Modulation of NR1 receptor by CaMKII plays an important role in
chronic itch development in mice
Nan-Qi Li, Yang Tang, Si-Ting Huang, Xue-Ting Liu, Li-Ping Zeng,
Hui Li, Li Wan
PII: S0361-9230(19)31003-2
DOI: https://doi.org/10.1016/j.brainresbull.2020.02.011
Reference: BRB 9864
To appear in: Brain Research Bulletin
Received Date: 13 December 2019
Revised Date: 22 February 2020
Accepted Date: 24 February 2020
Please cite this article as: Li N-Qi, Tang Y, Huang S-Ting, Liu X-Ting, Zeng L-Ping, Li H, Wan
L, Modulation of NR1 receptor by CaMKII plays an important role in chronic itch
development in mice, Brain Research Bulletin (2020),
doi: https://doi.org/10.1016/j.brainresbull.2020.02.011
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© 2020 Published by Elsevier.
Modulation of NR1 receptor by CaMKIIα plays an important
role in chronic itch development in mice
Nan-Qi Li1*
, Yang Tang1*
, Si-Ting Huang1
, Xue-Ting Liu2
, Li-Ping Zeng2
, Hui Li3
, Li Wan1
1Department of Pain Management, The State Key Clinical Specialty in Pain Medicine,
The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510260, P.R.
China.
2Guangdong Provincial Key Laboratory of Allergy & Clinic Immunology, Sino-French
Hoffmann Institute, The Second Affiliated Hospital, Guangzhou Medical University,
Guangzhou, 510260, P.R. China.
3Department of Anatomy, Histology and Embryology & K. K. Leung Brain Research
Centre, The Fourth Military Medical University, Xi’an, Shaanxi 710032, P.R. China.
*Correspondence should be addressed to: Dr. Li Wan, E-mail: [email protected]，
Running title: CaMKIIα modulate NR1 function in chronic itch.
Highlights
 NR1 subunit of the NMDA receptor involved in acute and chronic itch
 pNR1 facilitate histamine independent itch and chronic DNFB induced itch
 CaMKIIα modulate NR1 function to switch acute itch to chronic itch
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Abstract
Intractable scratching is the characteristic of chronic itch, which represents a great challenge
in clinical practice. However, the mechanism underlying chronic itch development is largely
unknown. In the present study, we investigated the role of NMDA receptor in acute itch and in
development of chronic itch. A mouse model was developed by painting DNFB to induce
allergic contact dermatitis (ACD). We found that the expression of pNR1, which is a subunit of
NMDA receptor, was significantly increased in the dorsal root ganglion in the DNFB model.
The DNFB-evoked spontaneous scratching was blocked by the NMDA antagonist D-AP-5, the
calcium-calmodulin-dependent protein kinase (CaMK) inhibitor KN-93, a CaMKIIα siRNA and
the PKC inhibitor LY317615. Moreover, activation of PKC did not reverse the CaMKIIα
knockdown-induced decrease in scratching, suggesting that PKC functions upstream of
CaMKIIα. Thus, our study indicates that modulation of NR1 receptor by CaMKIIα plays an
important role in the development of chronic itch.
Keywords: CaMKIIα, NMDA, GRP, itch, central sensitization
Introduction
Itch is an unpleasant somatosensation that provokes scratching and warns us of potential
threats to the body[5,45,53]
, Scratching behavior, an innoxious mechanical stimulus, inhibits the
itch sensation and spinal projection neurons[13,24,56]
. Moreover, scratching also intensifies skin
inflammation, which in turn provokes a more intense itch sensation and an uncontrollable
urge to scratch[47]
. Two kinds of peptides of the bombesin family and their receptors play an
important role in itch transmission. Gastrin-releasing peptide (GRP), a mammalian member of
the bombesin family of peptidesis, is an itch-specific neurotransmitter. It can specifically
activate GRP receptor (GRPR) and largely is restricted to the nonhistaminergic itch[1,29,52]
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including opioid-induced itch[30]
. Neuromedin B (NMB), another member of the bombesin
family of peptides, acts exclusively through NMBR to relay histamine dependent itch
information, whereas GRP can cross-activate NMBR as well in the spinal cord[60]. It was found
that NMBR interneurons are mostly glutamatergic and intermixed with GRPR neurons in
laminae I-II of the spinal cord[59]
. Histamine and NMB could activate the same synaptic
pathway, leading to the increased excitability of NMBR neurons to release glutamate onto
GRPR neurons which reflects enhanced glutamatergic transmission between NMBR neurons
to GRPR neurons. Moreover, NMBR neurons may function upstream of GRPR neurons via
glutamatergic transmission[56]
. However, the downstream of glutamatergic transmission in the
itch’s long-lasting mechanism is still unclear.
Glutamatergic receptors, particularly the N-methyl-D-aspartate receptor (NMDAR), participate
in the development of the sensory processing and transmission and are widely distributed
throughout the central and peripheral nervous system[7,23.,33,42,44,49,,55]
. NMDAR especially is
involved in synaptic plasticity and central sensitization in neuropathic pain. Antagonist
NMDAR reduces hyperalgesia and allodynia in animal models of neuropathic pain induced by
nerve injury and diabetic neuropathy[61]
. Activation of the NMDA receptor evokes Ca2+ influx
into neurons and initiates the intracellular cascades[41], resulting in neuroplastic changes in
the pain transmission pathway. It has been shown that itch and pain share a similar
mechanism, neuroplastic changes are the main mechanism underlying chronic pain and itch
development. Recently, Shen reported[61] that both NMDA antagonists, ketamine and
ifenprodil inbihited morphine-induced, pruritus-related ERK1/2 phosphorylation, which
underlines an intracellular cascade happened in an itch sensation for morphine intrathecal
injection. This study revealed the important effect of NMDA on central sensitization during
morphine-induced itch. Glutamate and NMDAR had been reported to contribute to histamine￾independent itch[1,4]
, and the indirect histamine-dependent NMB/GRP-NMBR-GRPR neuronal
pathway also has been mentioned[56]. However, the mechanism is still secret in which
glutamate is relesed from excitatory pruritic neurons with the NMDAR interaction and itch
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signal transmission .
