Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1-Expressing Neurons in Mouse Dorsal Root Ganglia

Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.

Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for INTRODUCTION Inflammation and peripheral nerve injury cause release of glutamate and other neurotransmitters/chemical mediators from sensory neurons, glia, and damaged cells in the spinal cord and peripheral tissue in rodents (Omote et al., 1998;Lawand et al., 2000;Liu and Salter, 2010;Miller et al., 2011). These mediators elicit firing in nociceptive neurons resulting in the induction of inflammatory and neuropathic pain. A similar phenomenon has been reported in humans: glutamate concentration in the synovial fluid of synovitis patients was 54 times higher than in non-arthritic control subjects .
Recent studies indicate that mGluR5 in DRG neurons contributes to activating and/or sensitizing transient receptor potential vaniloid type 1 (TRPV1) channel. TRPV1 channel is activated by capsaicin, noxious heat (>43 • C), low pH or various lipids, resulting in sensitization of sensory neurons in acute pain. TRPV1 activation via mGluR5 has been shown to produce an elevation in intracellular calcium concentration in a small number of rat and mouse DRG neurons (Crawford et al., 2000;Hu et al., 2002;Kim et al., 2009). TRPV1 activation is mediated by the production of prostaglandins via the phospholipase C (PLC) cascade in peripheral terminals of DRG neurons (Hu et al., 2002) and the production of diacylglycerol in central terminals (Kim et al., 2009). In addition, our previous work has revealed that mGluR5 biphasically modulates the intracellular calcium response induced by capsaicin, a TRPV1 agonist, in DRG neurons; mGluR5 activation potentiates the TRPV1-mediated intracellular calcium response, whereas subsequent cessation of mGluR5 activation depresses it, thus contributing to heat hyper-and hypoalgesia (Masuoka et al., 2015a,b).
Previous studies on the activation of mGluR5 receptors in the context of pain or inflammation have focused on short-term activation for a period of 5-20 min. However, glutamate remains elevated for much longer periods during inflammation. For example, a recent microdialysis study revealed that inflammation induced by formalin elevated extracellular glutamate concentration in the hind paw for over 3 h (Omote et al., 1998). In the present study, we show the long-term effects of group I mGluRs on DRG neurons. In particular, it was tested whether the proportion of functional TRPV1-expressing neurons are changed, thereby contributing to chronic heat hyperalgesia.

MATERIALS AND METHODS
All animal procedures were approved by the Ethics Committee of Kanazawa Medical University and animals were treated humanely, in accordance with the ''Guiding Principles for the Care and Use of Laboratory Animals'' set by the Japanese Pharmacological Society.

Calcium Imaging
Changes in intracellular calcium were measured with a fluorescent calcium indicator, as described previously (Yoshida et al., 2012;Masuoka et al., 2015b). For microscopic fluorometric measurement, cultured DRG neuronal cells were washed twice with ACSF and incubated for 45 min in the CO 2 incubator (37 ± 2 • C) in a solution of 3 µM of Fura-2-acetoxymethyl ester (Fura-2 AM; Dojindo Laboratories, Kumamoto, Japan) and 0.005% Cremophor EL (Sigma). After incubation, cells were washed in ACSF for 30 min and culture dishes were placed on the stage of an inverted microscope (ECLIPSE TE 300, Nikon, Tokyo, Japan) equipped with a 20× S-fluor objective. Fluorescence images were recorded and analyzed using a video image analysis system (ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan). Experimental agents were dissolved in ACSF and delivered by incubation in the CO 2 incubator and/or continuous perfusion in the recording chamber (2 mL/min) with a peristaltic pump. Image pairs were captured at 10 s intervals. Fura-2 fluorescence was recorded at an emission wavelength of 510 nm by exciting Fura-2 at 340 and 380 nm. The 340 to 380 nm fluorescence ratio (F340/F380) was used as a parameter of intracellular calcium concentration. To prevent the variance of neuronal population in DRG culture, cultures made from same mice at the same time were used in control and comparison groups in one experiment. Experiments were performed 4-6 times for each drug condition, with simultaneous recording of 20-40 neurons per iteration.

