Mas-related G-protein–coupled receptors inhibit
pathological pain in mice
Yun Guana,1, Qin Liub,1, Zongxiang Tangb, Srinivasa N. Rajaa, David J. Andersonc,d,2, and Xinzhong Dongb,e,2
aDepartment of Anesthesiology and Critical Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21205;bThe Solomon H. Snyder
Department of Neuroscience, andeHoward Hughes Medical Institute, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; andcDivision of
Biology, anddHoward Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125
Contributed by David J. Anderson, July 29, 2010 (sent for review May 10, 2010)
An important objective of pain research is to identify novel drug
inflammatory and neuropathic pain. Mas-related G-protein–coupled
receptors (Mrgprs) representalarge familyoforphanreceptors specif-
ically expressed in small-diameter nociceptive primary sensory neu-
rons. To determine the roles of Mrgprs in persistent pathological
pain states, we exploited a mouse line in which a chromosomal locus
spanning 12 Mrgpr genes was deleted (KO). Initial studies indicated
that these KO mice show prolonged mechanical- and thermal-pain
hypersensitivity after hind-paw inflammation compared with wild-
type littermates. Here, we show that this mutation also enhances
the windup response of dorsal-horn wide dynamic-range neurons,
an electrophysiological model for the triggering of central pain sensi-
of intrathecally applied bovine adrenal medulla peptide 8–22 (BAM 8–
22), an MrgprC11 agonist, on both inflammatory heat hyperalgesia
and neuropathic mechanical allodynia. Spinal application of bovine
adrenal medulla peptide 8–22 also significantly attenuated windup
in wild-type mice, an effect eliminated in KO mice. These data suggest
that members of the Mrgpr family, in particular MrgprC11, may con-
stitute an endogenous inhibitory mechanism for regulating persistent
pain in mice. Agonists for these receptors may, therefore, represent
a class of antihyperalgesics for treating persistent pain with minimal
side effects because of the highly specific expression of their targets.
dorsal-horn neuronal hyperexcitability (central sensitization) am-
plify ascending pain signals (1, 2), which, if uncontrolled, may lead
to various unremitting pain symptoms (e.g., tactile allodynia, ther-
of potential targets comprises a large family of orphan receptors
known as Mas-related G-protein–coupled receptors (Mrgprs).
Many Mrgprs (e.g., As, B4, B5, C11, and D) are expressed specifi-
cally on small-diameter, presumably nociceptive, nonpeptidergic
sensory neurons in the dorsal root ganglia (DRG) (6, 7). Recent
studies have begun to shed light on the physiological functions
served by Mrgprs, including mediation of nonhistaminergic itch by
MrgprA3 (8–11). Whether other Mrgprs mediate itch or regulate
persistent pathological pain states is not clear (12–14).
Examining the function of Mrgprs in vivo has been challenging,
because endogenous Mrgpr ligands have not been unequivocally
identified and deletion of a single Mrgpr gene may not cause
a detectable phenotype because of potential redundancy in the
Mrgpr gene family (7, 15). To overcome these problems, we gen-
erated a mouse line in which 12 intact Mrgpr coding sequences
(As, B4, B5, and C11) were simultaneously deleted; the resulting
mice are referred to as Mrgpr-clusterΔ−/−(KO) mice (11). The
deleted cluster contains most MrgprA and MrgprC genes and
represents ∼50% of the potentially functional Mrgpr repertoire in
mice (7). Importantly, the deleted Mrgprs are not required for
neuronal survival or fate determination of small-diameter sensory
neurons (11). Therefore, the KO mice may represent a useful tool
for studying the functions and determining the roles of Mrgprs in
pain in vivo. The KO mice respond normally to acute noxious
fter tissue inflammation or nerve injury, increased afferent
neuronal excitability (peripheral sensitization) and a state of
thermal, mechanical, and chemical stimuli compared with wild-
type (WT) littermates. However, they display prolonged mechan-
ical- and thermal-pain hypersensitivity after intraplantar injection
of complete Freund’s Adjuvant (CFA) or carrageenen, whereas
the development of neuropathic pain was similar between the two
genotypes (11). Findings from our previous work motivated us to
further examine the roles for Mrgprs in signaling and modulation
of persistent pain states with different origins (i.e., inflammation
and nerve injury) and investigate the underlying neurophysiologi-
cal mechanisms in vivo. We also tested the effect of intrathecal
administration of an MrgprC11 agonist, bovine adrenal medulla
peptide 8–22 (BAM 8–22), on mouse pain behavior. Our results
suggest that certain Mrgprs in mice may constitute endogenous
inhibitors of pathological pain. These data also suggest that ago-
nists, rather than antagonists, for MrgprC11 may represent a class
of antihyperalgesics for persistent pain.
Intense/Repetitive Noxious Input Activates an Endogenous Mrgpr
Mechanism to Counteract the Sensitization of Pain Responses. First,
we examined whether formalin-induced tissue injury leads to an
endogenous activation of Mrgprs to modulate spontaneous pain.
The formalin test is a unique model of persistent pain that
encompasses inflammatory, neurogenic, and central mechanisms
of nociception (16–18). Importantly, spontaneous pain responses
to two principally different stimuli, nociceptor activation (first
phase) and tissue inflammation (second phase), can be readily
revealed in the same test and separately analyzed. Formalin (2%;
5 μL) was injected into the plantar tissue of one hind paw. We
observed that spontaneous pain behavior in the second phase
(10–60 min post injection), which is driven largely by tissue in-
flammation and involves central sensitization of dorsal-horn
neurons (16), was significantly potentiated in KO mice compared
with WT littermates (Fig. 1A). Reflecting this behavioral phe-
notype, a greater increase of c-fos–expressing neurons in the
ipsilateral laminae I and II of lumbar (L4–L6) spinal segments in
KO mice than in WT mice was evident after an intraplantar
injection of formalin (Fig. 1B). In contrast, the first (acute) phase
of the formalin response (0–10 min post injection), which results
predominantly from a direct chemical stimulation of the C fiber-
afferent nociceptors, was not affected by the mutation (Fig. 1A).
