Functional interactions between NMDA receptors and TRPV1 in trigeminal
sensory neurons mediate mechanical hyperalgesia in the rat masseter muscle
Jongseok Leea, Jami L. Salomana, Gustave Weilanda, Q-Schick Auhb, Man-Kyo Chunga, Jin Y. Roa,b,⇑
aUniversity of Maryland School of Dentistry, Department of Neural and Pain Sciences, Program in Neuroscience, Baltimore, MA, USA
bKyung Hee University, School of Dentistry, Department of Oral Medicine, Seoul, Republic of Korea
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
a r t i c l ei n f o
Received 22 December 2011
Received in revised form 7 March 2012
Accepted 12 April 2012
a b s t r a c t
The NMDA and TRPV1 receptors that are expressed in sensory neurons have been independently demon-
strated to play important roles in peripheral pain mechanisms. In the present study, we investigated
whether the 2 receptor-channel systems form a functional complex that provides the basis for the devel-
opment of mechanical hyperalgesia. In the masseter muscle, direct application of NMDA induced a time-
dependent increase in mechanical sensitivity, which was significantly blocked when the muscle was pre-
treated with a specific TRPV1 antagonist, AMG9810. The NR1 subunit of the NMDA receptor and TRPV1
were coexpressed in 32% of masseter afferents in trigeminal ganglia (TG). Furthermore, NR1 and NR2B
formed protein-protein complexes with TRPV1 in TG as demonstrated by coimmunoprecipitation exper-
iments. Calcium imaging analyses further corroborated that NMDA and TRPV1 receptors functionally
interact. In TG culture, application of NMDA resulted in phosphorylation of serine, but not threonine
or tyrosine, residues of TRPV1 in a time course similar to that of the development of NMDA-induced
mechanical hyperalgesia. The NMDA-induced phosphorylation was significantly attenuated by CaMKII
and PKC inhibitors, but not by a PKA inhibitor. Consistent with the biochemical data, the NMDA-induced
mechanical hyperalgesia was also effectively blocked when the muscle was pretreated with a CaMKII or
PKC inhibitor. Thus, NMDA receptors and TRPV1 functionally interact via CaMKII and PKC signaling cas-
cades and contribute to mechanical hyperalgesia. These data offer novel mechanisms by which 2 ligand-
gated channels in sensory neurons interact and reinforce the notion that TRPV1 functions as a signal inte-
grator under pathological conditions.
? 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
There is a large body of evidence demonstrating that endoge-
nous release of glutamate in peripheral tissue after injury or
inflammation can exacerbate pain conditions and modulate func-
tional properties of nociceptors in skin, muscles, and joints
[33,46,58]. A higher level of endogenous glutamate in the masseter
muscle of temporomandibular disorder patients relative to healthy
subjects further suggests the involvement of peripheral glutamate
receptors (gluRs) in persistent muscle pain conditions . Human
and animal studies have consistently demonstrated the involve-
ment of gluRs such as the NMDA receptor (NMDAR) and the
metabotropic glutamate receptor 5 (mGluR5) in trigeminal sensory
neurons in acute pain and mechanical hyperalgesia arising from
orofacial muscle tissue [6,36,54,63]. However, the cellular mecha-
nisms by which these gluRs lead to heightened mechanical sensi-
tivity in deep craniofacial tissue are still unknown.
Mice lacking functional TRPV1 display impaired behavioral re-
sponses to noxious heat stimuli applied to cutaneous tissue while
responses to noxious mechanical stimuli remain intact [9,10].
More recent studies, however, indicate that TRPV1 also contributes
to the development of mechanical hyperalgesia. TRPV1 antagonists
effectively reduce mechanical hyperalgesia in rats with spinal
nerve ligation [13,14] as well as complete Freund’s adjuvant
(CFA)–induced mechanical hyperalgesia [19,24,50]. Aside from
cutaneous tissue, TRPV1 has been suggested to play an important
role in mechanical sensation in deep tissue such as the colon
. In muscle tissue, the direct injection of capsaicin significantly
lowers mechanical thresholds in both humans and rats [1,56], and
the blockade of TRPV1 attenuates mechanical hyperalgesia result-
ing from eccentric muscle contraction .
Activation of NMDARs results in an influx of Ca2+and invokes
Ca2+calmodulin-dependent protein kinase II (CaMKII), protein
kinase C (PKC), or protein kinase A (PKA) [18,22,41,68]. These
0304-3959/$36.00 ? 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
⇑Corresponding author at: Department of Neural and Pain Sciences, Program in
Neuroscience, University of Maryland Baltimore, School of Dentistry, 650 West
Baltimore Street, Baltimore, MA 21201, USA. Tel.: +1 410 706 6027; fax: +1 410 706
E-mail address: JRo@umaryland.edu (J.Y. Ro).
?153 (2012) 1514–1524
intracellular kinases directly modulate TRPV1 function, and phos-
phorylation of TRPV1 by such intracellular kinases has been sug-
gested as a major mechanism that accounts for sensitization of
TRPV1 [44,45,57]. However, there are no data on whether NMDAR
activation results in TRPV1 sensitization and phosphorylation in
sensory neurons. These observations led us to hypothesize that
the 2 important receptor/channel systems that have been indepen-
dently implicated in muscle pain and hyperalgesia (i.e., NMDAR
and TRPV1) functionally interact in nociceptors and these interac-
tions constitute the peripheral mechanisms underlying the devel-
opment of mechanical hyperalgesia. To test our hypothesis we
specifically investigated (1) whether the activation of peripheral
NMDARs leads to mechanical hyperalgesia via a TRPV1-dependent
manner, (2) whether the activation of NMDARs enhances TRPV1
function in TG neurons, (3) whether NMDAR activation leads to
TRPV1 phosphorylation in TG neurons, and (4) the intracellular
mechanisms involved in NMDAR-TRPV1 interactions.