In this study, we used both acute itch model and a mouse model of allergic contact dermatitis
(ACD), in which a prevalent skin disease triggered by direct skin contact with haptens or
allergens, induced by topical application of a chemical hapten, 2, 4-dinitrofluorobenzene
(DNFB) to clarify the role of NMDA receptor in itch transmission[37,48]. First, we observed the
chronic itch responses and the expression of NR1 mRNA in dorsal root ganglion (DRG) after
the administration of DNFB to C576L/BJ mice. Second, we observed the effects of NMDA
receptor agonists and antagonists on the acute itch behavior induced by pruritogens and on
chronic itch behavior in the DNFB model and compared the expression of NMDAR subunit of
pNR1 in the DNFB chronic itch model and in control mice. Finally, we studied the role of
CaMKIIα in central sensitization-hyperkinesis by administering CaMK inhibitor KN-39 or a
CaMKIIα siRNA. From our study, it shows that CaMKIIα plays an important role in allokenia,
and modulation of the NMDA receptor by CaMKIIα, resulting in central sensitization of the itch
pathway.
Materials and Methods
Animals Male mice between 7 and 12 weeks of age were used in the experiments.
C57BL/6J mice were purchased from the Guangdong Medical Laboratory Animal Center
(GDMLAC) (http://www.gdmlac.com.cn/index.php?q=en/node/373). All experiments were
performed in accordance with the guidelines of the National Institutes of Health and the
International Association for the Study of Pain and were approved by the Animal Studies
Committee at Guangzhou Medical University.
Drugs and reagents. Doses of drugs and injection routes are indicated in the figure
legends. DNFB was supplied by Xiya Reagents Inc. (Chengdu, China). GRP18-27 and NMB
were obtained from Qiangyao Biotech Co. Ltd. (Shanghai, China). KN-93 and NMDA were
purchased from MedChemExpress (Monmouth Junction, NJ, USA). The CaMKIIα siRNA was
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synthesized by RuiBo Co. Ltd. (Guangzhou, China), and in vivo jet-PEI® was obtained from
Polyplus-transfection (BIOPARC, Illkirch, France).
Itch models
Acute Itch of the Cheek model The skin on both cheeks of the mice was shaved 3 days
before the model was established. Mice received an i.t. injection of 0.05 nmol of NMDA 10
min before the CQ (50 μg), His (50 μg) or NMB (0.01 nmol) injections, according to the
experimental design. After the injection, each mouse was returned to the animal arena and
immediately video recorded for 30 min.
Chronic Itch-Allergic Contact Dermatitis (ACD) of the Nape model The ACD model
was established as previously described[18,22,58]. Briefly, the skin on stomach and nape of the
mice were shaved 3 days before model establishment. In the first week, 100 μl of 0.15%
DNFB (diluted in acetone) was painted on the skin of stomach for sensitization, and then 50
μl of 0.15% DNFB was painted on the nape twice a week for 4 sequential weeks.
Spontaneous scratching behaviors were recorded for 60 min on the next morning after DNFB
treatment for 20 hrs.
Behavioral tests Behavioral tests were videotaped (HDR-CX190, Canon) from the top of
the plastic animal arena. The videos were played back on a computer and the behaviors of
each animal were quantified by an observer who was blinded to the treatments and
genotypes. Hind limb scratching behavior towards the painted (nape) or injected area was
observed for 60 min with 5 min intervals. One bout of scratching was defined as the lifting of
the hind limb to the painted (nape) or injection site and then the return of the limb back to the
floor or to the mouth, regardless of how many scratching strokes occurred.
CaMKIIα siRNA treatment The sequence of the CaMKIIα siRNA was: forward, 5’-CAC CAC
CAU UGA GGA CGA AdTdT-3’ and reverse, 3’-dTdTGU GGU GGU AAC UCC UGC UU-5’. In
vivo jetPEI® was used as the transfection reagent and diluted with sterilized DEPC-treated
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PBS in ddH2O; the CaMKIIα siRNA was diluted with sterile DEPC-treated PBS in ddH2O to a
0.5 μg/ μl concentration. The PEI solution was mixed with the siRNA, and incubated at room
temperature for 20 min. Then, 2.5μg/10 μl of the CaMKIIα or control siRNA mixture was i.t.
injected into separate groups of mice that had been challenged with DNFB 3 times. The
siRNA and scrambled siRNA were injected into the DNFB-challenged mice twice a day for 5
consecutive days.
Acute scratching behavior after an intrathecal injection Scratching behaviors were
assessed as previously described[51-53]
. All behaviors were recorded during the light cycle.
Briefly, the injection area was shaved two days before the experiments. Prior to the
experiments, each mouse was placed in a plastic arena (10 × 11 × 15 cm) for 30 min and
allowed to acclimate. Mice were briefly removed from the chamber and intrathecally injected
in the lower back.