Neurons Viability Assay
To elucidate the neuronal death by glutamatergic drugs, cultured neurons were stained with a physiological solution containing 0.25% tripan blue (WAKO) for 5 min. Blue-stained and unstained neurons, which were recognized as dead and live neurons respectively, were counted in random fields under 200× magnification (DIAPHOT300, Nikon, Tokyo, Japan).

Whole-Cell Patch Clamp Recording
Cultured neurons were plated onto coverslips, transferred to the recording chamber, and superfused with ACSF. Neurons were visually identified using a 60× microscope objective (DIAPHOT300, Nikon, Tokyo, Japan). Pipettes for whole-cell recordings were made from borosilicate glass capillaries (1.5 mM outer diameter; World Precision Instruments Inc., Sarasota, FL, USA). Patch pipettes (4-6 MΩ) were filled with an internal solution containing 120 mM KCH 3 SO 3 , 5 mM KCl, 10 mM K-EGTA, 5 mM Na-HEPES, 3 mM Mg-ATP, and 0.4 mM Na-GTP (pH 7.4). Series resistance was 8-20 MΩ, which was monitored throughout recording. Membrane currents were recorded in a whole-cell configuration using an Axopatch-1D amplifier and pCLAMP 10 software (Axon Instruments, Foster City, CA, USA), digitized, and stored on a computer disk for offline analysis. Capsaicin and allylisothiocyanate (AITC)-induced current responses were recorded >5 min after the establishment of whole-cell configuration. Capsaicin current was induced by perfusion of 1 µM capsaicin for 15 s. More than 5 min after the capsaicin current disappeared, AITC currents were induced by perfusion of 500 µM AITC for 30 s. The extracellular solution was perfused at 3-4 mL/min.

Behavioral Tests
Pain responses to noxious heat stimuli were assessed in 8week-old C57BL/6J male mice (body weight; 20-24 g) by hot plate test. Mice were habituated to placement in experimental room for at least 30 min and on disconnected hot plate for 15 min before the test. The heated surface of the plate was kept at a temperature of 52 ± 0.2 • C (Model-DS 37, Ugo Basile, Comerio, Italy), as measured by a built-in digital thermometer, and verified by a surface thermometer. Mice were placed on the hot plate and surrounded by a clear acrylic cage (200mm diameter, 130 mm height). The latency to respond was measured as the time required for the mouse to lick, flick or bite its hind paw, or jump. After any of these responses, the mouse was immediately removed from the hot plate and returned to its home cage. Failure to respond within 20 s was grounds for removal from the test, a cutoff established to prevent tissue damage. All drugs were dissolved in saline and injected at 10 µl/paw. Control mice were injected with saline. Testing was performed three times in each mouse; 30 min prior to the intraplantar drug injection, 15 min after drug injection and 4 h after drug injection. To evaluate the changes in latency, the latency 30 min prior to the drug injection was subtracted from those observed 15 min, and 4 h after the treatment. Positive and negative values implied reduction and elevation of noxious heat sensitivity, respectively.

Statistical Analysis
Data were analyzed with SigmaPlot 13.0 Software (Systat Software Inc., San Jose, CA, USA). Results are expressed as mean ± standard error of the mean (SEM). The number of the cells examined are represented by ''n''. Parametric and nonparametric data were assessed using the one-way analysis of variance (ANOVA) and Kruskal-Wallis test, respectively. Chisquared tests were used to assess changes in the proportion of capsaicin-sensitive neurons. A p-value of < 0.05 was considered statistically significant.