2. Materials and methods
Adult male Sprague-Dawley rats (150 to 350 g; Harlan, IN, USA)
were used. All animals were housed in a temperature-controlled
room under a 12:12 light-dark cycle with access to food and water
ad libitum. All procedures were conducted in accordance with the
National Institutes of Health Guide for the Care and Use of Labora-
tory Animals (publication no. 80-23) and under a University of
Maryland approved Institutional Animal Care and Use Committee
2.2. Drug preparation
For behavioral experiments, NMDA, a specific agonist for the
NMDAR, and AP5, a competitive NMDAR antagonist, were dis-
solved in phosphate-buffered saline (PBS). AMG9810, a specific
antagonist for TRPV1, was dissolved in dimethyl sulfoxide (DMSO)
(100%). A PKC inhibitor, GF109203X, a specific CaMKII inhibitor,
KN93, a PKA activator, forskolin, and a specific PKA inhibitor,
KT5720, were dissolved in DMSO and diluted in PBS with final con-
centration of DMSO <1%. The concentration of mustard oil (MO)
For Ca2+imaging experiments, drugs were diluted to final con-
centrations in Ca2+imaging buffer (CIB): NMDA (500 lM), glycine
(10 lM), capsaicin (10 nM, 1 lM). Stock solutions were generated
as follows: NMDA 100 mM, pH 7 in CIB, glycine 100 mM in H2O,
and capsaicin 10 mM in ethanol. The final concentration of ethanol
GF109203X (10 lM), and a PKA inhibitor, KT5720 (1 lM), were
diluted to final concentrations in culture media from the stock
solution dissolved in DMSO. The final concentration of DMSO
was < 0.1%.
NMDA, AP5, forskolin, MO, and capsaicin were purchased from
Sigma (St. Louis, MO, USA), AMG9810, GF109203X, KN93, and
KT5720 from Tocris Bioscience (Ellisvile, MO, USA), and KN92 from
Calbiochem (San Diego, CA, USA).
KN92 andKN93 (10 lM),
2.3. Behavioral studies—assessment of mechanical sensitivity in
Noxious chemical or mechanical stimulation of the masseter
muscle evokes characteristic shaking of the ipsilateral hindpaw in
lightly anesthetized rats. We have previously described the use of
this behavior for testing mechanical sensitivity of the masseter
muscle [55,56]. This lightly anesthetized rodent paradigm allows
the delivery of a calibrated and reliable mechanical stimulus on
the masseter muscle or temporomandibular joint before and after
pharmacological manipulations, which is difficult in awake ani-
mals. Initially, rats (250 to 350 g) were anesthetized with an intra-
peritoneal injection of sodium pentobarbital (40 mg/kg). A level of
light anesthesia was determined by providing a noxious pinch to
the tail or the hindpaw with a serrated forceps. Animals typically
responded to the noxious pinch on the tail with an abdominal con-
traction and with a withdrawal reflex to the noxious pinch of a
hindpaw within 30 minutes after the initial anesthesia. Once the
animal reached this level, a metal clip calibrated to produce
600 g of force was applied 5 consecutive times to the tail, and
experiments were continued only after the animals showed reli-
able reflex responses to every clip application. A tail vein was con-
nected to an infusion pump (Harvard Apparatus, Pump11 Holliston,
Massachusetts) for continuous infusion of pentobarbital. The rate
of infusion was adjusted to maintain a relatively light level of anes-
thesia throughout the duration of the experiment (3 to 5 mg/h).
A baseline mechanical threshold for evoking the nocifensive re-
sponses was determined 15 minutes before drug injection using
the electronic von Frey (VF) anesthesiometer (IITC Life Science,
Inc, Woodland Hills, CA, USA). A rigid tip (2 mm diameter) attached
to the VF meter was applied to the masseter muscle until the ani-
mals responded with hindpaw shaking. The animal’s head was
rested flat against the surface of the table when pressing the
anesthesiometer on the masseter in order to provide stability.
The threshold was defined as the lowest force needed to evoke a
hindpaw response. Changes in masseter sensitivity were then as-
sessed at 15, 30, 45, 60, and 90 minutes after drug treatments. To
maintain the consistency of assessing behavioral responses, all
behavioral observations were made by 1 experimenter blinded to
the experimental conditions.
To test whether the activation of NMDAR induces mechanical
hypersensitivity, NMDA (10 mmol/40 lL) or the same volume of
vehicle was administered into the masseter muscle. To determine
that NMDA-induced behavioral responses are receptor mediated,
the masseter muscle was pretreated with AP5 (1 mmol/20lL)
5 minutes before the injections with NMDA in the same muscle.
To investigate the involvement of TRPV1 in NMDA-induced
mechanical hypersensitivity, AMG9810 (10, 100 nmol/10lL), or
the same volume of vehicle was administered in the masseter
5 minutes before the NMDA treatment. The high dose of
AMG9810 (100 nmol) was administered in the muscle contralat-
eral to the NMDA treatment to confirm that the antagonist’s effects
were mediated by blocking local TRPV1 receptors. To rule out the
possibility that the antagonist alone could produce analgesic re-
sponses, AMG9810 (100 nmol) was administered in the masseter
muscle without NMDA. Separate groups of animals received intra-
muscular MO (20%, 20 lL) alone or in combination with AMG9810
(100 nmol) to test the specificity of AMG9810. For kinase experi-
ments, GF109203X (100 nmol/20 mL), KN93 (0.1 and 1 mmol/
20 mL), KT5720 (30 nmol/20 mL), or the same volume of vehicle
was preadministered in the masseter 5 minutes before NMDA
injection in the same muscle. Finally, forskolin (50 nmol/20 mL)
was administered with either KT5720 (30 nmol/20 mL) or vehicle.
All injections were made directly in the midregion of the masseter
muscle with either a 25-mL or a 50-mL Hamilton syringe over 5 to
10 seconds. Experimental and control groups were randomized,
and all groups consisted of 6 to 8 rats per group.
The percent changes in VF thresholds after drug treatment were
calculated with respect to the baseline threshold and plotted
against time. The time-dependent mean percent changes in
mechanical thresholds normalized to the baseline threshold were
analyzed with a 2-way analysis of variance (ANOVA) with repeated
measures. All multiple-group comparisons were followed by a
J. Lee et al./PAIN
?153 (2012) 1514–1524
post-hoc test (Bonferroni). The significance of all statistical analy-
ses presented in this report was set to P < .05.