Next, we examined whether the enhanced inflammatory-pain
response in KO mice is also manifested at the cellular level of
spinal pain processing. Wide dynamic-range (WDR) neurons in
the deep dorsal horn are important for spinal pain processing and
are candidates for transmission cells in the gate theory of pain (2,
19, 20). They receive both innocuous and noxious sensory inputs
from the periphery and display A fiber- and C fiber-mediated
responses (A and C components, respectively) to a single in-
tracutaneous electrical stimulus with an intensity above C fiber-
Author contributions: Y.G., Q.L., S.N.R., D.J.A., and X.D. designed research; Y.G., Q.L., and
Z.T. performed research; Y.G., Q.L., Z.T., S.N.R., D.J.A., and X.D. analyzed data; and Y.G.,
Q.L., S.N.R., D.J.A., and X.D. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1Y.G. and Q.L. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com or xdong2@
| September 7, 2010
| vol. 107
| no. 36
activation threshold (Fig. 2A). Based on the axon-conduction
velocities, WDR neuronal responses to electrical stimuli in mice
were separated into a short latency A component (0–40 ms, ex-
cluding stimulus artifact) and a long latency C component (40–
250 ms) (20). Typically, the excitability of some WDR neurons
progressively increases in response to repetitive C fiber-afferent
stimulation, a short-term activity-dependent neuronal sensitiza-
tion called windup (21). Therefore, we recorded from WDR
neurons and examined whether the deletion of the Mrgpr cluster
would alter the excitability of WDR neurons. The effective
frequency of the electrical stimulation for inducing windup is
usually >0.3 Hz under physiological conditions, and a plateau
level of windup is often reached at a higher-frequency stimulation
of ∼1 Hz (20, 21). Accordingly, windup was examined by re-
petitive intracutaneous electrical stimuli (16 pulses, 3.0 mA/supra
C fiber-activation threshold, 2.0 ms) applied at 0.2 and 1.0 Hz,
with a minimum 10-min interval between each trial. We observed
that WDR neurons in WT mice (n = 23) showed windup
responses at the higher frequency of 1.0-Hz stimulation but rarely
at 0.2-Hz stimulation (Fig. 2 B and D), consistent with previous
studies (20, 21). To quantify the peak levels of windup, we mea-
sured the relative windup value, which is the averaged C com-
ponent responses to the last 10 (7–16) stimuli of the trial
normalized by the C component response to the first stimulus of
each trial (input value). The relative windup values were signifi-
cantly increased during 1.0- but not 0.2-Hz stimulation in WT
mice compared with the respective baseline (Fig. 2E). Strikingly,
unlikethecasein WTmice,manyWDRneuronsin KOmice(n=
30) exhibited windup at the normally ineffective 0.2-Hz stimula-
tion frequency (Fig. 2 B and D), and the relative windup value at
0.2-Hz stimulation in the KO group was significantly greater than
the baseline (Fig. 2E). Importantly, the windup responses at both
0.2- and 1.0-Hz stimulation frequencies were significantly greater
in KO than in WT mice (P < 0.05) (Fig. 2E). The mean recording
depth of WDR neurons did not differ between WT (450 ± 23 μm)
and KO mice (441 ± 22 μm, P > 0.05). In contrast with their
differences in windup, the acute responses of WDR neurons to
graded intracutaneous electrical stimulation (0.05–5.0 mA, 2.0
ms) were comparable between the WT and KO mice. The
threshold and population stimulus-intensity response (S-R)
functions of the C component were also similar in the two groups
(Fig. 2C and Table 1). These data suggest that one or more
Mrgprs within the KO cluster function to limit the extent of in-
creased WDR neuronal excitability in response to repetitive C
andNeuropathicPaininWTbutNotKOMice.One gene within the KO
cluster that can potentially modulate pain responses is MrgprC11.
An endogenous agonist of MrgprC11 is a 22-amino acid peptide
calledBAM 22(6,7);itbelongs tothefamilyofendogenousopioid
peptides and is derived from the proenkephalin A gene. In-
terestingly, the N terminus of BAM 22 binds and activates opioid
receptors, whereas the C terminus of the peptide activates mouse
MrgprC11, rat MrgprC, and human MrgprX1. Previous studies
have shown that a truncated BAM 22, which lacks its N terminus
(BAM 8–22), specifically activates Mrgprs but not opioid receptors
cells (9, 11, 14). In rats, several rodent MrgprC agonists, including
BAM 8–22 and γ2-melanocyte-stimulating hormone (γ2-MSH),
have been reported to have both pro- and antipain effects (12, 13,
22–25). Because these previous studies did not use transgenic
hanced pain responses after intraplantar formalin injection. (A) KO mice
expressed enhanced spontaneous pain responses in the second phase of for-
malin-induced pain but responded normally in the first (acute) phase.
(B) Transverse sections of L4–L5 spinal cord from WT and KO mice were stained
with anti–c-fos antibody 3 h after intraplantar formalin injection (2%, 5 μL).
Ten sections were chosen randomly from each mouse (three mice per geno-
type). c-fos–positive nuclei are indicated by arrows. KO mice had significantly
more c-fos–positive cells than did WT mice, indicating that the KO mice had
greater neuronal activation. Data are expressed as mean ± SEM.