Long-Term Activation of mGluR1/5 Increases the Proportion of Capsaicin-Sensitive Neurons
To evaluate the effect of long-term activation of glutamate receptors on TRPV1 responses, cultured DRG neurons were incubated for 4 h in ACSF containing glutamate (30 µM) or quisqualate (10 µM), AMPA receptor and mGluR1/5 agonist ( Figure 1A). TRPV1-mediated responses were induced by perfusion of 0.5 µM capsaicin, a TRPV1 agonist. Responses were assessed by measuring changes in intracellular calcium concentration with Fura-2 AM. Perfusion of capsaicin caused clear intracellular calcium elevation in a subset of DRG neurons ( Figure 1B). Long-term exposure to quisqualate appeared to produce an increase in the proportion of DRG neurons sensitive to capsaicin (Figure 1C). Figure 1D shows the changes in the cumulative curve for intracellular calcium responses induced by capsaicin after glutamate or quisqualate Frontiers in Cellular Neuroscience | www.frontiersin.org treatment. In the figure, the Y axis represents the cumulative frequency of recording neurons arranged in ascending order to capsaicin responses (∆F340/F380). In control group, more than half of the DRG neurons (54.2%) showed little to no response to capsaicin (∆F340/F380 ratio < 0.15). However, glutamate and quisqualate treatment increased the proportion of capsaicin-sensitive neurons to 72.0 and 67.9%, respectively. Data are summarized in Figure 1E. The proportion of capsaicin-sensitive neurons significantly increased after treatment with glutamate (30 µM; 67 out of 93 neurons, 72.0%, p < 0.001 compared to control group) or quisqualate (10 µM; 55 out of 81 neurons, 67.9%, p < 0.01), while 76 out of 166 neurons (45.8%) responded to capsaicin in control DRG neurons. This increase occurred in a concentrationdependent manner after treatment with glutamate (3-30 µM, Figure 1E). Although we analyzed the magnitude of capsaicininduced maximal response normalized to KCl, there was no significant difference in the amplitudes for either of ligand concentration (p = 0.069: Figure 1F). We performed tripan blue staining after 30 µM glutamate or 10 µM quisqualate treatment for 4 h ( Figure 1G). Long-term treatment with these drugs did not cause neuronal death in the DRG culture.
In the next experiment, DRG neurons were treated with 10 µM quisqualate for 4 h, followed by recording capsaicin responses in the absence of quisqualate (Figure 2A). An increase in proportion of capsaicin-sensitive neurons was observed even after the washout of quisqualate ( Figure 2B). The increased proportion of capsaicin-sensitive neurons associated with quisqualate (from 47.5% in control to 67.7% following quisqualate application) was significantly antagonized by treatment with 100 µM of the selective mGluR1 antagonist CPCCOEt, (78 out of 155 neurons, 50.3%, p < 0.01), but not by 50 µM of the selective mGluR5 antagonist MPEP (90 out of 157 neurons, 57.32%, p = 0.177; Figure 2B). However, treatment with NBQX (10 µM), a selective AMPA receptor antagonist, did not affect the quisqualate-related increase in capsaicin-sensitive neurons (95 out of 145 neurons, 65.5%, p = 1.00). In addition, DHPG, a selective mGluR1/5 agonist, significantly increased the proportion of capsaicin-sensitive neurons (95 out of 123 neurons, 77.2%, p < 0.01; Figure 2B). These results indicate that the increase in proportion of capsaicin-sensitive cells was caused by mGluR1 activation, and only partially by activation of mGluR5.

Activation of mGluR1/5 Increases the Proportion of Capsaicin-Sensitive Neurons in a Time-Dependent Manner
To examine the time-dependency of the observed increases in the proportion of capsaicin-sensitive neurons, 10 µM quisqualate was applied to the DRG culture for 1-4 h ( Figure 3A). Quisqualate time-dependently increased number of capsaicinsensitive neurons, and the increases became significant for drug application durations lasting over 2 h (51 out of 85 neurons, 60.0%, p < 0.05; Figure 3B). This increase in the proportion of capsaicin-sensitive neurons was sustained even after 2 h of washes and incubation in the absence of quisqualate (67 out of 122 neurons, 54.9%, p < 0.05; Figure 3B). We next examined whether the observed increase in capsaicin-sensitive neurons was related to gene expression. Actinomycin D (5 µg/mL), a transcription inhibitor, completely inhibited the quisqualateinduced increase in capsaicin-sensitive neurons (actinomycin D alone: 48 out of 110 neurons, 43.6%, vs. actinomycin D + quisqualate: 34 out of 93 neurons, 36.6%; p = 0.379; Figure 3C).