2.4. Immunohistochemistry for TRPV1 and NR1 in masseter afferents
Fast Blue (FB) (2%; 10 lL; Sigma) was injected in the masseter to
retrogradely label TG muscle afferents in 3 rats. FB was injected
into multiple sites in the masseter muscle using aseptic tech-
niques. To avoid any leakage of the tracer, the injection needle
was left in place for 1 to 2 minutes before it was slowly retracted.
The injection site was then covered with petroleum jelly. After
allotting 7 days for the FB to label the masseter afferents, the ani-
mals were transcardially perfused with cold PBS followed by 4%
paraformaldehyde in PBS (250 mL; pH 7.3 to 7.4; Sigma). The right
TG from each rat was extracted and postfixed for 90 minutes,
placed in 30% sucrose solution at 4?C overnight, and sectioned cor-
onally at 12 lm. Every eighth section was collected and mounted
on gelatin-coated slides for double-labeling immunohistochemis-
try. After blocking, the sections were incubated overnight with pri-
mary antisera for the NR1 subunit of the NMDAR (1:100; goat
polyclonal; Santa Cruz, CA), and incubated with Daylight 594 don-
key antigoat antiserum for 60 minutes at 37?C for immunofluores-
cence. The NR1 antibody is directed against the human C-terminus
of the NMDAf1 receptor subtype. After staining for NR1, sections
were incubated overnight with primary antisera for TRPV1
(1:1000; rabbit polyclonal; Neuromics, Edina, MN), followed by
60 minutes with Daylight 488 goat antirabbit antiserum (1:250;
Jackson Immunoresearch West Grove, PA) for immunofluores-
cence. This antibody corresponds to amino acid residues 4 to 21
of TRPV1. The primary antibody for TRPV1 or NR1 was omitted
from the processing of selected sections to control for nonspecific
TRPV1-, NR1-, and FB-positive cells were counted from 8 repre-
sentative sections per ganglion from 3 TGs. The somatotopic distri-
bution of FB-positive cells within the TG was assessed to ensure
that the FB did not spread to other divisions of the TG. Trigeminal
and facial motor nuclei were also evaluated as positive and nega-
tive controls for FB labeling, respectively. Only the labeled neurons
that showed a clear nucleus were included in the counting. The
percentages of masseter afferents labeled with TRPV1 and/or NR1
were calculated and presented as mean ± standard error of the
2.5. Calcium imaging
TG extracted from naïve male rats weighing between 150 and
200 g were minced in Dulbecco’s modified Eagle medium/F12 con-
taining horse serum and penicillin/streptomycin/glutamine on ice,
and were then incubated in media containing collagenase (type XI,
Sigma) for 30 minutes at 37?C. After mechanical dissociation, the
cells were incubated for 2 minutes in trypsin (0.05%)/ethylenedi-
aminetetraacetic acid (0.1%) in PBS. The cells were then separated
by Percoll and plated on poly-L-ornithine and laminin-coated glass
coverslips. Two rats (4 TGs) were used for 1 culture, and cultures
were used for experimentation 48 hours later.
Primary TG cultures were loaded with 20 lL of 1 mM Fura2-AM
and 2 lL of 20% pluronic acid for 40 minutes at 37?C in a buffer
containing NaCl 130, KCl 3, 0.5 MgCl2, CaCl20.9, 4-(2-hydroxy-
ethyl)-1-piperazineethanesulfonic acid (HEPES) 10, sucrose 10,
NaHCO31.2 (in mM, pH 7.45, 320 mOsm adjusted with mannitol).
After a 15-minute wash period for de-esterification, dual images
(510-nm emission) were collected every 2 seconds using NIS Ele-
ments (Nikon Melville, NY). The Fura response (F) was defined as
the ratio of emissions measured during excitation at 340 and
380 nm. The CIB used during recording was as follows in mM: NaCl
130; KCl 3; CaCl22.5; HEPES 10; sucrose 10; NaHCO31.2; pH 7.45;
and 320 mOsm. We omitted Mg2+from the buffer to facilitate the
response of NMDARs.
2.6. Recording protocol
An initial submaximal concentration of capsaicin (10 nM) was
applied to assess the basal responses across individual cells and
culture preparations. Next, vehicle (CIB) or 500 lM NMDA/10 lM
glycine was applied for 3 minutes. One minute after the NMDAR
agonist application, a second application of 10 nM capsaicin was
made. A submaximal concentration of capsaicin was used to min-
imize the desensitizing effects of repeated capsaicin applications,
and also to ensure that sensitization can be clearly observed while
ceiling effects are avoided. In the control condition, after the sec-
ond 10 nM capsaicin application, NMDA/glycine was applied to
identify NMDAR positive cells. At the end of the experiment, a
supramaximal concentration of capsaicin (1 lM) was applied to
confirm the identification of all TRPV1 neurons in both experimen-
tal and control conditions.
2.7. Data analysis
We calculated the changes in Fura response (DF, F minus base-
line) in every cell. Baseline was defined as the average of the 5 data
points before a given stimulus application. A neuron was consid-
ered to be a responder if the Fura response to NMDA or any of
the 3 capsaicin applications was above threshold.
Threshold was determined after a similar method as in Chung
et al. . A histogram was generated from a population of neu-
rons to a given stimulus protocol. The DF > 2 SD from the peak
DF of nonresponding neurons was classified as responsive. The
DF of the first and second capsaicin application were analyzed with
2-way ANOVA with repeated measures, followed by a Bonferroni
To confirm that the NMDA-induced Ca2+responses are indeed
mediated by functional NMDAR, we treated cells with either AP5
(125 lM) or vehicle with NMDA (500 lM NMDA/10 lM glycine).