Immunohistochemistry Immunohistochemical (IHC) staining was performed using
previously described methods[48,49,52]. DRG tissues of L2-L5 were sectioned at a 20 µm
thickness. Free floating sections were incubated in blocking solution containing 2% donkey
serum and 0.1% Triton X-100 in PBS (PBS-T) for 2 hrs at room temperature. Sections were
incubated with primary antibodies overnight at 4°C, washed three times with PBS-T,
incubated with the secondary antibodies for 2 hrs at room temperature and washed three
times. Sections were mounted on slides with Fluoromount G (Southern Biotech) and
coverslipped. The following primary antibodies were used: rabbit anti-GRP (1:500; 20073,
Immunostar), rabbit anti-CGRPα (1:3000; AB1971, Millipore), rabbit anti-NR1 (1:100; goat
polyclonal; Santa Cruz). The NR1 antibody is directed against the C-terminus of the human
NMDARζ1 receptor subtype. Guinea pig anti-Substance P (1:1000; Ab10353, Abcam) and
guinea pig anti-TRPV1 (1:1000; GP14100, Neuromics) antibodies were also employed. The
secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and
included DyLight 488-conjugated goat anti-rabbit antiserum (1:500, 1.25 µg/mL), DyLight 594-
conjugated goat anti-rabbit antiserum, Alexa Fluor 488-conjugated avidin (0.33 µg/mL), and
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DyLight 594-conjugated goat anti-guinea pig antiserum (1:500, 2 mg/mL). Images were
captured using a Nikon Eclipse Ti-U microscope or a Leica TCS SPE confocal microscope.
RT-PCR. RT-PCR was performed as previously described[31] using the Fast-Start Universal
SYBR Green Master Mix (Roche Applied Science). All samples were assayed in duplicate
(heating at 95°C for 10 sec and at 60°C for 30 sec). Data were analyzed using the
comparative CT method (StepOne Software version 2.2.2.), and the expression of target
mRNAs was normalized to the expression of Gapdh. The following primers were used: Nr1,
5′-CTAATCAACGGCAAGAACGA-3′/5′-GCTTGACATACACGAAGGGTT-3′; Nr2b, 5′-
CATCACAACAATCCCGGCAG-3′/5′-GGCTGACACCACTGGCTTAT-3′; Nr2c, 5′-
AGAACTGGGGCAACAATCG-3′/5’-GAATAGCGGGAGAAATGGG-3′; Nr2d, 5′-
TCGCCGTCACAGTTTTCA-3′/5′-ACTTCAGGGGTGGGTATTGC-3′; Grp, 5’-
TGGGCTGTGGGACACTTAAT-3’/5’-GCTTCTAGGAGGTCCAGCAAA-3’; Grpr, 5’-
TGATTCAGAGTGCCTACAATCTTC-3’/5’-CTTCCGGGATTCGATCTG-3’; Trpv1, 5’-
ACCACGGCTGCTTACTATCG-3’/5’-CGGAAATAGTCCCCAACGGT-3’; Tac1, 5’-
GTGGCCCTGTTAAAGGCTCT-3’/5’-GCTCACCAGGAAGGCATCA-3’; Calca, 5’-
TGCCCATTAGTCCAACAAAGGA-3’/5’-TGGGCTGCTTTCCAAGATTGA-3’; Calcb, 5’-
GGACACTTCACTCTCGCTGT-3’/5’-CCATATGCTGCTGAGAGCCA-3’; CamkIIα, 5’-
TGGGTTTGGCTCTTGTATGGA-3′/5′-AAGAAAACAGTGCAGACAGGAGATC-3′; and Gapdh,
5’-CCCAGCAAGGACACTGAGCAA-3’/5’-TTATGGGGGTCTGGGATGGAAA-3’.
Western Blot Western blot analyses were performed as previously described[30]
. Briefly,
tissues were homogenized at 4°C with an electric homogenizer (Qiagen, USA) in a buffer
containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and
RIPA with complete protease inhibitor cocktail (Selleck, USA). The homogenate was
centrifuged at 800 × g for 15 min at 4°C and the supernatant was collected. Protein
concentrations were determined using a Micro-BCA protein assay kit (Pierce, Cat. 23235,
Rockford, IL, USA), and protein samples were diluted with the 2× electrophoresis sample
buffer, boiled for 10 min and cooled to room temperature. For western blotting, proteins were
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electrophoretically transferred from resolving gels to nitrocellulose membranes (0.2 μm, Bio￾Rad) in transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH 8.3) and 20% methanol] for 1 hr
at 100 V using a Bio-Rad Trans-blot tank apparatus at 48°C. Blotted membranes were rinsed
twice with 10 mM PB containing 0.09% NaCl (pH 7.4), and blocked for 1 hr at room
temperature with a solution of 10 mM PB plus 0.09% NaCl (pH 7.4), 0.3% Tween-20 (Bio￾Rad, Cat 170-6531), 3% teleostean gelatin (Sigma, cat. G7765) and 0.3% milk (svelty nonfat),
all of which were purchased from Bio-Rad. Blots were incubated overnight at 4°C plus 1 hr at
room temperature with a mouse CaMKIIa monoclonal antibody, (Cell Signaling Technology,
diluted 1:1000) and goat anti-GAPDH (Proteintech, China, diluted 1:10000) after stripping with
the stripping buffer (Dingguobio, China). After four 10min washes with a solution containing
0.2% Tween-20, 3% gelatin, 10 mM PB and 0.09% NaCl (pH 7.4), the membrane was
incubated with HRP-conjugated goat anti-mouse (Proteintech, China, diluted 1:5000) and
HRP-conjugated goat anti-rabbit (Proteintech, China, diluted 1:5000) secondary antibodies
diluted in PBS-T for 2 hrs at room temperature. After four washes with 10 mM PB plus 0.09%
NaCl/0.1% Tween-20 at room temperature, bound antibodies were visualized with
ImageQuant LAS4000 mini using an enhanced chemiluminescence kit (Thermo Fisher, MA
USA).