KO mice display stronger dorsal-horn neuronal activation and en-
input. (A) A WDR neuron displayed typical A component (0–40 ms) and
C component (40–250 ms) responses in response to suprathreshold in-
tracutaneous electrical stimulation. This unit from a KO mouse showed pro-
gressive increases in C component (windup) in response to repetitive electrical
stimulation of0.2 Hz(16 pulses, 3.0 mA, 0.5 ms).(B) Histogramsshowresponses
of WDR neurons from KO and WT mice to 0.2-Hz stimulation. The KO neuron
displayed windup, but the WT neuron did not. Bin size is 50 ms. APs, action
30).(D) C componentsofthe responses torepetitivewindup-inducing electrical
the stimulation number of each trial. (E) The averaged C components of the
responses to the last 10 stimuli (7–16) of 0.2- and 1.0-Hz stimulation were sig-
nificantly higher in KO than WT mice. Windup data are normalized to the re-
± SEM. **P < 0.01 vs. the input value;#P < 0.05 vs. WT group.
| www.pnas.org/cgi/doi/10.1073/pnas.1011221107 Guan et al.
animals that lack Mrgprs and the specificity of these agonists for
Mrgprs has not been fully established, it has been difficult to de-
termine whether the effects of these peptides in vivo are mediated
Because the spinal cord is an important site for pain modu-
lation and Mrgprs are likely expressed on the central as well as
the peripheral terminals of DRG neurons (12, 13), we examined
the effects of intrathecal BAM 8–22 (1 mM, 5 μL) on pain be-
havior in WT and KO mice. This peptide on its own elicited mild,
short-lived (less than 5 min) scratching and tail-biting behavior in
some WT mice, but this response was similar in KO mice, sug-
gesting that it is not mediated by Mrgprs (or at least not by those
contained within the deleted cluster). We next examined the
ability of BAM 8–22 to modulate persistent inflammatory pain
by examining enhanced pain sensitivity to a noxious heat stim-
ulus as monitored by the Hargreaves test 24 h after intraplantar
injection of CFA (6 μL, 50%) into one hind paw. In the absence
of BAM 8–22, thermal hyperalgesia was comparable between the
two genotypes: the paw-withdrawal latencies (PWL) of the ipsi-
lateral hind paw in KO and WT mice were 4.3 ± 0.7 s and 3.3 ±
0.3 s, respectively. In WT mice, a single intrathecal injection of
at 30 min postinjection, increasing the PWL by 1.9-fold compared
with predrug baseline (Fig. 3A, WT, n = 13, ipsilateral paw). In
contrast, this antihyperalgesic effect of BAM 8–22 was not ob-
served in KO mice (Fig. 3A, KO, n = 10), indicating that it is
Mrgpr-dependent. In addition, intrathecal BAM 8–22 did not
paw to acute radiant heat in either group (Fig. 3A, contralateral
paw). These data suggest that Mrgprs (most likely, Mrgpr C11)
play a role in mediating the effect of exogenously administered
BAM 8–22 to attenuate inflammatory thermal hyperalgesia in
To determine whether intrathecal BAM 8–22 also affects acute
thermal nociception, we performed tail-immersion tests in naïve
WTandKOmice.The tail-flicklatenciesafter intrathecalinjection
of BAM 8–22 were not significantly different from the respective
predrug values in either WT (n = 10) or KO mice (n = 10) (Fig.
3B). This finding is in line with the observation that the peptide did
not significantly change the sensitivities to acute radiant heat
(Hargreaves test) in WT (n = 15) and KO (n = 14) (Fig. 3B) mice.
In addition, thermal sensitivities were comparable between the two
groups before and after BAM 8–22 treatment (Fig. 3B).
There are substantial differences between neuropathic-pain and
inflammatory-pain states (e.g., etiology, pathology, and treatment
strategy) (4, 26–28). Because mechanical allodynia is the most
common and disabling stimulus-evoked symptom of neuralgia and
is often difficult to treat, we determined whether BAM 8–22 can
also reduce neuropathic mechanical allodynia in mice. We sub-
the sciatic nerve is ligated loosely with a suture. The effect of this
manipulation on mechanical-pain sensitivity was tested at 14–18
d afterinjury by measuringpaw-withdrawal frequency topunctuate
mechanical stimuli of different strengths. Intrathecal injection of
BAM 8–22 (0.5 mM, 5 μL) significantly attenuated CCI-induced
mechanical-pain hypersensitivity to both low-force (0.07 g) and
mice (Fig. 3C) (n = 7). This antihyperalgesic effect of the peptide
was eliminated in KO mice (Fig. 3C) (n = 8), although the de-
velopment of mechanical allodynia itself was not affected by the
mutation. BAM 8–22 did not significantly change paw-withdrawal
responses on the uninjured side (Fig. 3D, contralateral paw). This
result suggests that the antiallodynic effect of intrathecal BAM 8–
22 under neuropathic conditions is also mediated by Mrgprs.
Spinal Application of BAM 8–22 Attenuates Windup in WT but Not KO
Mice. Next, we asked whether the antihyperalgesic effect of BAM
8–22 can be seen at the level of central nociceptive processing.