The proportions of cells showing NMDA-induced responses were
compared between AP5- and vehicle-treated groups with the v2
We have also conducted additional calcium imaging experi-
ments to test the specificity of AMG9810. To confirm that
AMG9810 does not directly modulate NMDAR, we treated the cells
with AMG9810 (100 nmol) or vehicle with NMDA (500 lM NMDA/
10 lM glycine). As a positive control we tested the same concen-
tration of AMG9810 with capsaicin (100 nmol). The proportions
of cells showing NMDA- or capsaicin-induced responses were com-
pared between AMG9810- and vehicle-targeted groups with thev2
2.8. Immunoprecipitation and coimmunoprecipitation
Dissociated TG cultures were lysed with RIPA lysis buffer (cell
signaling). The lysates were centrifuged at 12,000 rpm at 4?C for
20 minutes to remove cellular debris. The supernatant was incu-
bated with anti-TRPV1 polyclonal antibody (Calbiochem) at 4?C
overnight, and then with protein A/G-Sepharose beads (Santa Cruz)
for 2 hours. Lithium Dodecyl Sulfate (LDS) sample buffer including
Sodium Dodecyl Sulphate (SDS) was added to elute proteins from
the protein A/G beads. Each sample was separated by 4% to 12%
NuPAGE gel (Invitrogen) electrophoresis and subjected to immu-
noblotting. To determine the level of p-Ser, p-Thr, or p-Tyr, the
membranes were blocked and incubated with antiphosphoserine
(1:1000; Santa Cruz, Billerica, Massachusetts) or antiphosphothre-
onine (1:1000; Calbiochem) or antiphosphotyrosine (1:1000;
J. Lee et al./PAIN
?153 (2012) 1514–1524
immunoglobulin G Horseradish Peroxidase (HRP) (1:5000), and
then developed with the Enhanced Chemiluminescence (ECL)
detection kit (Amersham Bioscience, Piscataway, NJ). The mem-
branes were stripped and reprobed with anti-TRPV1 (1:1000;
Calbiochem) to determine the amount of immunoprecipitated pro-
teins. After immunoblotting, the bands on the membrane were
scanned and quantified with an image analyzer (Image J software)
and normalized to that of TRPV1, which served as the loading con-
trol. The data were subjected to either a 1-way ANOVA or Kruskal-
Wallis analysis, depending on the outcome of the normality test.
All multiple-group comparisons were followed by a post-hoc test.
Data are shown as mean ± standard error of the mean obtained
from 6 to 8 repeated experiments. For coimmunoprecipitation
(co-IP) of NMDARs and TRPV1, intact TG were extracted and pro-
cessed according to the protocol described earlier. For this experi-
ment, the concentration of NP-40 (1%) in the lysis buffer was
diluted to 0.5%, and the tissue incubation time was adjusted to
2 hours for anti-NR1 and 4 to 6 hours for anti-NR2B. The following
antibodies were used: NR1 (1:500; Millipore) or NR2B (1:500;
Millipore) and TRPV1 (1:1000; Calbiochem). The specificity of
these antibodies was confirmed in previous studies [3,47,69].
Two TG were used for each co-IP experiment.
3.1. NMDAR-mediated mechanical hyperalgesia involves TRPV1
The baseline mechanical threshold that evoked the nocifensive
response ranged from 501 to 582 g in lightly anesthetized rats,
which was similar to those we previously reported [36,56]. There
were no significant differences in the baseline mechanical thresh-
olds between any of the groups we examined. We first confirmed
that direct activation of NMDARs in the masseter muscle evokes
a time-dependent increase in mechanical sensitivity (Fig. 1A).
There was a significant group effect (F = 65.04, P < .001) and a sig-
nificant time effect (F = 33.59, P < .001). Masseteric injection of
NMDA (10 lmol/40 lL) significantly decreased the mechanical
threshold reaching the peak effect at 15 minutes (?39% ±2.6) and
gradually returning to the baseline level within 2 hours after the
injection (Fig. 1A). The vehicle injection in the same manner did
not alter the mechanical sensitivity of the masseter muscle. A pre-
treatment of the muscle with a systemically low dose of AP5
(1 lmol) , a competitive NMDAR antagonist, completely pre-
vented the development of NMDA-induced masseter hyperalgesia,
indicating that the responses are produced specifically by the acti-
vation of local NMDARs.
We then examined whether the NMDA-induced mechanical
hyperalgesia was altered by pretreatment of the muscle with a
specific TRPV1 antagonist, AMG9810. There was a significant group
effect (F = 37.7, P < .001) and a significant time effect (F = 44.8,
P < .001) (Fig. 1B). The NMDA-induced mechanical hyperalgesia
was significantly blocked when 100 nmol of AMG9810 was pread-
ministered in the masseter. A lower dose of AMG9810 (1 nmol) did
not significantly block the NMDA-induced responses indicating a
dose-dependent effect of the antagonist (Fig. 1B). The vehicle did
not alter the NMDA-induced mechanical hyperalgesia. The higher
dose of AMG9810 (100 nmol) administered in the contralateral
masseter muscle failed to attenuate the NMDA-induced masseter
hypersensitivity, thus suggesting that the AMG9810 produced its
effects via blocking local TRPV1 (Fig. 1C). The same dose of
AMG9810 injected alone did not alter masseter sensitivity. The
pretreatment with all drugs was made 5 to 10 minutes before
the NMDA injection.
Because it is possible that AMG9810 at 100 nmol can produce
nonspecific effects such as desensitization of nociceptive afferents,
we conducted additional experiments in which AMG9810 was
used to block behavioral responses induced by MO (20%), a TRPA1
agonist. Fig. 1D shows that there was only a significant time effect
(F = 67.8, P < .001), without a significant group effect (F = 1.5,
P > .05). The dose of AMG9810 that significantly attenuated the
NMDA-induced responses failed to block the effects of MO, sug-
gesting that AMG9810 did not produce nonspecific effects, but by
specifically antagonizing TRPV1. Together these behavioral data
showed that functional interactions between peripheral NMDARs
and TRPV1 mediate the development of mechanical hyperalgesia.
3.2. NMDAR and TRPV1 are coexpressed in TG neurons and form
We have previously shown that NMDA receptor subunits NR1,
NR2A, and NR2B are expressed in TG, and that TRPV1 is expressed
in distinct populations of TG neurons that innervate masseter
afferents [37,56]. Here, we sought to investigate the extent to
which NMDARs and TRPV1 are coexpressed in masseter afferents.
NR1 positive neurons were more abundant in TG as the NR1 was
expressed in all size TG neurons, compared with TRPV1-positive
neurons that were predominantly expressed in small TG neurons.
Of the 1827 TRPV1-positive TG neurons we examined, 96% coex-
pressed NR1. These data are consistent with the ubiquitous expres-
sion of NR1 in TG .