Increases in the Proportion of Capsaicin-Sensitive Neurons in Transient
Receptor Potential Ankyrin Type 1 (TRPA1)-Expressing Neurons TRPA1 as well as TRPV1 are recognized as multiple irritant sensors in the sensory neuron that contribute to the pain response (Guimaraes and Jordt, 2007;Zurborg et al., 2007). Recent studies revealed that TRPA1 interacts with TRPV1 and contributed to the development of inflammatory pain (Akopian, 2011). To clarify the relationship with TRPA1-positive neurons in the increase of the functional TRPV1-expressing neurons population, we examined the proportion of capsaicin-sensitive neurons in TRPA1-expressing neurons before and after chronic application of quisqualate. To identify DRG neurons expressing TRPA1, AITC (a TRPA1 agonist) was perfused before capsaicin application ( Figure 4A). Figure 4B shows the representative calcium mobilization detected with Fura-2 loaded in DRG neurons. Application of AITC (200 µM) and/or capsaicin (0.5 µM) induced intracellular calcium elevation in a distinct population of DRG neurons. The proportions of neurons that responded to AITC and/or capsaicin are shown in Figure 4C. Treatment with quisqualate for 4 h increased the proportion of capsaicin-sensitive neurons from 45.0% (control; 68 out of 151 neurons) to 59.5% (quisqualate; 97 out of 163 neurons, p < 0.05), in contrast, there are no significant changes in the proportion of AITC-sensitive neurons (control; 13.9% vs. quisqualate; 18.4%, p = 0.34). Among the neurons that responded to AITC, the increase in the capsaicin-sensitive population was drastic (23.8%; 5 out of 21 neurons to 73.3%; 22 out of 30 neurons, p < 0.01). These results implicate that the increase in capsaicin-sensitive population was mainly derived from AITCsensitive neurons.

Long-Term Application of Quisqualate Increases Capsaicin-Induced Current Density in AITC-Sensitive Neurons
To identify changes in the current density of TRPV1 after long-term activation of mGluR1/5, capsaicin -induced current were measured in AITC-sensitive and -insensitive neurons after quisqualate treatment for 4 h (Figure 5). Whole-cell recordings were conducted on cultured DRG neurons, and the membrane potential was held at −70 mV. Capsaicin at a concentration of 1 µM was rapidly perfused into the recording chamber for 15 s and then washed out with ACSF for more than 2 min. To check AITC responsiveness, 500 µM AITC was perfused into the chamber for 30 s. Representative recordings from control and quisqualate-treated DRG neurons are shown in Figures 5A,B, respectively. Although the current response to capsaicin in AITC-sensitive DRG neurons tends to be smaller than in AITC-insensitive DRG neurons (Figure 5A), quisqualate seems to increase the current response of capsaicin in AITCsensitive neurons (Figure 5B). Quisqualate treatment caused significant increase in current density induced by capsaicin in AITC-sensitive neurons (p < 0.05; Figure 5C), but not AITCinsensitive neurons (p = 0.081; Figure 5C).