Because intrathecal BAM 8–22 inhibited both inflammatory and
neuropathic pain, we postulated that MrgprC11 agonists might
attenuate spinal neuronal sensitization involved in persistent
pain. We examined the effects of topical spinal application of
BAM 8–22 (0.1 mM, 30 μL) on the windup of WDR neuronal
responses to repetitive noxious inputs. We used 0.5-Hz stimula-
tion frequency, because it can induce windup but does not satu-
rate the response (21). Therefore, it can be used to examine the
effect of either facilitatory or inhibitory drug action on windup. In
WT mice (n = 25), windup to 0.5-Hz stimulation was significantly
attenuated after spinal superfusion with BAM 8–22 (Fig. 4 A–C,
WT), consistent with the antihyperalgesic effect of BAM 8–22 in
our behavioral studies. In KO mice, by contrast, the effect of
BAM 8–22 was not simply eliminated but rather, reversed: the
mediated action potentials in WDR neurons
The thresholds and latency of the first A and C fiber-
14.0 ± 0.8
14.8 ± 0.6
14.2 ± 0.8
12.1 ± 0.7
14.5 ± 0.6
12.9 ± 0.6
124 ± 5
108 ± 6
114 ± 5
115 ± 4
126 ± 3
118 ± 4
0.23 ± 0.04
0.26 ± 0.03
0.16 ± 0.02
0.17 ± 0.03
0.17 ± 0.02
0.19 ± 0.03
2.0 ± 0.3
2.3 ± 0.2
1.5 ± 0.3
1.7 ± 0.4
1.7 ± 0.5
1.9 ± 0.6
pain and neuropathic pain in WT but not KO mice. (A) Intrathecal (i.th.) in-
jection ofBAM 8–22 (1mM, 5μL) significantly alleviated thermal hyperalgesia
in the ipsilateral hind paw 24 h after intraplantar injection of CFA (6 μL, 50%)
in WT (n = 12) but not KO mice (n = 10). BAM 8–22 did not affect PWL of the
contralateral hind paw in either group. (B) The same dose of BAM 8–22 did
not significantly change the tail-flick latency in the tail-immersion test (50 °C)
in WT (n = 10) or KO mice (n = 10). In addition, the tail-flick latencies were not
significantly different between the two groups at pre- and postdrug con-
ditions. PWL of the contralateral hind paw to radiant heat (Hargreaves test)
in the CFA experiment was similar before and after intrathecal BAM 8–22
injection in both groups. (C) BAM 8–22 (0.5 mM, 5 μL, i.th.) also attenuated
mechanical-pain hypersensitivity induced by CCI of the sciatic nerve in WT
mice but not KO mice. The PWF of the ipsilateral hind paw to low-force (0.07
g) and high-force (0.45 g) punctuate stimulation was significantly increased
from the preinjury levels in both KO and WT mice 14–18 d postinjury. BAM
8–22 significantly reduced the PWF of the ipsilateral hind paw in response to
low- and high-force stimuli in WT mice (n = 7) but not KO mice (n = 8) after
30 min. (D) BAM 8–22 did not significantly reduce the PWF of the contralat-
eral hind paw in either group. Data are expressed as mean ± SEM. *P < 0.05
and **P < 0.01 vs. preinjury value;##P < 0.01 vs. predrug value.
Intrathecal injection of BAM 8–22 inhibits persistent inflammatory
Guan et al.PNAS
| September 7, 2010
| vol. 107
| no. 36
peptide significantly increased the input value and C component
responses of WDR neurons to 0.5-Hz stimulation (Fig. 4A, cf
WT vs. KO, black squares). Population S-R functions of C fiber-
mediated responses of WDR neurons to graded electrical stim-
ulation were similar before and after drug administration in WT
mice (n = 25) (Fig. 4D). However, in KO mice, BAM 8–22 sig-
nificantly increased the number of C component responses to
graded electrical stimulation at intensities >1.0 mA (n = 17) (Fig.
4D). Nevertheless, the threshold and latency of the first C fiber-
mediated action potential was unaffected by administration ofthe
peptide in either WT or KO mice (Table 1). These results suggest
that the inhibitory effects of BAM 8–22 on windup, a measure of
short-term neuronal hyperexcitability, are mediated by Mrgprs.
Furthermore, deletion of Mrgprs unmasks a potentiating effect of
the peptide on the same response mediated by another class
Receptors Within a Cluster of Mrgprs May Mediate an Endogenous
Inhibitory Mechanism for Persistent Pain and Hyperalgesia. Our
previous behavioral studies indicated that deletion of the Mrgpr
gene cluster exaggerates inflammatory-pain responses while leav-
question of the underlying neurobiological mechanism(s). Intense
nociceptor activation through C fiber-evoked responses in dorsal-
horn neurons results in a state of central sensitization manifested
as an increased neuronal response to subsequent stimuli (2, 29).
The windup phenomenon in WDR neurons reflects an activity-
Like many biological functions, the windup response to changes in
stimulation frequency mayberepresentedby a sigmoidalfunction.
We postulate that a facilitation of windup by removal of endoge-
nous inhibition is manifested as a reduction in the effective-
frequency threshold for eliciting this response from 1.0- to 0.2-Hz
stimulation. A similar phenotype was observed in mice lacking the
μ-opioid–receptor gene (20). The facilitated windup in KO mice is
consistent with the enhanced inflammatory- and formalin-pain
behavioral phenotype. In contrast to windup, the acute responses
of WDR neurons to graded electrical stimulation (e.g., S-R func-
tion and threshold) were not significantly different between the
two genotypes. These electrophysiological data suggest that the
encoding of acute noxious stimuli by WDR neurons is largely in-
tact in KO mice, consistent with the observation that KO mice
respond to nociceptive mechanical- and thermal-pain in a manner
similar to their WT littermates (Fig. 3 B–D).
Together, our behavioral and electrophysiological experiments
provide complementary lines of evidence that an endogenous
mechanism mediated by a cluster of Mrgprs inhibits persistent
pain behavior and a spinal electrophysiological correlate of
hyperalgesia, presumably by acting on sensory afferent fibers in
the dorsal horn. Apparently, this Mrgpr-mediated pain inhibition
is not tonically active but is triggered only by intense noxious
inputs (e.g., by inflammation or repetitive electrical activation of
C fibers), wherein it functions to attenuate pathological pain se-
verity and counteract dorsal-horn neuronal sensitization.
ligand that mediate this inhibitory influence in WT mice remain to
be determined, although MrgprC11 and BAM 22 are potential
candidates. A previousstudyhas shown that BAM22 expression is
CFA-induced hind-paw inflammation (32). Future studies are
needed to determine whether changes in the expression of Mrgprs
also occur after tissue inflammation and nerve injury.