Results
Increased Nr1 mRNA expression after chronic itch development
In order to observe the role of NMDA receptor in chronic itch, we established the chronic itch
model by painting 0.15% DNFB on the back of nape twice a week for 4 weeks after the initial
sensitization of the C57 mice to verify this hypothesis. From challenge 2, mice showed
gradually increased scratching behaviors that peaked at approximately challenge 4 and
gradually decreased to the baseline at challenge 6 (Figure 1. A). This spontaneous scratching
behavior was consistent to our previous results obtained using the DNFB model. After the
success of chronic itch model, real-time RT-PCR methods was used to detect the mRNAs
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expression in the DRG neurons and found that the Grp and Trpv1 mRNAs were dramatically
upregulated in the DRG of the chronic itch model by 25-, and 3.8-fold, respectively, compared
to the control mice (Figure 2.B, and C). The level of the Tac1 mRNA (Figure 2. D), which
encodes substance P and had been reported facilitate to itch-scratching cycle in PAG
neurons[16] was increased by 3.8-fold in DRG neurons after the establishment of the chronic
itch model in our experiment, which underline a itch sensation role of Tac1 in peripheral nerve
system. The levels of the Calca and Calcb mRNAs, which encode the two CGRP-IR peptides
CGRPα and CGRPβ and are mainly involved in heat and itch sensation in spinal neurons, they
were significantly increased (3.16- and 1.98-fold, Figure 2. H and I), particularly Calca, which
is closely related to noxious heat sensitivity and itch sensation after chronic itch
development[36]
. In itch transmission, Carstens et al.[8]
reported that substance P and CGRP,
the two neuropeptides contributing to pruritus, released by primary afferent neurons and
partially expressed by the TRPV1 population, ablation of dorsal horn neurons expressing the
neurokinin-1 receptor resulted in reduced responses to pruritogens in rats. Using an animal
model of atopic dermatitis, it was reported that the increased release of CGRP and substance
P during scratching and a decreased sensitivity to itch after specific deletion of CGRP-positive
neurons[26,36] demonstrated the important role of CGRP and SP/Tac1 in the itch sensation.
The NR1 subunit of the NMDA receptor is a structural subunit that may modulate the effect of
NMDA on the central sensitization to pain. As we expected, the levels of the Nr1 mRNA
(Figure 2. E) exhibited a 2.37-fold increase in the chronic itch DNFB model, but the
expression of Nr2b mRNA (Figure 2. G) which is mainly involved in the central sensitization
for pain sensation and Nr2d mRNA (Figure 2. H) was not significantly increased. Based on
the increased Nr1 mRNA expression with other increased itch related genes, the results
indicate NR1 may also modulate the itch sensation and play a role in chronic itch
development. The increased CGRP and Tac1/SP may have a synergistic effect on the NMDA
mechanism in chronic itch. Journal Pre-proof
Increased levels of phosphorylated NMDAR (pNR1) after chronic itch development
To verify the role of NMDA receptor in chronic itch transmission, we also assessed NR1
expression in ACD mice and C57 control mice. Consistent with a previous report[35] we found
that phosphorylated NR1 (pNR1) was expressed in small and large diameter DRG neurons,
and pNR1 levels were significantly increased in chronic itch model mice (Figure 2. B).
Furthermore, we detected the co-expression of pNR1 with GRP (Figure 2. A), CGRP (Figure
2. E), TRPV1 (Figure 3. A) and SP (Figure 3. E) in C57 mice. In our study, the expression of
GRP was 4.5% in wild type (WT) mice and increased to 17% (Figure 2. A) after chronic itch
was induced, the results were consistent with our previous report[58]
. Meanwhile, the
expression of GRP in neurons overlapped with several pNR1-positive neurons in DNFB
control mice (6.5%, Figure 2. C and D), and the number of pNR1-positive neurons tripled
(16.3%) after chronic itch development. CGRP has long served as a molecular marker of
peptidergic nociceptive neurons, responds to noxious heat and mechanical stimuli, and
transmits heat and itch sensations[4,36]
. Previously, 10% to 36% of CGRP-expressing DRG
neurons were shown to be IB4-positive[4]. In the present study, CGRP-expressing neurons
were sparse in the DRG (14%) (Figure 2. E), and a few neurons overlapped with pNR1-
positive neurons in control mice (Figure 2. G), but the number was significantly increased
after chronic itch development in DNFB model mice (24.8% CGRP-positive neurons and
12.4% overlay with pNR1 neurons) (Figure 2. G, H). Recent research has shown that genetic
ablation of CGRPα-expressing sensory neurons reduced sensitivity to noxious heat, capsaicin
and itch (histamine and chloroquine) and impaired thermoregulation but did not impair
mechanosensation or β-alanine itch-stimuli associated with nonpeptidergic sensory neurons
[30]
. TRPV1 expression was also observed in the DNFB model, and the percentage of TRPV1-
positive neurons increased from 4.7% to 22.8% (Figure 3. A, D). Moreover, 15% of TRPV1-
positive neurons were pNR1-positive (Figure 3. B, C). Substance P is another peptidergic
transmitter in nociceptive neurons in the DRG. Consistent with other reports, the SP￾expressing cells in the DRG was 7% in C57 mice and increased to 22.6% in DNFB model
mice (Figure 3. E, H), and the population of SP neurons that were pNR1-positive increased
from 5% to 15% in chronic itch model mice (Figure 3. F, G). The co-expression of pNR1 with
GRP, CGRP, SP and TRPV1 in DRG neurons imply that the NR1 may facilitate itch sensation
in chronic situation.