Intraplantar Injection of Quisqualate Causes Hyperalgesia via mGluR Activation
We reported that TRPV1 activation is necessary to pain response to noxious heat in hot plate test, because 5 -iodoresiniferatoxin, a selective TRPV1 antagonist inhibits the heat response (Masuoka et al., 2015b). In a previous report, we showed that quisqualate at a dose of 4 nmol/paw significantly decreased the latency of heat response 15 min after the treatment. To test the longterm activation of mGluR1/5 on noxious heat sensitivity, higher doses of quisqualate or DHPG were injected into the foot soles of mice. Hot plate tests were subsequently performed 15 min and 4 h after the injection (Figure 6A). Fifteen minutes after quisqualate administration, we observed a significant decrease in the latency to shake, lick, or jump on the hot plate at doses of 20 and 40 nmol/paw (p < 0.05; Figure 6B). DHPG also decreased the latency at doses of 20 and 40 nmol/paw (p < 0.05). The quisqualate-induced heat hyperalgesia was abolished by MPEP (25 nmol/paw; p < 0.05) but not by CPCCOEt (50 nmol/paw; p = 0.986; Figure 6C). Decreased response latency persisted even 4 h after the quisqualate and DHPG injection at doses of 40 and 60 nmol/paw, respectively (p < 0.05; Figure 6D). This effect at 4 h was completely antagonized by CPCCOEt (p < 0.05) or MPEP (p < 0.05; Figure 6E). Finally, the effects of PD98059 and SB203580 on quisqualate (40 nmol/paw) induced heat hyperalgesia were examined. Decreased response latency 4 h after the quisqualate injection was antagonized by the concurrent injection of PD98059 (10 nmol/paw; p < 0.05) or SB203580 (0.2 nmol/paw; p < 0.05, Figure 6G). There was no significant effect on decreased latency 15 min after the injection by either kinase inhibitor ( Figure 6F).

