C chemokine receptor type 2 (CCR2) antagonists on these changes in nocifensive behaviors were evaluated. Exogenous CCL2 was added to medullary dorsal horn slices to evaluate its contributory role in the induction of extracellular signal-regulated kinase (ERK) activation ex vivo. We found a significant increase in the expression of CCL2 and glial fibrillary acidic protein (GFAP), corresponding well to the nocifensive behaviors after ETM. In addition, application of recombinant CCL2 led to ERK activation, which could be attenuated effectively by pretreatment with CCL2-neutralizing antibody ex vivo. The magnitude of the nocifensive behavior could be reduced by medullary CCR2 antagonists in a dose-dependent manner. Therefore, the astrocytic CCL2 is actively involved in the development and maintenance of tooth-movement pain and thus may be a potential target for analgesics in orthodontic nocifensive responses control., (© 2013 Eur J Oral Sci.)

Prednisolone is a member of synthetic glucocorticoids which are widely used to treat chronic inflammatory diseases. In this study, neuronal degeneration and cell death, and glial reaction were investigated in the rat trigeminal ganglion (TG) and brainstem after subcutaneous injection of prednisolone for 7 days. Expression of c-Jun activating transcription factor 3 and caspase-3 was absent or infrequent in the TG, and cranial sensory and motor nuclei of saline- and prednisolone-treated animals. In these animals, distribution of calcitonin gene-related peptide-immunoreactive (-IR) neurons and nerve fibers was similar in the brainstem. In addition, the number of Iba1- and glial fibrillary acidic protein (GFAP)-IR cells with some processes in the brainstem was barely affected by prednisolone treatment. However, the treatment increased ramification of Iba1-IR processes in the subnucleus caudalis of the trigeminal sensory complex. Prednisolone scarcely influenced the morphology of GFAP-IR cells in the brainstem. Expression of p38 mitogen-activated protein kinase was very rare in the brainstem of saline- and prednisolone-treated animals. The present study suggests that microglia are activated by prednisolone in the subnucleus caudalis of the trigeminal sensory complex. The glucocorticoid may affect nociceptive transmission in the brainstem.

Temporomandibular joint (TMJ) arthritis is a common cause of orofacial pain. In the present study, the modulatory effects of N-methyl-d-aspartate receptors (NMDA-Rs) and magnesium were investigated in TMJ arthritis hypernociception. Male Wistar rats received an intra-articular injection of carrageenan (Cg) in the TMJ, and mechanical hypernociception was measured. The NMDA-R antagonist, MK-801, and magnesium chloride (MgCl₂ ) were administered before arthritis induction. Magnesium deficiency was promoted by feeding rats a synthetic magnesium-free diet for 9 d before injection of Cg. The Cg induced mechanical hypernociception that lasted for 120 h. MK-801 inhibited this hypernociceptive state. MgCl₂ pretreatment prevented Cg-induced hypernociception and altered the nociceptive threshold in the absence of Cg. Magnesium deficiency increased hypernociception and induced spontaneous hypernociceptive behavior. TMJ arthritis increased the expression of mRNA for all NMDA-R subunits and immunostaining of phosphorylated NR1 (phospho-NR1). MgCl₂ inhibited expression of NR2B mRNA and phospho-NR1 immunostaining and increased expression of NR3 mRNA. Magnesium deficiency increased expression of both NR1 and NR3 mRNAs and phospho-NR1 immunostaining in the trigeminal subnucleus caudalis. We found that magnesium modulates nociceptive behavior and induces NMDA-R subunit rearrangement in the subnucleus caudalis. The present results may lead to a better understanding of central processing in the nociceptive trigeminal pathway and the development of new approaches to treat orofacial pain with a TMJ origin., (© 2013 Eur J Oral Sci.)