NMDA facilitated GRP, CQ and NMB-induced scratch but not His-induced scratch
The increased NR1 expression in the chronic itch model prompted us to determine whether
NR1 plays a role in acute itch. In this study, we initially observed the effect of a noneffective
low dose (0.01 nmol) of NMDA, a specific agonist for NMDAR, by i.t. injecting it into C57
mice. After the i.t. injection, we did not observe itching, but an NMDA-induced biting behavior
was observed at 30 min(data not shown). A histamine-independent component is a
characteristic of chronic itch, and thus we postulated that NMDA may not enhance the
histamine-dependent itch. We first administered an i.t. injection of 0.05 nmol of NMDA to C57
mice to verify our hypothesis. At this dose, NMDA does not evoke any scratches (noneffective
dose), but a little bit biting behavior on their tails, then, we i.t. pre-injected 0.05 nmol NMDA
and 10 min later administered i.d. injections of histamine (50 μg). We found the scratching
behaviors were not significantly increased compared to the single histamine injection group,
consistent with our hypothesis (Figure 4. A). However, non-effective low dose of NMB i.t.
injection in 0.05nmol with 0.05nmol NMDA can induce a significantly increased scratching
behaviors (Figure 4. B) which is considered a histamine-dependent pruritogen[56]. Based on
these results, NMDA facilitates NMB-induced itch rather than histamine induced itch. To
further demonstrate the role of NR1 in itch processing, the CQ- and GRP-induced itch was
observed in NMDA treated mice. The CQ induces histamine-independent itchiness by
activating Mas-related G-protein-coupled receptor A3 (MrgprA3)[29]
. After the i.d. injection of
50 μg of CQ, the mice only showed moderate scratching behaviors, but when the i.t. injection
of 0.05 nmol NMDA was administered prior to CQ, the mice showed significantly increased
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scratching behaviors (Figure 4. D), these results suggest a facilitated effect of NMDA to CQ
induced itch. GRP evoked scratching behaviors is by pharmacologic activation of GRPR,
which is essential for itch processing at the spinal level[52,53]. In our study, a single i.t. injection
of 0.005 nmol of GRP only induce a weak scratching behavior. However, after a prior i.t.
injection of 0.05 nmol of NMDA, significantly increased scratching behaviors were observed in
C57 mice (Figure 4. C). Together, these results suggest a facilitating effect of NMDA on
histamine-independent itch and NMB-induced itch, but not histamine-induced itch.
CaMKIIα maybe a major activator of the switch from acute itch to chronic itch
development
Since NMDAR activation has been shown to trigger a CaMKII-mediated signaling pathway[17]
we presumed that the CaMK or CaMKII antagonist would influence NMDA-facilitated GRP
induced itch. Consistent with our hypothesis, the NMDA-facilitated GRP induced itch was
blocked by an i.t. injection of 0.1 nmol of the CaMK antagonist KN-93 (Figure 5. A) and this
NMDA-facilitated GRP induced itch also can be blocked by 3 μg of the NMDA-specific
antagonist D-AP-5 (Figure 5. C), which indicating that CaMKII may function as an upstream
signal of NMDA receptors and this facilitated scratching is NMDA dependent. In order to
clarify whether NMDA facilitated GRP induced itch is related to the PKC function, 5 nmol of
the PKC antagonist LY317615 was i.t. injected before NMDA and GRP i.t injection, the results
showed that NMDA facilitated GRP-evoked scratching was significantly inhibited (Figure 5. B),
suggesting that NMDA may function downstream of PKC. Although our results showed that
CaMKII inhibition influenced the effect of NMDAR on facilitating GRP induced scratching, we
wished to determine whether this facilitatory effect was blocked by direct CaMKII
downregulation using a CaMKIIα siRNA. First, we observed the effect of an i.t. injection of the
CaMKIIα siRNA on chronic spontaneous scratching from challenge 3 to challenge 4 for 5
sequential days and observed that chronic itch behavior was significantly decreased after 3
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days of injections, and much more significantly inhibited after 5 days of injections (Figure 5. G
and H). This reduced spontaneous scratching was not reversed by the administration of 5
nmol of the PKC-specific agonist staurosporine (Figure 5. I), suggesting that PKC is located
upstream of CaMKII. Despite the decrease in chronic itch behavior after the siRNA injection,
we did not clearly determine whether a pre-injection of CaMKIIα siRNA before DNFB
challenge can completely blocked the transformation of acute itch to a chronic situation. We
changed the i.t injection protocol from beginning at challenge 3 to before the challenge and
found that the siRNA-treated DNFB chronic itch model mice showed few scratching behaviors
(Figure 5. G), and the expression of CaMKIIα protein was virtually lost in siRNA-treated DNFB
mice (Figure 5. L). From above results, it suggests that CaMKII may be a major activator of
the switch from acute itch to chronic itch. Based on our results, we postulate that CaMKIIα
may be an important target for inhibiting the transformation from acute itch to a chronic
situation. Further studies are needed to confirm this hypothesis.
Discussion
Although chronic itch development is closely related to GRPR expression and function, the
progression of acute itch to a chronic status remains elusive. The present study demonstrated
that the NR1 subunit of the NMDA receptor not only participates in GRP, CQ and NMB￾induced itch but is also involved in chronic itch development, and this itch sensitization is
closely related to CaMKII modulation. Several lines of evidence support this conclusion. First,
the i.t. injection of an NMDA-specific agonist can significantly evoke scratching behavior in
mice injected with a non-effective low dose of NMB, and histamine-independent GRP and
CQ, and this scratching behavior can be blocked by the NMDA-specific antagonist D-AP-5.
Second, in chronic itch ACD mice, the phosphorylated subunit of NMDAR NR1 (pNR1), which
has been implicated in central sensitization for itch, is significantly increased along with
increases in GRP expression. Finally, the facilitating effect of NMDA on GRP induced itch and
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chronic itch of ACD can be inhibited by a CaMK antagonist KN-93 and by CaMKIIα siRNA,
respectively. It is known that CaMKII inhibitors can reduce the phosphorylation of NMDA
receptors to inhibit long-term potentiation (LTP) [9] which is the mainly mechanism of central
sensitization. Therefore, CaMKII might be involved in central sensitization for itch by
phosphorylating NR1 function. These results suggest that NMDA plays a role in NMB-induced
and histamine-independent itch and ACD chronic itch, and this itch sensation is mediated by
CaMKIIα.