DISCUSSION
In the present study, we evaluated TRPV1 function by measuring intracellular calcium responses to capsaicin in cultured DRG neurons. Application of non-selective glutamatergic receptor agonists for 4 h increased the proportion of capsaicin-sensitive neurons. This effect was inhibited by the administration of a selective mGluR1 antagonist and only partially by an mGluR5 antagonist. These results indicate that long-term activation of mGluR1 and mGluR5 increase the population of DRG neurons expressing functional TRPV1. In addition, this phenomenon is likely to depend on the expression of TRPV1 and/or proteins modulating TRPV1 sensitivity such as TRPV1b (Vos et al., 2006), and A-kinase anchoring protein (Jeske et al., 2009), because the enhanced neuron recruitment persisted up to 2 h following cessation of mGluRs activation and was completely inhibited in the presence of a transcription inhibitor, actinomycin D. It has been reported that mGluR1/5 activates several intracellular signaling molecules, phosphatidyl inositol 3 (PI3)-kinase, PKC, p38 MAPK, striatal-enriched protein tyrosine phosphatase (STEP), and caspase-3 (O'Riordan et al., 2006;Young et al., 2008;Chen et al., 2013). Kwon et al. (2014) reported that TRPV1 expression in DRG neurons is under the control of p38 MAPK phosphorylation in inflammatory sites. In present study, we also confirmed that the increase in capsaicin-sensitive neuron was suppressed by application of PLC, PKC, MEK and p38 MAPK inhibitors. Therefore, it was suggested that persistent activation of mGluR1/5 enhanced MEK and p38 MAPK pathways via PLC and PKC activation, resulted in increased numbers of DRG neurons expressing functional TRPV1. Recently, an inflammatory mediator prostaglandin E2, whose metabotropic receptors EP1-4 were expressed in DRG neurons, was found to facilitate the amount of TRPV1 expression, and the percentage of TRPV1-positive neurons in the rat neuropathic pain model (Ma et al., 2010). In addition, activation of the muscarinic acetylcholine receptor M2 decreased the TRPV1 transcript expression, and the number of capsaicin-responsive neurons in mice DRG (De Angelis et al., 2014). Therefore, metabotropic receptors might be one of the important regulators of functional TRPV1 expression in primary sensory neurons.
Although short-term stimulation of mGluR5 in cultured DRG neurons potentiated intracellular calcium influx via TRPV1 activation (Masuoka et al., 2015b), the magnitude of individual intracellular calcium elevation induced by capsaicin was not changed after long-term activation of glutamate receptors. These results suggest that the augmentation of the TRPV1-mediated intracellular response by mGluR5 disappeared after long-term stimulation of glutamate receptors. Direct phosphorylation of TRPV1 via PKC activation has been shown to acutely sensitize the TRPV1 response (Bhave et al., 2003), which is related to potentiation of TRPV1-mediated intracellular calcium responses by short-term stimulation of mGluR5. On the other hand, work in Xenopus oocytes has shown that the PKC activated by mGluR5 can simultaneously phosphorylate several targets of mGluR5a (S613, S881, S890 etc.), resulting in mGluR5 desensitization (Gereau and Heinemann, 1998). Therefore, the persistent stimulation of mGluR5 may desensitize acute mGluR5-TRPV1 interaction.
It has been shown that TRPV1 expression in nociceptive DRG neurons is increase in peripheral neuropathy in rodents (Hudson et al., 2001;Rashid et al., 2003;Biggs et al., 2007;Kim et al., 2008). For instance, TRPV1 protein in glabrous skin is overexpressed in Fabry disease model mice showing peripheral neuropathy (Lakomá et al., 2014). Kwon et al. (2014) found that heat hyperalgesia in inflammatory pain correlates to expression of TRPV1, a molecule working as a sensor of noxious heat in sensory neurons. Inflammatory pain induced by complete Freund's adjuvant increases artemin, resulting in increase of TRPV1/A1-positive DRG neurons (Ikeda-Miyagawa et al., 2015). In the present study, we show that the population of functional TRPV1-expressing neurons dramatically increased in TRPA1-expressing DRG neurons, and TRPV1 current density significantly increased in TRPA1expressing neurons. The TRPA1 channel was originally proposed to be a multiple irritant sensor and a noxious cold sensor (Guimaraes and Jordt, 2007;Zurborg et al., 2007). Thus, it is compelling that the increase in TRPV1 activity in TRPA1positive neurons augments centripetal flow of inputs associated with pain, even when rodents are exposed to the same noxious heat stimuli.
TRPV1-TRPA1 interactions in peripheral neurons are important for adaptive development of inflammatory pain system (Akopian, 2011). Bandell et al. (2004) reported that one of the dominant inflammatory mediators, bradykinin, could indirectly activate TRPA1, likely via production of diacylglycerol. It has also been known that the pharmacological desensitization of TRPA1-mediated responses in sensory neurons lacking TRPV1 is more pronounced than in neuron expressing TRPV1 (Akopian et al., 2007). Taken together, these lines of evidence suggest that the co-expression of TRPV1 and TRPA1 as a consequence of mGluR1/5 activation may lead to a slower desensitization of bradykinin responses in sensory neurons, and this, in turn, may enhance the development of inflammatory hyperalgesia.
Disruption of protein kinase A (PKA) or PKC-mediated TRPV1 sensitization attenuates λ-carrageenan-induced heat hyperalgesia (Fischer et al., 2013). In addition, increase of TRPV1/TRPA1 expression induced artemin, a neurotrophic factor, caused heat hyperalgesia (Ikeda-Miyagawa et al., 2015). We employed a hot plate test to test whether the increase in functional TRPV1-expressing neurons induced by mGluR1/5 affects noxious heat sensitivities in the periphery. Intraplantar injections of high-dose quisqualate induced persistent heat hyperalgesia for 4 h in the present study. Hyperalgesia 15 min after the injection was attenuated by mGluR5 antagonist, but not by mGluR1 antagonist. This result confirms that the acute phase of hyperalgesia is caused by augmentation of the TRPV1 response by mGluR5, without changes in the proportion of TRPV1-expressing neurons, as we previously reported (Masuoka et al., 2015b). On the other hand, hyperalgesia 4 h after the injection was inhibited by both mGluR1 and mGluR5 antagonists. In addition, the MEK and p38 MAPK inhibitors selectively attenuated hyperalgesia 4 h after the injection. This suggests that chronic heat hyperalgesia is related to the increase in the proportion of functional TRPV1-expressing neurons following mGluR1 and mGluR5 activation.
In conclusion, long-term activation of mGluR1/5 produces an increase in the number of DRG neurons expressing functional TRPV1, which may contribute to the development of chronic heat hyperalgesia. Persistent elevation of glutamate release in the periphery may be crucial for understanding sensory disorders induced by inflammation and tissue damage.

AUTHOR CONTRIBUTIONS
TM designed and performed the research, analyzed the data and wrote the article. MK, JY and NI performed research and analyzed data. JY, TI, IM, NK and MN wrote and revised the article.