Of the 111 FB-positive cells from 3 rats, 81.6% ± 4% were NR1
positive and 34% ± 2% were TRPV1 positive. The percentage of FB-
positive masseter afferents that coexpress NR1 and TRPV1 was
32% ± 3%. Fig. 2A shows examples of masseter afferents that con-
tain both NR1 and TRPV1. These data suggest that most masseter
afferents that express TRPV1 also contain NR1, and that functional
interactions between the 2 receptor systems can occur at the
Fig. 1. The effects of intramuscular injection of NMDA and AMG9810 on masseter mechanical sensitivity. (A) Changes in mechanical sensitivity after NMDA, phosphate-
buffered saline, or AP5 followed by NMDA administration in the masseter muscle. (B) NMDA-induced changes in mechanical sensitivity after pretreatment of the masseter
with AMG9810, a specific TRPV1 antagonist, or vehicle. (C) The effects on AMG9810 administered in the masseter contralateral to the NMDA treatment or AMG9810 alone on
masseter mechanical sensitivity. (D) The effects of AMG9810 (100 nmol) on mustard oil-induced mechanical hyperalgesia. (⁄P < .05 in group effects with respect to vehicle
condition in 2-way analysis of variance;#P < 0.05 to the baseline in post-hoc test; n = 6 to 8 for each group).
J. Lee et al./PAIN
?153 (2012) 1514–1524
We then utilized co-IP techniques to demonstrate protein-pro-
tein interactions between NMDARs and TRPV1. Immunoprecipita-
tion of TG extracts using a TRPV1 antibody resulted in a clear
immunoblot stained with an NR1 antibody (Fig. 2B). The mem-
branes were then stripped and blotted with the TRPV1 antibody
to confirm immunoprecipitation of TRPV1. The reverse immuno-
precipitation in which TRPV1 was blotted after immunoprecipita-
tion of TG extracts using the NR1 antibody confirmed the co-IP of
the 2 receptors. Because functional NMDARs require both NR1
and NR2 subunits, we performed a similar experiment with an
NR2B antibody. As with NR1, NR2B also coimmunoprecipitated
with TRPV1 in TG extracts. These data provided evidence that
NMDARs and TRPV1 are not only coexpressed in the same TG neu-
rons, but also that they form protein-protein complexes.
3.3. NMDA potentiates capsaicin responses in a subset of trigeminal
To further explore functional interactions between NMDARs
and TRPV1, we examined whether NMDA can enhance capsaicin-
induced Ca2+responses in dissociated TG neurons. The application
of a low concentration of capsaicin (10 nM) typically produced a
small increase in Ca2+responses in a subpopulation of neurons.
To examine whether activation of NMDARs enhances capsaicin-
evoked responses, we applied vehicle or NMDA with glycine before
the second application of 10 nM of capsaicin and compared the
relative amplitude of the responses after the first and second appli-
cation of capsaicin. In the vehicle-treated group, we applied NMDA
after the second application of capsaicin to identify neurons
responding to both capsaicin and NMDA so that we could compare
vehicle- and NMDA-treated groups only in those subpopulations.
The proportions of neurons responding to NMDA only, capsaicin
only, or both NMDA and capsaicin were similar across the 2 treat-
ment groups: vehicle [2% (20), 88% (792), and 9% (84) of 896 cells,
and NMDA [2% (18), 84% (821), and 14% (140) of 979 cells], respec-
tively. Neurons responding to both capsaicin and NMDA showed
mixed responses to the repeated application of capsaicin. The per-
centages of neurons showing increasing or decreasing responses
were similar between vehicle, 55% (46 of 84) and 45% (38 of 84),
respectively, and NMDA, 49% (69 of 140) and 51% (71 of 140),
respectively, treated groups.
The cells with a low Ca2+influx during the initial capsaicin
treatment tended to show increasing responses to the second cap-
saicin treatment in both vehicle- and NMDA-treated groups. Exam-
ples of cells showing this type of responses are shown in Fig. 3A
and B. Among the cells that showed increasing responses, re-
sponses to the initial application of capsaicin were similar between
the vehicle- (0.06 ± 0.02) and NMDA-treated groups (0.06 ± 0.01).
However, the NMDA-treated group showed a significantly greater
enhancement of the response after the second application of capsa-
icin compared with the vehicle-treated group (vehicle: 0.15 ± 0.02,
NMDA: 0.27 ± 0.03, P < .001, Fig. 3C).
The cells with higher initial Ca2+responses tended to show
decreasing responses in both vehicle- and NMDA-treated groups.
Among the cells that showed decreasing responses, responses to
both the initial and second applications of capsaicin were not sig-
nificantly different between the vehicle- and NMDA-treated
groups (vehicle n = 38: cap1 0.27 ± 0.06, cap2 0.06 ± 0.02; NMDA
n = 71: cap1 0.24 ± 0.04, cap2 0.09 ± 0.02, P = .993).
To confirm that NMDA-induced Ca2+responses are receptor
mediated, we applied NMDA in the presence of a specific NMDAR
antagonist, AP5 or its vehicle control. The percentage of cells
Fig. 2. NR1 and TRPV1 expression and coimmunoprecipitation in trigeminal ganglia (TG). (A) Immunohistochemical staining of TG sections showing Fast blue–labeled muscle
afferents, TRPV1- and NMDA-labeled neurons as indicated. The arrows indicate muscle afferents that coexpress both NR1 and TRPV1. Scale bar: 50 lm. (B) Left: Immunoblot
(IB) using anti-TRPV1 or anti-NR1 antibody after immunoprecipitation (IP) of TG extract with anti-NR1 antibody. Right: Reverse IP with anti-TRPV1 antibody and IB with anti-
NR1 or anti-TRPV1 antibody. (C)The same IB-IP protocols were used to show coimmunoprecipitation of TRPV1 and NR2B subunit.
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responding to NMDA under vehicle and AP5 conditions were
20.78% of 255 cells, and 2.9% of 343 cells, respectively (v2= 39.4,
P < .001), confirming that the NMDA-induced responses can be
effectively blocked when NMDA receptors are pharmacologically
inhibited. The AP5 application alone rarely produced responses.