NMDAR has long been considered important for central sensitization in chronic pain, it
consists of many subunits, which are categorized into three subtypes (NR1, NR2, and NR3)
and expressed in a broad area of the peripheral and central nervous system[9-11,38]. It has
been shown that the CaMK inhibitor KN-93 could decrease allodynia and hyperalgesia by
regulating the phosphorylation of NMDA receptor subunits NR1, which are present in the cell
bodies and central terminals of both small- and large-diameter dorsal root ganglion
neurons[35]. Our study is also showed the same expression cell type, which is consistent with
these results. Except for increased pNR1 expression, CGRP and SP also are up-regulated
after chronic DNFB treatment. Studies have demonstrated that SP plays an important role in
cold noxious pain, tissue injury-induced pain, and inflammation induced hyperalgesia.
Subsequently, acute noxious heat- and tissue injury-induced pain involves CGRP
transmission[43]
. However, the genetic disruption of the substance P receptor gene did not
result in differences in acute heat pain[61]. These studies demonstrated that substance P is not
required for heat pain transmission, but CGRP involved in noxious heat pain transmission. In
our study, the increased scratching and expression of SP/Tac1 and CGRP also related no
effect on heat sensitivity with the Hargreaves test (Figure S1. A), which partly coincides with
the research mentioned above. However, the enhanced mechanical pain threshold may be
due to the increased NR1 (Figure S1. B), in which the GRPR function is facilitate to inhibit
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noxious stimulation-induced pain behavior.
NMDAR is considered a potential target in the treatment of itch[3]
. But how do the NMDA
receptor NR1 subunits exert it’s function in itch? Haddadi[20] recently reported that a non￾effective dose of the NMDAR agonist NMDA (75 mg/kg) potentiates the scratching behavior
provoked by subeffective doses of CQ (200 µg/site). In contrast, combined pretreatment
consisting of subeffective doses of the NMDAR antagonist MK-801 and the NOS agonist L￾NAME can decrease CQ-induced scratching behavior, which indicates that NMDA plays a role
in histamine-independent scratching. However, many studies, including our previous studies,
have shown that an i.d. injection of 200 µg of CQ can induce a dramatic increase in itch
behavior. Moreover, in our experiment, we found that neither a noneffective low dose (0.01
nmol) nor a high dose (1 nmol) of NMDA could induce any scratching behavior; however, a
dramatic increase in itching behavior was obtained by the combination of the non-effective
dose of NMDA administered via the i.t. route with an i.d or i.t. injection of CQ or GRP or NMB,
our results support the theory that NMDA exerts a facilitating effect on histamine-independent
and histamine related itch, the former is related to intractable itch in clinical practice. Except
the GRP and CQ induced itch, in which had been considered GRPR and MrgprA3 function
related. Inoue et al [23] reported that scratching behaviors evoked by CQ could be inhibited by
the NMDA receptor antagonists D-AP-5 and CP101,606 and that intracisternal NMDA- and
GRP-induced robust scratching could be reduced by the GRP receptor antagonist RC-3095.
This research demonstrated a functional association between GRPR and the NMDA receptor
and showed that NMDA receptor activation occurs upstream of the GRP-GRPR pathway.
From our NMDA facilitate GRP, CQ and NMB induce itch, it may imply a facilitated effect of
NMDA on GRPR function in itch sensitization. The activation of NMDAR induce an influx of
Ca2+ and invokes Ca2+ calmodulin-dependent protein kinase II (CaMKII), protein kinase C
(PKC) activation[38,41] Journal Pre-proof
, thereafter to phosphorylate the GRPR neuron to influence itch behavior
Itch can be classified as either acute or chronic according to the course duration. Acute itch is
a daily experience that can usually be abolished by briefly scratching near the itching area.
Chronic itch can be debilitating, and local scratching often provides little relief and can even
exacerbate the problem. Many pruritogens, such as CQ, histamine, 5-HT, ET-1, SLIGRL,
BAM-22, GRP, NMB and BNP, can induce acute scratches, and each of these induce
histamine-dependent or histamine-independent scratching[6,10,59]
. However, the i.d. or i.t.
injection of NMDA at either a non-effective dose (0.01 nmol) or a high dose (1 nmol) cannot
induce any scratching but does induce biting or grooming behavior, however, these biting
behavior cannot be excluded completely with itch behavior[34] and also need further research.
Although our behavior experiments administration of NMDA alone does not show itching
characteristics after peripheral or lower central injection, but it can increase scratching in a
low dose of GRP (0.005 nmol, does not exert an effect), CQ and NMB. This finding together
with other researches suggest that NMDA plays an important role in histamine-independent
and NMB related histamine itch. Moreover, in chronic situations, pNR1 expression in the DRG
and spinal cord is increased in the chronic itch DNFB model, phosphorylation is the
mechanism by which ionic glutamate receptor activated. Neuroplastic changes constitute the
basis of central sensitization[12,39,52]
. There are researches showed Serine/threonine or/and
tyrosine kinase activation-induced phosphorylation of NR1/NR2 subunit have shown to play
crucial roles in the occurrence and progress of synaptic plasticity, and those C terminal
domains are the substrates of protein kinase A, protein kinase C (PKC), calcium/calmodulin
dependent protein kinase II (CaMKII), and extracellular regulated protein kinase (ERK) [52]
which combined with our research indicates that NMDA might play a role in chronic itch
development and it’s function is may from the phosphorylation by PKC, CaMKII and/or ERK.