BAM 8–22 Inhibits Persistent Pain and Spinal Neuronal Sensitization
Through Activation of Mrgprs. In the current study, intrathecal ad-
ministration of BAM 8–22 suppressed thermal hyperalgesia in-
duced by hind paw inflammation in WT mice. This finding is in
accordance with previous observations in rats that intrathecal ad-
ministration of BAM 8–22 suppresses inflammatory pain and spi-
nal c-fos expression induced by intraplantar formalin injection (22,
32, 33) and diminishes NMDA-evoked nocifensive behaviors (12).
Importantly, the antihyperalgesic effect of intrathecal BAM 8–22
was not observed in KO mice, suggesting that the effect is Mrgpr-
dependent. Peripheral-nerve injury-induced mechanical allodynia
also was attenuated by intrathecal BAM 8–22 in WT mice but not
in KO mice. These findings show that spinal administration of
BAM 8–22 attenuates both thermal- and mechanical-pain hyper-
sensitivity in mice under different pathological conditions (tissue
inflammation and nerve injury), presumably through activation of
Mrgprs at central terminals of DRG neurons. Among the 12 de-
leted Mrgprs in Mrgpr-clusterΔ−/−mice, MrgprC11 likely con-
tributes most to the inhibitory effect, because BAM 8–22 is
a specific agonist of MrgprC11 (11, 14). However, additional
experiments that use mice with a single MrgprC11 deletion and
MrgprC11 rescue in cluster KO mice will be needed to directly
determine the role of MrgprC11 in the antihyperalgesic effect.
Because MrgprC11 is an ortholog of human MrgprX1, which can
also be activated by BAM 8–22 (6), BAM 8–22 delivered through
a spinal route may represent a promising treatment for patholog-
ical pain states. Notably, BAM 8–22 is unlikely to compromise
protective physiological pain, because it did not affect baseline
nociception (e.g., tail immersion test and paw withdrawal thresh-
old/latency on the uninjured side).
WDR neuronal response to 0.5-Hz stimulation were plotted as a function of
stimulus number before and after BAM 8–22 administration. (B) The aver-
aged C component responses for the last 10 stimuli during 0.5-Hz stimulation
in WT mice were normalized by the respective response evoked by the first
stimulation of each trial (input value). The relative windup in WT mice was
significantly decreased by BAM 8–22 compared with the predrug level. Be-
cause of a significant increase of input in KO mice after BAM 8–22 treat-
ment, windup data were not normalized. (C) The histograms show an
example of the inhibitory effect of BAM 8–22 on the windup of a WDR
neuron in WT mice at 0.5-Hz stimulation. The windup response was sub-
stantially attenuated 10–30 min after BAM 8–22 application and was par-
tially recovered 10–30 min after saline washout. Bin size is 50 ms. (D) BAM
8–22 (0.1 mM, 30 μL) significantly increased the C component response to
graded electrical stimulation at intensities of 2.0–5.0 mA in KO (n = 17) but
not WT mice (n = 25) at 10–30 min after spinal topical application. Data are
expressed as mean ± SEM. *P < 0.05 and **P < 0.01 vs. the predrug condi-
tion;#P < 0.05 vs. the input value.
BAM 8–22 inhibits windup in WT mice. (A) The C components of
| www.pnas.org/cgi/doi/10.1073/pnas.1011221107 Guan et al.
inhibited windup in WT mice but not in KO mice. These results
suggest that the inhibitory action of BAM 8–22 on windup in WT
receptor antagonists, and adenosine) that alleviate chronic pain
inhibition of windup by BAM 8–22 correlates well with its anti-
hyperalgesiceffect onbehavior. Unlikewindup,thethreshold and
S-R functions for C fiber-mediated responses in WDR neurons to
22 in WT mice, consistent with the fact that BAM 8–22 did not
affect baseline nociceptive-pain sensitivity in WT mice.
In KO mice, BAM 8–22 increased, rather than decreased, the C
fiber-mediated responses of WDR neurons to graded electrical
stimulation at intensities >1 mA, indicating a facilitatory effect of
BAM 8–22 on neuronal response to acute noxious input in the
absence of the Mrgpr cluster. This excitatory action of BAM 8–22
mechanism or by Mrgpr family members that were not part of the
deleted cluster. However, we do not think that BAM 8–22 should
increase acute peripheral-pain sensitivity in KO mice, because the
threshold for activation of the C component in WDR neurons did
likely caused by activation of a receptor(s) expressed in the spinal
cord (e.g., on WDR neurons themselves, interneurons, or
on pain transmission uncovered by our experiments may help to
reconcile some of the previous conflicting reports regarding the
effect of this peptide on pain behavior (13, 39).
Potential Mechanisms for Mrgpr-Mediated Pain Inhibition. Our study
suggests that activation of certain Mrgprs normally suppresses the
WDR windup response and also attenuates behavioral hyper-
algesia. This might imply an inhibitory action of the relevant en-
dogenous ligand on the primary sensory neurons that express
these receptors. However, we previously observed an Mrgpr-
dependent increase in intracellular calcium levels after BAM 8–
22 treatment in DRG cells, which we proposed may underlie the
itch response induced by intradermal injection of BAM 8–22 in
WT mice (11). Although BAM 8–22 may activate different Mrgpr
family members in the periphery versus at central terminals, it is
alsopossible thatthis peptide mightexert differenteffects on pain
and other sensations through the same receptor when applied at
different locations. For example, capsaicin, which excites DRG
neurons and induces intense burning pain in the periphery
through the receptor TrpV1, depressespresynaptic excitation and
inhibits pain when applied centrally (40, 41). This effect could
result from presynaptic terminal depolarization, which would
decrease the amplitude of action potentials and induce pre-
cellular activities differently at the cell body (e.g., pattern of ac-
tion-potential firing) than at central terminals (e.g., neurotrans-
mitter release). Such differences could occur as a result of
disparate distributions and compartmentalization of Mrgprs, in-
tracellular signalingmachinery(e.g.,GqandGi),andreceptors or
channels (e.g., calcium channels) whose activity may be modu-
lated by Mrgpr activation (43, 44). In light of the important roles
of high voltage-activated (HVA) calcium channels in excitatory
neurotransmitter release and pain inhibition (24, 44–47), it would
be interesting to examine whether BAM 8–22 inhibits HVA cal-
cium current in small DRG neurons through MrgprC11.