We conducted additional experiments with NMDA application
in the presence of a specific TRPV1 antagonist, AMG9810 (1 lmol)
or its vehicle control to provide additional evidence for the speci-
ficity of AMG9810. The percentage of cells responding to NMDA
under vehicle and AMG9810 conditions were 13.5% and 9.25% of
281 cells, respectively (v2= 2.0, P > .05). These data confirmed that
AMG9810 does not directly modulate the NMDA-induced re-
sponses. The percentage of cells responding to capsaicin under
vehicle and AMG9810 conditions were 65.5% of 183 cells and
36% of 259 cells, respectively (v2= 12.5, P < .001). Thus, the
concentration of AMG9810 that failed to block NMDA responses
effectively antagonized TRPV1 receptors. The AMG9810 applica-
tion alone rarely produced responses.
These data reinforced the notion of functional interactions
between NMDARs and TRPV1 in a subpopulation of TG neurons,
3.4. NMDA phosphorylates TRPV1 at specific residues in TG
To examine whether the activation of NMDARs leads to the
phosphorylation of TRPV1, we examined the changes in TRPV1
phosphorylation in sensory neurons after the application of NMDA.
It is not feasible to perform such analysis at the primary afferent
terminal level because the neural elements in the muscle tissue
are too low to detect meaningful biochemical changes. Instead,
we assessed the relative level of phosphorylated TRPV1 in cultured
TG neurons 15, 30, and 45 minutes after the application of NMDA
(200 lM),a timecourse comparable
The NMDA application caused a time-dependent elevation in
serine phosphorylation (p-Ser) of TRPV1 (Fig. 4A). A significant in-
crease in p-Ser could be observed at 15 minutes and 30 minutes,
the time points at which mechanical hyperalgesia was most prom-
inent after NMDA injection in the in vivo condition. The total
TRPV1 expression level did not change during this time course.
The same concentration of NMDA did not produce a significant
alteration in phosphorylation of TRPV1 at either threonine or tyro-
sine residues (Fig. 4B and C). These data provide support for
NMDAR-TRPV1 interactions and that NMDAR activation leads to
TRPV1 phosphorylation at specific sites.
3.5. CaMKII and PKC mediate NMDAR-TRPV1 interactions in TG
Serine phosphorylation of TRPV1 can be mediated by various
[5,16,30,51,53,65]. Because NMDAR activation has been shown to
invoke a CaMKII-mediated signaling pathway , we first exam-
ined whether the NMDA-mediated phosphorylation of serine resi-
dues in TRPV1 involves the CaMKII pathway. The significant
increase in p-Ser of TRPV1 15 minutes after NMDA application
was blocked when TG cultures were pretreated with a specific
CaMKII inhibitor, KN93 (Fig. 5A). The same concentration of
KN92, an inactive analog of KN93, failed to block the NMDA-in-
duced increase in p-Ser of TRPV1 (Fig. 5B). Consistent with the bio-
chemical data, the NMDA-induced mechanical hyperalgesia was
dose-dependently blocked when KN93, but not vehicle, was pread-
ministered in the same muscle (Fig. 5C). There was a significant
group effect (F = 12.06, P < .01) and a significant time effect
(F = 41.1, P < .001). The injection of KN93 alone did not alter the
mechanical sensitivity in the muscle (data not shown). Collectively,
these data provide evidence that activation of NMDARs invokes a
CaMKII signaling cascade that results in serine phosphorylation
of TRPV1 in TG.
We performed similar experiments to examine whether PKC is
also involved in NMDAR-TRPV1 interactions. The significant in-
crease in p-Ser of TRPV1 15 minutes after NMDA application was
blocked when TG cultures were pretreated with a PKC inhibitor,
GF109203X (Fig. 6A). The same concentration of vehicle failed to
block the NMDA-induced increase in p-Ser of TRPV1 (Fig. 6B).
Again, consistent with the biochemical data, the NMDA-induced
mechanical hyperalgesia was dose-dependently blocked when
GF109203X, but not vehicle, was preadministered in the same
muscle (Fig. 6C). There was a significant group effect (F = 23.02,
P < .001) and a significant time effect (F = 41.39, P < .001). The
injection of GF109203X compound alone did not alter the mechan-
ical sensitivity (data not shown). These data provide evidence that,
in addition to the CaMKII signaling pathway, activation of NMDARs
also invokes a PKC signaling cascade that results in serine phos-
phorylation of TRPV1 in TG.
Finally, we examined whether NMDAR-TRPV1 interaction also
involves a PKA signaling pathway. The NMDA-induced increase
in p-Ser of TRPV1 was not significantly blocked when the TG was
pretreated with a specific PKA inhibitor, KT5720 (Fig. 7A). The dose
of KT5720 was chosen based on the literature . The NMDA-in-
duced mechanical hyperalgesia was partially but significantly re-
duced when the muscle was pretreated with KT5720 (Fig. 7B).
The same dose of KT5720 almost completely blocked the
CaMKII, PKC,and PKA
Fig. 3. The effects of NMDA on capsaicin-induced responses in trigeminal ganglia neurons. Representative traces show Fura ratio from trigeminal ganglia neurons in NMDA-
treated (A) and vehicle-treated (B) groups. (C) Averaged changes in Fura response by first and second application of 10 nM capsaicin in NMDA or vehicle-treated groups.
⁄P < .05 in 2-way analysis of variance.