Objectives: To determine the expression levels of nociceptin following experimental tooth movement., Materials and Methods: A total of 72 male Sprague-Dawley rats were divided into two groups: sham and experimental groups. For the experimental group, closed coil springs were used to mimic orthodontic force (80 g) between upper incisors and first molars, and the rats were killed at 0 hours, 4 hours, 12 hours, 1 day, 2 days, 5 days, 7 days, 10 days, and 14 days. All of these procedures were similar for the sham group, except for that no force was applied. The four rats killed at 0 hours without any intervention were used as the baseline control in each group. Trigeminal nucleus caudalis from both the ipsilateral and contralateral sides of force applications were obtained for immunostaining., Results: Nociceptin was expressed in both the ipsilateral and contralateral sides of each group. Its expression levels started to increase on day 2, peaked on day 7, and returned to baseline on day 10 in the experimental group, while expression levels started to decrease on day 1 and returned to baseline on day 10 in the sham group. Moreover, the expression levels were similar between the ipsilateral and contralateral sides in each group., Conclusion: The expression levels of nociceptin were elevated following experimental tooth movement. The anesthetic agent used in this study (chloral hydrate) may have an antagonism with nociceptin. Due to bilateral innervation of anterior teeth and bilateral projection of nerve fibers, the expression levels of nociceptin were similar between ipsilateral and contralateral sides.

Several lines of evidence suggest that the hypothalamus is involved in trigeminal pain processing. However, the organization of descending hypothalamic projections to the spinal trigeminal nucleus caudalis (Sp5C) remains poorly understood. Microinjections of the retrograde tracer, fluorogold (FG), into the Sp5C, in rats, reveal that five hypothalamic nuclei project to the Sp5C: the paraventricular nucleus, the lateral hypothalamic area, the perifornical hypothalamic area, the A11 nucleus and the retrochiasmatic area. Descending hypothalamic projections to the Sp5C are bilateral, except those from the paraventricular nucleus which exhibit a clear ipsilateral predominance. Moreover, the density of retrogradely FG-labeled neurons in the hypothalamus varies according to the dorso-ventral localization of the Sp5C injection site. There are much more labeled neurons after injections into the ventrolateral part of the Sp5C (where ophthalmic afferents project) than after injections into its dorsomedial or intermediate parts (where mandibular and maxillary afferents, respectively, project). These results demonstrate that the organization of descending hypothalamic projections to the spinal dorsal horn and Sp5C are different. Whereas the former are ipsilateral, the latter are bilateral. Moreover, hypothalamic projections to the Sp5C display somatotopy, suggesting that these projections are preferentially involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in rats. Therefore, our results suggest that the control of trigeminal and spinal dorsal horn processing of nociceptive information by hypothalamic neurons is different and raise the question of the role of bilateral, rather than unilateral, hypothalamic control.

Background: Obesity is a risk factor associated with several pain syndromes. However, the mechanisms underlying the association between obesity and pain are not known. The aim of this study was to test the hypothesis that obesity enhances neuronal responses to nociceptive stimulation within the trigeminal nucleus caudalis (TNC)., Methods: Male and female C57BL/6J mice were fed a high-fat or regular diet from the time of weaning until 20 weeks of age. We then quantified neuronal activation by measuring Fos immunoreactivity within the TNC in response to a facial injection of a low dose of capsaicin (1 μg/10 μL)., Results: We found that 0.01% capsaicin did not significantly increase Fos immunoreactivity in control mice fed a regular diet. In contrast, this low dose of capsaicin caused a 3.3-fold increase in Fos in the TNC in obese mice (p < 0.001)., Conclusions: These results support the hypothesis that diet-induced obesity in mice enhances nociceptive processing within the TNC. Diet-induced obesity may be a useful model for mechanistic studies. Future studies will improve our understanding of how obesity may contribute to trigeminal pain by sensitizing the trigeminal nociceptive system., (© 2012 European Federation of International Association for the Study of Pain Chapters.)

Calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid-1 (TRPV1) play an important role in the development of pain and migraine pathogenesis. Increase in plasma CGRP levels is associated with delayed migraine-like attacks in migraine patients. Although several lines of evidence have indicated a key role of CGRP in migraine pain, its mechanisms remain unclear. In this study, we aimed to investigate the functional role of CGRP on trigeminal nociceptive pathway by determining the alteration in TRPV1 levels in trigeminal ganglion (TG) and the activation of trigeminal nucleus caudalis (TNC) of rat. Post intravenous injection of CGRP (600ng/kg) at 60min significantly increased the levels of TRPV1, CGRP, phosphorylated protein kinase C and phosphorylated cyclic AMP responsive element-binding protein in TG of rats. The number of small and medium TRPV1 and CGRP positive immunostaining neurons accompanying with co-localization of TRPV1 with CGRP neurons were significantly increased in TG of CGRP-injected rats. The sustained increase in c-Fos expression in TNC neurons was also observed in CGRP-injected rats. These results indicate that CGRP may participate in trigeminal nociceptive system sensitization by induced increase in TRPV1 and CGRP levels in TG neurons and activation of the central neurons in TNC., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To examine the hypothesis that glial activation would regulate the expression of the N-methyl-D-aspartate receptor subunit 1 (NR1) in the trigeminal subnucleus caudalis (Sp5C) after temporomandibular joint (TMJ) inflammation., Methods: Inflammation of TMJ was produced in rats by injecting 50 μL complete Freund's adjuvant (CFA) into unilateral TMJ space. Sham control rats received incomplete Freund's adjuvant injection. Mechanical nociception in the affected and non-affected TMJ site was tested by using a digital algometer. Fractalkine, fluorocitrate, and/or MK801 were intracisternally administrated to examine the relationship between astroglial activation and NR1 upregulation., Results: CFA TMJ injection resulted in persistent ipsilateral mechanical hyperalgesia 1, 3, and 5 days after CFA injection. The inflammation also induced significant upregulation of CX3C chemokine receptor 1 and glial fibrillary acidic protein (GFAP) beginning on day 1 and of NR1 beginning on day 3 within the ipsilateral Sp5C. Intracisternal administration of fluorocitrate for 5 days blocked the development of mechanical hyperalgesia as well as the upregulation of GFAP and NR1 in the Sp5C. Conversely, intracisternal injection of fractalkine for 5 days exacerbated the expression of NR1 in Sp5C and mechanical hyperalgesia induced by TMJ inflammation. Moreover, once daily intracisternal fractalkine administration for 5 days in naïve rats induced the upregulation of NR1 and mechanical hyperalgesia., Conclusions: These results suggest that astroglial activation contributes to the mechanism of TMJ pain through the regulation of NR1 expression in Sp5C., (Wiley Periodicals, Inc.)

Background: In the orofacial region, limited information is available concerning pathological tongue pain, such as inflammatory pain or neuropathic pain occurring in the tongue. Here, we tried for the first time to establish a novel animal model of inflammatory tongue pain in rats and to investigate the roles of metabotropic glutamate receptor 5 (mGluR5)-extracellular signal-regulated kinase (ERK) signaling in this process., Methods: Complete Freund's adjuvant (CFA) was submucosally injected into the tongue to induce the inflammatory pain phenotype that was confirmed by behavioral testing. Expression of phosphorylated ERK (pERK) and mGluR5 in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were detected with immunohistochemical staining and Western blotting. pERK inhibitor, a selective mGluR5 antagonist or agonist was continuously administered for 7 days via an intrathecal (i.t.) route. Local inflammatory responses were verified by tongue histology., Results: Submucosal injection of CFA into the tongue produced a long-lasting mechanical allodynia and heat hyperalgesia at the inflamed site, concomitant with an increase in the pERK immunoreactivity in the Vc and C1-C2. The distribution of pERK-IR cells was laminar specific, ipsilaterally dominant, somatotopically relevant, and rostrocaudally restricted. Western blot analysis also showed an enhanced activation of ERK in the Vc and C1-C2 following CFA injection. Continuous i.t. administration of the pERK inhibitor and a selective mGluR5 antagonist significantly depressed the mechanical allodynia and heat hyperalgesia in the CFA-injected tongue. In addition, the number of pERK-IR cells in ipsilateral Vc and C1-C2 was also decreased by both drugs. Moreover, continuous i.t. administration of a selective mGluR5 agonist induced mechanical allodynia in naive rats., Conclusions: The present study constructed a new animal model of inflammatory tongue pain in rodents, and demonstrated pivotal roles of the mGluR5-pERK signaling in the development of mechanical and heat hypersensitivity that evolved in the inflamed tongue. This tongue-inflamed model might be useful for future studies to further elucidate molecular and cellular mechanisms of pathological tongue pain such as burning mouth syndrome.