Alternatively, either of two circuit mechanisms could also ex-
plain the inhibitory role of Mrgprs in persistent pain. First,
MrgprC11 ligands may activate DRG neurons that synapse onto
inhibitory interneurons located in the substantia gelatinosa (lam-
ina II) of the spinal cord. Such a mechanism would be consistent
with the fact that these ligands increase intracellular calcium in
cultured DRG neurons (11). This local inhibitory circuit has been
identified by previous studies (48) and has been suggested to re-
ceive input from relatively large-diameter, more rapidly conduct-
(diameter = 20–25 μm) that centrally project to lamina II (9, 49),
Mrgpr-expressing neurons may be engaged in this inhibitory
circuitry. Alternatively, the activation of Mrgprs may lead to the
release of inhibitory neurotransmitters or neuromodulators onto
way. PKC-γ interneurons, which may contribute to pathological
pain, might also play a role in BAM 8–22-induced pain inhibition
and should be investigated in future studies (50–52).
In summary, the present results together with previous data
(11) suggest that certain Mrgprs (mouse MrgprC11 and human
MrgprX1) may constitute an inhibitory mechanism for patho-
logicalpain andspinal neuronal sensitization, although additional
molecular mechanisms. Because human MrgprX1 expression is
restricted to DRG neurons, specific agonists of this receptor
might provide relief from chronic pain while producing few side
effects. If so, then such agonists could represent a class of anal-
gesic agents for the treatment of patients with chronic pain.
Production of Mrgpr-ClusterΔ−/−Mice. ThedeletionofaclusterofMrgprgenes
in themouse germline was described in a previousstudy (11). Briefly, chimeric
M Mrgpr-clusterΔ−/−(KO) mice were produced by blastocyst injection of
positive embryonic stem cells. The KO mice were generated by mating chi-
meric mice to C57BL/6 mice. Mice were backcrossed to C57BL/6 mice for at
least five generations. The Mrgpr-clusterΔ−/−mice were fertile, appeared
healthy, and were indistinguishable by their behavior and appearance from
the WT littermates.
CCI Model of Neuropathic Pain in Mice. A CCI at the left sciatic nerve was in-
duced in adult male mice. Inhalation anesthesia was induced with a constant
level of isoflurane (2.0%) delivered through a nose cone. Under aseptic
conditions, the left sciatic nerve at the middle thigh level was separated from
the surrounding tissue and loosely tied with three nylon sutures (9-0 non-
absorbable monofilament; S&T AG). The distance between two adjacent
ligatures was around 0.5 mm. None of the mice displayed autotomy or
exhibited marked motor deficits.
Behavioral Studies. All behavioral tests were performed by an experimenter
blinded to the genotype. The mice used in the tests were 2- to 3-mo-old males
(20–30 g). All experiments were performed under the protocol approved by
the Animal Care and Use Committee of the Johns Hopkins University School
Formalin test. Formalin (5 μL 2% formalin in PBS) was injected into the plantar
region of one hind paw, and spontaneous pain behavior (licking and biting)
was recorded for 60 min as previously described (23, 37).
CFA-induced heat hyperalgesia. Theintraplantarregion ofonehindpaw ofeach
mouse was injected with 6 μL 50% CFA solution in saline. Thermal-pain sen-
sitivity was assessed by recording PWL on exposure to a defined radiant-heat
Tail-immersion test. Mice were gently restrained in a 50-mL conical tube that
the mice voluntarily entered. The protruding one-third of the tail was then
dipped into a 50 °C water bath. Latency to respond to the heat stimulus with
vigorous flexion of the tail was measured three times and averaged.
CCI-induced mechanical allodynia. Mechanical sensitivity was assessed with the
von Frey test by the frequency method (53). Two calibrated von Frey mono-
filaments (low force = 0.07 g; high force = 0.45 g) were used. Each von Frey
filament was applied perpendicularly to the plantar side of each hind paw for
∼1 s; the stimulation was repeated 10 times to both hind paws. The occur-
rence of paw withdrawal in each of these 10 trials was expressed as a percent
response frequency: PWF = (number of paw withdrawals/10 trials) × 100%.
Drug and Intrathecal Injection. BAM 8–22 was purchased from Tocris and sus-
pended in 0.9% saline. The drug was injected intrathecally under brief iso-
thespinous and transverse processes.Theneedlewas moved carefullyforward
to the intervertebral space. A tail flick indicated that the tip of the needle was
inserted into the subarachnoid space.