J. Lee et al./PAIN
?153 (2012) 1514–1524
mechanical hyperalgesia induced by a direct injection of forskolin,
a PKA activator (Fig. 7C). These data suggest that NMDAR-induced
mechanical hyperalgesia may involve PKA pathway, but the activa-
tion of that pathway does not result in TRPV1 phosphorylation in
The current study demonstrated that the NMDAR and TRPV1
functionally interact in rat trigeminal sensory neurons and that
such interactions are essential for the development of mechanical
hyperalgesia in the masseter muscle. Several lines of evidence sup-
port this conclusion. First, the NR1 subunit of the NMDAR and
TRPV1 are coexpressed in a subset of trigeminal afferents that
innervate the masseter muscle, and form protein-protein com-
plexes in TG neurons. Second, NMDAR activation enhances capsa-
icin-induced responses in a subset of TG neurons. Third, NMDAR
activation leads to phosphorylation of specific residues in TRPV1
in TG via intracellular kinases that have been implicated in sensi-
tization. Finally, NMDA-induced mechanical hyperalgesia in the
masseter muscle requires TRPV1, and the inhibition of kinases that
mediate TRPV1 phosphorylation in TG attenuates NMDA-induced
mechanical hyperalgesia. Especially noteworthy is that the
NMDA-induced phosphorylation of TRPV1, which we demon-
strated in native sensory neurons, correlated with our behavioral
responses, and that the pharmacological blockade of kinases pro-
duced corroborating biochemical and behavioral responses. These
results strongly suggest that NMDARs and TRPV1 are functionally
linked in peripheral terminals of nociceptors in the muscle tissue.
These data offer novel mechanisms by which 2 distinct ligand-
gated channels in nociceptors interact and form the underlying cel-
lular basis for the development of mechanical hyperalgesia.
The significance of our data is that these 2 important ligand-
gated ion channels, which have been independently implicated in
muscle pain and hyperalgesia, interact and may operate as func-
tional units, which bearsimportant scientificand clinical
Fig. 4. The effects of NMDA treatment on the phosphorylation of TRPV1 in cultured trigeminal ganglia neurons. (A-C) Immunoblots using anti-p-Ser (A), p-Thr (B), or p-Tyr (C)
antibody after immunoprecipitation using anti-TRPV1 antibody (top). The samples were collected at the indicated time points after the treatment with vehicle or NMDA
(200 lM). Immunoblots using anti-TRPV1 antibody in the same gel in the upper panel (bottom). Averaged relative p-Ser/TRPV1, p-Thr/TRPV1, or p-Tyr/TRPV1 are also shown.
(⁄P < .05 in 1-way analysis of variance; n = 6 to 8 for each time point).
Fig. 5. The involvement of CaMKII in NMDA-induced serine phosphorylation of TRPV1 and mechanical hyperalgesia. (A) Examples of immunoblots and averaged relative p-
Ser/TRPV1 after the NMDA (200 lM) treatment with and without KN93 (10 lM). (B) Examples of immunoblots and averaged relative p-Ser/TRPV1 after the NMDA treatment
with and without KN92 (10 lM), an inactive analog of KN93. The samples were collected at the 15-minute time point, during which the NMDA-induced serine
phosphorylation of TRPV1 was most prominent (⁄P < .05 in 1-way analysis of variance; n = 6 to 8 per group in A and B). (C) The effects of KN93 pretreatment on NMDA-
induced mechanical hyperalgesia (⁄P < .05 in group effects with respect to vehicle condition in 2-way analysis of variance;#P < .05 to the baseline in post-hoc test; n = 6 to 8
J. Lee et al./PAIN
?153 (2012) 1514–1524
implications. Our data provide molecular mechanisms of possible
interactions between glutamate receptors and capsaicin receptors
that have been suggested in animal as well as human experimental
muscle pain models. For example, injecting glutamate into cranio-
facial deep tissues significantly enhances capsaicin-induced noci-
ceptor activity and lowers mechanical thresholds . Similarly,
capsaicin injections followed by glutamate in human tendon tissue
significantly facilitate pain responses and decrease pressure pain
An important implication of these data is that the NMDAR is
activated upstream to TRPV1 activation under injury or inflamma-
tory conditions. We conducted additional behavioral experiments
to test the possibility that TRPV1 is activated upstream to NMDAR.
The mechanical hyperalgesia induced by the direct injection of
capsaicin in the masseter was significantly inhibited when the
muscle was pretreated with AP5 (data not shown), suggesting that
the functional interactions are bidirectional. The directionality of
interactions between TRPV1 and NMDARs could be governed by
various factors such as the availability of endogenous ligands, the
expression levels of TRPV1 and NMDARs, and the requisite intra-
cellular machineries. It is likely that the intracellular mechanisms
underlying the interactions in each direction are different from
Therefore, although it is also plausible that the NMDAR func-
tions as a downstream target of TRPV1 activation, we thought
the present data may more realistically reflect pathological condi-
tions for the following reasons. First, it is well established that ex-
cess glutamate is released in peripheral tissue, including muscle
tissue, upon injury or inflammation from numerous sources
[33,34,42,46,48,49], which supports our data as physiologically
tenable. Similar physiologically relevant endogenous agonists for
TRPV1 in muscle tissue after injury may also be released, but they
are yet to be fully characterized. Second, there is increasing evi-
dence that TRPV1 functions as a downstream inflammatory signal
Fig. 6. The involvement of protein kinase C in NMDA-induced serine phosphorylation of TRPV1 and mechanical hyperalgesia. (A) Examples of immunoblots and averaged
relative p-Ser/TRPV1 after the NMDA (200 lM) treatment with and without GF109203X (10 lM). (B) Examples of immunoblots and averaged relative p-Ser/TRPV1 after the
NMDA treatment with and without dimethyl sulfoxide (0.1%), the vehicle control for GF109203X. The samples were collected at the 15-minute time point (⁄P < .05 in 1-way
analysis of variance; n = 6 to 8 per group in A and B). (C) The effects of GF109203X pretreatment on NMDA-induced mechanical hyperalgesia (⁄P < .05 in group effects with
respect to vehicle condition in 2-way analysis of variance;#P < .05 to the baseline in post-hoc test; n = 6 to 8 per group).
Fig. 7. Protein kinase A is not involved in NMDA-induced TRPV1 phosphorylation. (A) KT5720, a protein kinase A inhibitor, or vehicle control failed to prevent the NMDA-
induced elevation of p-Ser of TRPV1 (⁄P < .05 in 1-way analysis of variance; n = 6 to 8 per group in A and B). (B, C) NMDA-induced mechanical hyperalgesia was only partially
attenuated at the dose that completely blocked the forskolin induced mechanical hyperalgesia (⁄P < .05 in group effects with respect to vehicle condition in 2-way analysis of
variance;#P < .05 to the baseline in post-hoc test; n = 6 to 8 per group).