The PLCβ3/IP3/Ca2+ pathway also plays an important role in acute itch, and the intracellular
Ca2+ signal has been confirmed to participate in itch sensation and central sensitization[4,30]. In
our experiment, the itching behavior was significantly increased, similarly to the expression of
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GRP, CGRP, and pNR1 proteins in chronic situations. These findings suggest that
neuroplastic changes occurred in the DRG. Most importantly, these enhanced itching
behaviors can be blocked by the CaMK antagonist KN-93 or CaMKII siRNA, it verifies that
CaMKII may phosphorylate the NMDA to facilitate the GRP induced itch, and may functions
as upstream of the NMDA -GRP-GRPR signaling pathways.
CaMKIIα siRNA can block spontaneous scratches and CaMKIIα mRNA expression in the
dorsal root ganglion (Figure 4. K), and these inhibitory effects cannot be reversed by the
PKC-specific activator staurosporine, which suggests that CaMKIIα is located upstream of
PKC. It showed that PKC can directly phosphorylate the NR1 subunit[2,54]
, and the PKC-cell
adhesion kinase β/proline-rich tyrosine kinase 2-Src signaling cascade can indirectly
upregulate the NMDAR function[21,54]
. Our research, though, clarifies the role of CaMKIIα in
the modulation of the NR1 function in chronic itch development. Except histamine itself, the
histamine-dependent mechanism may also be involved in the development of DNFB chronic
itch due to the itch behavior observation: with the i.p. injection of chlorpheniramine, an
antihistamine agent into DNFB-treated mice, it showed very significantly decreased itch
behavior( Figure S2. A). However, the antihistamine-to-dry-skin AEW model mice did not
show significant differences compared to saline control mice (Figure S2. B), which suggests a
histamine-dependent mechanism may be involved in DNFB-induced chronic itch. Altogether,
we speculate that the skin releases inflammatory mediators, such as glutamate, that might
directly activate or sensitize presynaptic NMDA receptors in the peripheral nervous systems,
which would activate intracellular calcium and CaMKIIα, the latter of which phosphorylates the
NMDA receptor to exert a facilitating effect on GRPR function. GRPR neurons are the final
output in the spinal cord, resulting in the central sensitization to itch. The inhibition of CaMKIIα
might reverse the facilitating effect of NMDA on the GRP/GRPR pathaway and abrupt central
sensitization development. Accordingly, the modulation of NMDA by CaMKIIα plays an
important role in chronic itch development. A medicine targeting CaMKIIα may be an
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important candidate for chronic itch therapy. Although our research has demonstrated NR1
participates in chronic itch, how it has been activated and which site is phosphyralated are the
key points. Whether CREB is involved in a modulated NMDA receptor function still needs
more research to determine.
In conclusion, NMDA facilitates histamine-independent and NMB-induced itch, as well as
regulates chronic itch with CaMKIIα. The inhibition of CaMKIIα blocks hyperkinesis
(allokinesis) and chronic itch development.
Abbreviations GRPR, gastrin-releasing peptide receptor; GRP, gastrin-releasing
peptide; NMDA, N-methyl-D-aspartic acid; KO, knockout; CGRP, calcitonin gene-related
peptide; GPCRs, G protein-coupled receptors; TRP, transient receptor potential; TRPV1,
transient receptor potential cation channel, subfamily V, member 1; DRG, dorsal root
ganglion; CQ, chloroquine; NMB, neuromedin B; i.t., intrathecal/intrathecally injection; SP,
substance P; SPR, substance P receptor, NMDARζ, NMDA receptor ζ; CaMKIIα, calcium￾calmodulin-dependent protein kinase IIα; PBS, phosphate-buffered saline; IHC,
immunohistochemistry; RT-PCR, reverse transcription polymerase chain reaction.
Declarations
Ethics approval and consent to participate: All the experiments were performed in
accordance with the guidelines of the National Health Commission and were ethics approved
by the Animal Studies Committee at Guangzhou Medical University.
Consent for publication: All of authors are consent for publication of this paper.
Availability of data and material: Data sharing applicable to this article.
Competing interests: There were no competing interests in this study.
Funding: The research was supported by the National Natural Science Foundation of China
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(No. 81301948) to X. T. Liu， by the National Natural Science Foundation of China (No.
81771182) to L Wan, by Natural Science Foundation of Guangdong Province
(No.2016A030313599) to L Wan.
Authors’ contributions: Author contributions as follows: L.W. designed the experiments,
N.Q.L. performed behavioral, immunohistochemistry and molecular biology experiments. Y. T.
performed immunohistochemistry and molecular biology experiments. S.T.H. performed
behavioral experiment. X.T.L. and L. P.Z.performed molecular biology experiments and
analysis, H.L. contributed to the design and experiment, L.W. wrote the manuscript and N.Q.L
participated in the writing.
Authors’ information (optional): N.Q.L., Y.T. and S.T.H. are undergraduate student of
Anesthesiology and engage in chronic itch and pain mechanism research. X.T.L and L.P.Z.
are the fellow of Guangdong Provincial Key Laboratory of Allergy & Clinic Immunology, Sino￾French Hoffmann Institute, The Second Affiliated Hospital, Guangzhou Medical University,
both of them mainly engage in the itch research related to allergy. H.L. is the vice dean of
Department of Anatomy, Histology and Embryology & K. K. Leung Brain Research Centre, The Fourth
Military Medical University, he is a senior scientist in brain anatomy and function research and
also a senior itch research scientist in China. L.W. is the dean of Department of Pain
Medicine, State Key Clinical Specialty in Pain Medicine, the Second Affiliated Hospital,
Guangzhou Medical University, she is major chronic itch and pain mechanism research and
also involved in clinical pain management.
Conflict of interest
The authors declare no conflict of interests.