Electrophysiological Recording of WDR Neurons Electrophysiologicalrecording
of WDR neurons in the dorsal horn of the spinal cord was performed by an
paralyzed with pancuronium bromide (0.15 mg/kg i.p.) during neurophysio-
logical recording. Throughout the experiment, anesthesia was maintained
Guan et al.PNAS
| September 7, 2010
| vol. 107
| no. 36
withaconstantlevelofisoflurane(1.5%)carriedinmed-air. Aspinalunitwith Download full-text
a cutaneous receptive field located in the plantar area of the hind paw was
located by applying mechanical stimuli. WDR neurons were defined as those
that responded to both innocuous and noxious mechanical stimuli and that
had increasing rates of response to increasing intensities of stimuli. Electrical
stimuli were applied through a pair of fine needles inserted s.c. across the
of individual neurons were obtained by using fine-tip (<1.0 μm) paralyn-
was applied directly to the exposed surface of the spinal cord at the recording
segment in a volume of 30–50 μL after predrug tests. The effects of BAM 8–22
on the spontaneous activity of WDR neurons were examined within 0–10 min
after application. The evoked neuronal responses were recorded 10–30 min
after drug application. Only one neuron in each animal was used to test the
drug effects. The postdrug responses were compared with the predrug
responses, allowing each neuron to act as its own control.
Data Analysis. The number of action potentials evoked by graded electrical
stimuli was compared between two genotypes by a two-way mixed-model
Student t test was used to compare the recording depth,activation threshold,
latency of the first A fiber-mediated response, and latency of the first C fiber-
mediated response between the two groups. For windup, the raw data were
the number of action potentials in the C component evoked by each stimulus
in a train of repetitive electrical stimuli. Because the number of action
potentials in the C component varies among WDR neurons, the raw data for
each neuron were normalized to the first response in each trial (input) and
and averaged C component responses to the last 10 stimuli (7–16) of the trial
between thetwo genotypesandbetween pre- andpostdrug conditions. Data
are presented as mean ± SEM. P < 0.05 was considered significant.
ACKNOWLEDGMENTS. We thank Claire Levine, MS, for editing the manu-
script and Yixun Geng for assistance with the mice. D.J.A and X.D. are
investigators at the Howard Hughes Medical Institute. The work was
supported by the Johns Hopkins Blaustein Pain Research Fund Award (to
Y.G. and X.D.), an Alfred P. Sloan Neuroscience grant (to X.D.), and a
Whitehall Foundation grant (to X.D.). This work was also supported by
National Institutes of Health Grants NS26363 (to S.N.R.), NS048499 (to D.J.A.),
and NS054791 and NS58481 (to X.D.).
1. Julius D, Basbaum AI (2001) Molecular mechanisms of nociception. Nature 413:203–210.
2. Woolf CJ, Salter MW (2000) Neuronal plasticity: Increasing the gain in pain. Science
3. Costigan M, Scholz J, Woolf CJ (2009) Neuropathic pain: A maladaptive response of
the nervous system to damage. Annu Rev Neurosci 32:1–32.
4. Baron R (2006) Mechanisms of disease: Neuropathic pain—a clinical perspective. Nat
Clin Pract Neurol 2:95–106.
6. Lembo PM, et al. (2002) Proenkephalin A gene products activate a new family of
sensory neuron—specific GPCRs. Nat Neurosci 5:201–209.
7. Dong X, Han S, Zylka MJ, Simon MI, Anderson DJ (2001) A diverse family of GPCRs
expressed in specific subsets of nociceptive sensory neurons. Cell 106:619–632.
8. Rau KK, et al. (2009) Mrgprd enhances excitability in specific populations of
cutaneous murine polymodal nociceptors. J Neurosci 29:8612–8619.
9. Liu Q, et al. (2007) Molecular genetic visualization of a rare subset of unmyelinated
sensory neurons that may detect gentle touch. Nat Neurosci 10:946–948.
10. Cavanaugh DJ, et al. (2009) Distinct subsets of unmyelinated primary sensory fibers
mediate behavioral responses to noxious thermal and mechanical stimuli. Proc Natl
Acad Sci USA 106:9075–9080.
11. Liu Q, et al. (2009) Sensory neuron-specific GPCR Mrgprs are itch receptors mediating
chloroquine-induced pruritus. Cell 139:1353–1365..
12. Chen T, Hu Z, Quirion R, Hong Y (2008) Modulation of NMDA receptors by intrathecal
administrationof the sensoryneuron-specific
13. Grazzini E, et al. (2004) Sensory neuron-specific receptor activation elicits central and
peripheral nociceptive effects in rats. Proc Natl Acad Sci USA 101:7175–7180.
14. Han SK, et al. (2002) Orphan G protein-coupled receptors MrgA1 and MrgC11 are
distinctively activated by RF-amide-related peptides through the Galpha q/11
pathway. Proc Natl Acad Sci USA 99:14740–14745.
15. Zylka MJ, Dong X, Southwell AL, Anderson DJ (2003) Atypical expansion in mice of
the sensory neuron-specific Mrg G protein-coupled receptor family. Proc Natl Acad Sci
16. Coderre TJ, Melzack R (1992) The contribution of excitatory amino acids to central
sensitization and persistent nociception after formalin-induced tissue injury.
J Neurosci 12:3665–3670.
17. Seal RP, et al. (2009) Injury-induced mechanical hypersensitivity requires C-low
threshold mechanoreceptors. Nature 462:651–655.
18. Calignano A, La Rana G, Giuffrida A, Piomelli D (1998) Control of pain initiation by
endogenous cannabinoids. Nature 394:277–281.
19. Melzack R, Wall PD (1965) Pain mechanisms: A new theory. Science 150:971–979.
20. Guan Y, Borzan J, Meyer RA, Raja SN (2006) Windup in dorsal horn neurons is
modulated by endogenous spinal mu-opioid mechanisms. J Neurosci 26:4298–4307.
21. Herrero JF, Laird JM, López-García JA (2000) Wind-up of spinal cord neurones and
pain sensation: Much ado about something? Prog Neurobiol 61:169–203.
22. Hong Y, Dai P, Jiang J, Zeng X (2004) Dual effects of intrathecal BAM22 on
nociceptive responses in acute and persistent pain—potential function of a novel
receptor. Br J Pharmacol 141:423–430.