The purpose of this study was to assess the temporal relationship between pancreas transplant and the development of electrophysiological changes in the sciatic and caudal nerves of alloxan-induced diabetic rats. Nerve conduction studies were performed in diabetic rats subjected to pancreas transplantation at 4, 12, and 24 weeks after diabetes onset, using non-diabetic and untreated diabetic rats as controls. Nerve conduction data were significantly altered in untreated diabetic control rats up to 48 weeks of follow-up in all time points. Rats subjected to pancreas transplantation up to 4 and 12 weeks after diabetes onset had significantly increased motor nerve conduction velocity with improvement of wave amplitude, distal latency, and temporal dispersion of compound muscle action potential in all follow-up periods (P<0.05); these parameters remained abnormal when pancreas transplantation were performed late at 24 weeks. Our results suggest that early pancreas transplant (at 4-12 weeks) may be effective in controlling diabetic neuropathy in this in vivo model., (© Georg Thieme Verlag KG Stuttgart · New York.)

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-HT(3) receptors in the contralateral Vi/Vc with direct microinjection of the select 5-HT(3) 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 transient receptor potential ankyrin 1 (TRPA1) is implicated in the mechanical and cold hyperalgesia following inflammation and nerve injury. Its expression has been presumed to be confined to primary afferent terminals. Here, we show that TRPA1 is expressed in astrocytes in the superficial laminae of the rat trigeminal caudal nucleus by use of electron microscopic immunoperoxidase and immunogold labeling techniques. Immunoreactivity for TRPA1 was consistently observed in somata and process of astrocytes and was weaker than that in presumed nociceptive primary afferent terminals, but increased significantly in the fine process of astrocyte in rats with experimental inflammation of the temporomandibular joint. Thus, we provide ultrastructural evidence that TRPA1 is expressed in astrocytes in the brain stem and propose a novel pathway of its involvement in the central mechanism of inflammatory hyperalgesia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Gamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN)., Methods: We utilize biochemical (superfused synaptosomes loaded with [(3) H]GABA) and morphological (immunofluorescence experiments with specific antibody) techniques., Results: Our results show that GLU potentiates the release of [(3) H]GABA evoked by 9, 15 and 30 mM [K(+)](e); 15 mM [K(+)](e)-evoked [(3) H]GABA release was also reinforced by domoate and kainate (KA), two naturally occurring GLU-receptor agonists. The enhancement of 15 mM [K(+)](e)-evoked [(3) H]GABA release produced by 100 μM KA was abolished by NBQX, a mixed AMPA/KA receptor antagonist, but was not affected by GYKI52466, a selective AMPA receptor antagonist. ATPA, a selective agonist for KA receptors containing the GLUK1 subunit, had no effect on depolarization-induced [(3) H]GABA release, and UBP310, which selectively antagonizes these same receptors, failed to reverse the KA-induced potentiation of 15 mM [K(+)](e)-evoked [(3) H]GABA release. The KA-induced potentiation was also unaffected by concanavalin A (10 μM), a positive allosteric modulator of GLUK1- and GLUK2-containing KA receptors. Immunofluorescence experiments revealed that GABAergic nerve terminals in the TCN differentially expressed GLUK subunits, with GLUK2/3-positive terminals being twice more abundant than GLUK1-containing synaptosomes., Conclusions: These findings indicate that pre-synaptic KA receptors facilitating GABA release from TCN nerve terminals mainly express GLUK2/GLUK3 subunits, supporting the notion that different types of KA receptors are involved in the various stages of pain transmission., (© 2012 European Federation of International Association for the Study of Pain Chapters.)