Acknowledgements: We thank W. J. J for initial help with project and technical support. We
also thank Yan-Gang Sun for comments.
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Figure Legend
Figure.1. Chronic itch behavior test and qRT-PCR comparing gene expression in DRGs
from ACD model mice.
(A)Spontaneous scratching was induced by 0.15% DNFB sensitization and challenged for 6
times. From the challenge 2, the mice showed a gradually increased scratches and up to the
peak around challenge 4, and gradually decreased to the baseline at challenge 6.
Significantly increased scratching behavior was showed at challenge 3, 4, and 5 when
compared to challenge 1, 2 and 6, the X axis means challenge times. * P<0.05;*** P<0.001,
One-way ANOVA in A. (B-I) qRT-PCR analysis of select transcripts Gadph, as an internal
control showed stable expression among all animals and qRT-PCR data from DRGs were
normalized with Gadph. PCR products of grp(B), tac1(C), nr1(E) were all significantly
increased in ACD model mice compared to their control group, while trpv1(D), calca (H) and
calcb (I) were all very significantly increased in ACD mice group, but the expression of Nr2b
(F) and Nr2d (G) had no significantly difference in DNFB painting ACD mice group compared
to their control group. n=4, *P<0.05, ** P<0.01, ***P<0.001, unpaired t test in (A-G). Error
bars represent SEM.
Figure. 2. GRP, pNR1 and CGRP expression in DRG in chronic DNFB model
(A-C). Immunohistochemistry (IHC) showed the GRP+ neurons(green) (A) and pNR1+
neurons (red) (B) in DRGs of ACD model mice was significantly increased compared with
control mice (D), and the increased GRP+ neurons overlay with pNR1 neurons (yellow) (C),
Scale bar, 20µm; (E-H). IHC showed CGRP+ neurons (green) (E) and pNR1+ neurons (F)
colocalize (yellow) (G) in DRGs, and the number of positive neurons increased significantly in
DRGs of ACD model mice (H). n=4, *** P<0.001, unpaired t test. Scale bars: 20µm.
FigureJournal Pre-proof . 3. TPRV1, SP and pNR1 expression in DRG in chronic DNFB model
(A-D).IHC showed TRPV1+ neurons (green) (A) and pNR1+ neurons (B) colocalize (yellow)
(C) in DRGs, and the number of positive neurons increased significantly in DRGs of ACD
model mice compared to WT mice (D). (E-H). The number of SP+ neurons (E) in DRGs of
ACD model mice were significantly increased and overlay with pNR1 (yellow) (F, G)
comparing to WT mice (H). n=4. * P<0.05, ***P<0.001, unpaired t test. Scale bars: 20µm.
Figure. 4. NMDA facilitate GRP, CQ and NMB induced scratches rather than histamine,
induced scratch
(A). 0.05nmol NMDA i.t. injection cannot facilitate 50ug histamine i.d. injection induced
scratches in cheek model, when compared to histamine 50ug i.d. injection alone, n=5, P>0.05,
unpaired t test. Error bars represent SEM. (B). 0.05nmol NMDA i.t. injection can significantly
increase the scratching behavior after NMB 0.05nmol i.d. injection, n=5, *P <0.05, unpaired t
test. Error bars represent SEM. (C).50ug CQ i.d. injection induced scratching in cheek model
can be facilitated by 0.05nmol NMDA i.t. injection, comparing with CQ i.d. injection alone, n=5,
*P<0.05, unpaired t test. Error bars represent SEM. (D). pre-injection of 0.05nmol NMDA i.t.
to C57 mice can significantly increase the GRP induced scratches n=6, *P<0.05, paired t
test. Error bars represent SEM.
Figure. 5. Inhibition of CaMKII, PKC or NMDA function can significantly reduce NMDA
facilitated GRP induced itch or chronic itch behavior
(A-C). Non-effective NMDA (0.05nmol, i.t.) facilitate non-effective dose of GRP (0.005nmol,
i.t.) inducing significant increased scratching behaviors (red bar) when compared to GRP
(0.005nmol, i.t.) alone (blue bar), and attenuated by intrathecal (i.t.) injection of CaMKII
antagonist KN-93 (5nmol, i.t.) (green bar) (A), by PKC specific inhibitor LY317615 (5nmol, i.t.)
(green bar) (B) and by NMDA antagonist D-AP-5 (3ug, i.t.) (green bar) (C), respectively. (D￾F). Spontaneous scratching induced by painting DNFB in ACD mice was significantly inhibited
by i.t. injection of KN-93 (5nmol, i.t.) (D), by LY317615 (5nmol. i.t.) (E) and by D-AP-5 (3ug,
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i.t.) (F), respectively. (G-I). 2.5 µg/10µl CaMKIIα siRNA i.t. injection twice a day for 4 to 5 days
can significantly inhibit DNFB induced spontaneous scratches in ACD model (G and H), and
PKC specific agonist straurosporine (5 nmol) cannot reverse this inhibit effect (I). Error bars
represent SEM, n=6. *P<0.05, ** P<0.01, one-way ANOVA followed by Bonferroni post-test in
A-C, paired t test in D-F, unpaired t test in G-I. (J). 2.5 µg/10µl CaMKIIα siRNA pre￾administrate (i.t. injection twice a day for 3 days) before DNFB challenge can significantly
inhibit DNFB challenge (3 times) induced spontaneous scratches in ACD model, *P<0.05,
unpaired t test. (K). camkIIα mRNA expression in spinal cord L5 level was significantly
decreased after sequential 5 days CaMKIIα siRNA i.t. injection in DNFB challenge 4 mice,
*P<0.05, unpaired t test; (L)Western blot showed the significantly decreased of CaMKIIα
protein in spinal cord in siRNA treated mice when compared to DNFB model. Journal Pre-proof