J. Lee et al./PAIN
?153 (2012) 1514–1524
integrator after the activation of G-protein coupled receptors
(GPCRs) . Our data add to these observations in that signaling
cascades invoked by activation of ligand-gated ion channels also
converge onto TRPV1.
Functional interactions between glutamate receptors and
TRPV1 have been suggested at the level of the spinal cord. In-
creased glutamate release due to enhanced presynaptic Ca2+sig-
naling afterTRPV1 activation
nociceptors could result in prolonged activation of NMDARs in
postsynaptic dorsal horn neurons [43,60]. The mechanisms de-
scribed in the previously mentioned studies, however, are intercel-
lular rather than intracellular mechanisms. A recent study
demonstrated that DAG produced upon mGluR5 activation directly
activates TRPV1 in the same neuron in a membrane-delimited
manner, a mechanism that has been proposed to contribute to
the modulation of synaptic transmission in the substantia gelatin-
osa neurons of the spinal cord . In this study, we demonstrated
that NR1 subunits and TRPV1 coexpress in a subset of trigeminal
sensory neurons and that the 2 receptors form protein-protein
complexes. At present, we do not know the precise nature of their
association because co-IP data alone do not establish direct physi-
cal interactions. It is also not known whether the functional inter-
actions between the 2 receptor systems require such physical
interactions. Our data, however, provided novel information that
NMDAR and TRPV1 reside in close proximity within the same cell,
and suggest mechanisms relevant for functional interactions
resulting from intracellular changes initiated by NMDAR in a micro
Phosphorylation of TRPV1 has been considered as a major
mechanism that accounts for TRPV1 sensitization, and various sec-
ond messenger pathways have been associated with TRPV1 phos-
phorylation [4,5,27,28,30,40,67]. It is well known that different
kinases phosphorylate different residues of TRPV1. For example,
PKC phosphorylates TRPV1 at Ser-502, Ser-800, and Thr-704,
whereas PKA activation results in the phosphorylation of Ser-
502, Ser-116, Thr-144, and Thr-370 residues [5,40,44,53]. CaMKII
activation also leads to phosphorylation of TRPV1 at Ser-502,
Thr-370, and Thr-704 . It is also well known that activation
of GPCRs, such as neurokinin, bradykinin, prostaglandin, and TrkA
receptors, sensitize TRPV1 via pathways involving PKC, PKA, and
CaMKII [27,35,59,62,64,70]. However, it is unclear whether the
activation of these receptors phosphorylates a specific residue in
TRPV1 by invoking a specific kinase pathway. The present study re-
vealed that activation of NMDARs results in phosphorylation of
TRPV1, primarily at serine residues through the activation of PKC
and CaMKII, but not PKA, pathways.
Activation of NMDARs increases the influx of Ca2+, which in-
vokes multiple intracellular signaling cascades, including activa-
tion of CaMKII [2,62]. The NMDAR-CaMKII cascade functionally
coupled to acid-sensing ion channels (ASICs) has been shown to
contribute to acidotoxicity during ischemia . Specifically, CaM-
KII-induced phosphorylation of a specific serine residue in ASICs
plays an essential role in ischemia-induced cell death in the pres-
ence of excess glutamate, an example of CaMKII-mediated mecha-
nisms of channel-channel interactions. CaMKII is expressed in both
peptidergic and nonpeptidergic TRPV1-positive DRG neurons .
Blockade of CaMKII effectively reduces capsaicin-induced CGRP
release in TG neurons . Our behavioral data corroborated the
biochemical data by demonstrating that NMDA-induced masseter
hypersensitivity is attenuated by a CaMKII inhibitor. Together,
these data allow us to postulate the NMDAR-CaMKII-TRPV1 cas-
cade as an underlying factor for the development of masseter
Induction of the PKC signaling pathway after NMDAR activation
has been widely demonstrated in the central nervous system
[22,61,66]. In DRG neurons, activation of NMDARs enhances the
at thecentral terminalsof
activity of voltage-dependent Ca2+channels through PKC [11,39].
Our data provided additional evidence that NMDAR activation in
TG sensory neurons invokes PKC, which targets TRPV1 at serine
residues. We have previously shown that the activation of another
glutamate receptor, mGluR5, in the masseter muscle results in
mechanical hyperalgesia via PKC . Thus, it seems that both
the NMDAR and mGluR5 activated by excess glutamate released
under pathological conditions can recruit PKC, which then modu-
lates the activity of other pronociceptive molecules such as TRPV1.
Hu et al.  showed mGluR5 activation in DRG neurons in-
creases TRPV1 function in a PKA- but not PKC-dependent manner.
However, there are no data on whether the activation of NMDARs
results in PKA activation in sensory neurons. The NMDA-induced
mechanical hyperalgesia was partially attenuated by a PKA inhib-
itor at the dose that almost completely abolished forskolin-in-
duced hyperalgesia. These data suggest that NMDAR activation
may also recruit the PKA pathway. Although TRPV1 is a substrate
for PKA, our biochemical data showed that NMDAR-mediated in-
crease in TRPV1 phosphorylation does not involve PKA. Therefore,
the NMDAR-PKA pathway may converge on other channels such as
TRPA1 (unpublished observations). Collectively, our data suggest
that the NMDAR engages TRPV1 in specific ways, primarily by
CaMKII and PKC rather than PKA.
This work establishes that the 2 prominent channels in nocicep-
tive circuitry cooperate as functional units. Our data also open up
the possibility that other nonspecific cationic channels such as
P2X and ASICs might also be communicating with TRPV1 via spe-
cific or common intracellular signaling pathways. Given that sig-
nals arising from GPCRs converge onto TRPV1 and TRPA1 ,
our data further reinforce the notion that TRP channels are at the
core of forming functional units in nociceptive signaling under
pathological conditions. In a broader context, it would be interest-
ing to explore whether similar interactions also take place in the
central nervous system. Recently, TRPV1 is shown to be present
in hippocampus,dentate gyrus,
[12,21,23], brain areas enriched in glutamate receptors.
and nucleus accumbens
Conflict of interest statement
There is no conflict of interest to declare.
The authors thank Youping Zhang and Gregory Haynes for tech-
nical assistance. This study was supported by National Institutes of
Health grant RO1 DE16062 (J.Y.R.).
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