23. Chen T, Cai Q, Hong Y (2006) Intrathecal sensory neuron-specific receptor agonists
bovine adrenal medulla 8-22 and (Tyr6)-gamma2-MSH-6-12 inhibit formalin-evoked
nociception and neuronal Fos-like immunoreactivity in the spinal cord of the rat.
24. Chen H, Ikeda SR (2004) Modulation of ion channels and synaptic transmission by
a humansensory neuron-specific G-protein-coupled
heterologously expressed in cultured rat neurons. J Neurosci 24:5044–5053.
25. Honan SA, McNaughton PA (2007) Sensitisation of TRPV1 in rat sensory neurones by
activation of SNSRs. Neurosci Lett 422:1–6.
26. Basbaum AI (1999) Distinct neurochemical features of acute and persistent pain. Proc
Natl Acad Sci USA 96:7739–7743.
27. Scholz J, Woolf CJ (2007) The neuropathic pain triad: Neurons, immune cells and glia.
Nat Neurosci 10:1361–1368.
28. Rodriguez Parkitna J, et al. (2006) Comparison of gene expression profiles in
neuropathic and inflammatory pain. J Physiol Pharmacol 57:401–414.
29. Melzack R, Coderre TJ, Katz J, Vaccarino AL (2001) Central neuroplasticity and
pathological pain. Ann N Y Acad Sci 933:157–174.
30. Vikman KS, Kristensson K, Hill RH (2001) Sensitization of dorsal horn neurons in
a two-compartment cell culture model: Wind-up and long-term potentiation-like
responses. J Neurosci 21:RC169.
31. Li J, Simone DA, Larson AA (1999) Windup leads to characteristics of central
sensitization. Pain 79:75–82.
32. Cai M, Chen T, Quirion R, Hong Y (2007) The involvement of spinal bovine adrenal
medulla 22-like peptide, the proenkephalin derivative, in modulation of nociceptive
processing. Eur J Neurosci 26:1128–1138.
33. Zeng X, Huang H, Hong Y (2004) Effects of intrathecal BAM22 on noxious stimulus-
evoked c-fos expression in the rat spinal dorsal horn. Brain Res 1028:170–179.
34. Ikeda H, et al. (2006) Synaptic amplifier of inflammatory pain in the spinal dorsal
horn. Science 312:1659–1662.
35. Costigan M, Woolf CJ (2002) No DREAM, no pain. Closing the spinal gate. Cell 108:
36. De Felipe C, et al. (1998) Altered nociception, analgesia and aggression in mice
lacking the receptor for substance P. Nature 392:394–397.
37. Knabl J, et al. (2008) Reversal of pathological pain through specific spinal GABAA
receptor subtypes. Nature 451:330–334.
38. Chen L, Huang LY (1992) Protein kinase C reduces Mg2+ block of NMDA-receptor
channels as a mechanism of modulation. Nature 356:521–523.
39. Ndong C, et al. (2009) Role of rat sensory neuron-specific receptor (rSNSR1) in
inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway.
40. Kusudo K, Ikeda H, Murase K (2006) Depression of presynaptic excitation by the
activation of vanilloid receptor 1 in the rat spinal dorsal horn revealed by optical
imaging. Mol Pain 2:8.
41. Caterina MJ, et al. (2000) Impaired nociception and pain sensation in mice lacking the
capsaicin receptor. Science 288:306–313.
42. MacDermott AB, Role LW, Siegelbaum SA (1999) Presynaptic ionotropic receptors and
the control of transmitter release. Annu Rev Neurosci 22:443–485.
43. Li W, Thaler C, Brehm P (2001) Calcium channels in Xenopus spinal neurons differ in
somas and presynaptic terminals. J Neurophysiol 86:269–279.
44. Delmas P, Abogadie FC, Buckley NJ, Brown DA (2000) Calcium channel gating and
modulation by transmitters depend on cellular compartmentalization. Nat Neurosci
45. Callaghan B, et al. (2008) Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type
calcium channels in rat sensory neurons via GABAB receptor activation. J Neurosci 28:
46. Gerevich Z, et al. (2004) Inhibition of N-type voltage-activated calcium channels in rat
dorsal root ganglion neurons by P2Y receptors is a possible mechanism of ADP-
induced analgesia. J Neurosci 24:797–807.
47. Altier C, Zamponi GW (2004) Targeting Ca2+ channels to treat pain: T-type versus
N-type. Trends Pharmacol Sci 25:465–470.
48. Lu Y, Perl ER (2003) A specific inhibitory pathway between substantia gelatinosa
neurons receiving direct C-fiber input. J Neurosci 23:8752–8758.
49. Zylka MJ, Rice FL, Anderson DJ (2005) Topographically distinct epidermal nociceptive
circuits revealed by axonal tracers targeted to Mrgprd. Neuron 45:17–25.
50. Neumann S, Braz JM, Skinner K, Llewellyn-Smith IJ, Basbaum AI (2008) Innocuous, not
noxious, input activates PKCgamma interneurons of the spinal dorsal horn via
myelinated afferent fibers. J Neurosci 28:7936–7944.
51. Miraucourt LS, Dallel R, Voisin DL (2007) Glycine inhibitory dysfunction turns touch
into pain through PKCgamma interneurons. PLoS ONE 2:e1116.
52. Malmberg AB, Chen C, Tonegawa S, Basbaum AI (1997) Preserved acute pain and
reduced neuropathic pain in mice lacking PKCgamma. Science 278:279–283.
53. Guan Y, Yaster M, Raja SN, Tao YX (2007) Genetic knockout and pharmacologic
inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced
mechanical hypersensitivity in mice. Mol Pain 3:29.
| www.pnas.org/cgi/doi/10.1073/pnas.1011221107 Guan et al.