, 968 (2013);
Santosh K. Mishra and Mark A. Hoon
The Cells and Circuitry for Itch Responses in Mice
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to the content of substituted Fe in Mn3O4. The
potential of Mn3-xFexO4as a function of the Fe
theory calculations, which indicated that a solid
solution of Mn3-xFexO4can occur between Mn3O4
and Fe3O4(fig. S20) and that their average re-
action voltages are proportional to the amount of
x ≤ 3 (table S2) (20). Our results suggest that the
potentials of multicomponent spinel electrode
materials can be easily tuned by galvanic replace-
ment reaction and subsequent thermal treatment.
Figure 3D shows cycle performances of hol-
low Mn3-xFexO4NCs. Mn1.1Fe1.9O4exhibited
first discharge and charge capacities of 1339 and
984 mAh g−1, respectively. Mn1.1Fe1.9O4exhib-
ited a high reversible capacity of ~1000 mAh g−1
with almost no fading up to 50 cycles. The revers-
ible capacities and cyclic stabilities of Mn2.0Fe1.0O4
and Mn1.5Fe1.5O4were comparable to those of
Mn1.1Fe1.9O4.Most of the samplesshowed high-
er specific capacities and better cyclic stabilities
than those of carbon-coated solid Mn0.8Fe2.2O4
NCs (fig. S21) (20). These good cyclic stabilities
can be attributed to their hollow structure, which
provides extra free space for alleviatingthe struc-
tural strain caused by the large volume change
(26, 27) and additional sites for lithium ion stor-
age (28). Mn0.6Fe2.4O4and Mn0.3Fe2.7O4, which
are close to Fe3O4, showed less cyclic stability
than the other samples, indicating that multi-
metallic composition contributes to the enhanced
exhibited the highest rate capability (fig. S22)
(20), which is due to the improvement in its elec-
tronic conductivity resulting from the mixed va-
lency of the multicomponent Mn3-xFexO4(30).
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Acknowledgments: We acknowledge financial support by the
Research Center Program of the IBS in Korea (T.H. and Y.-E.S.);
the WCU (R31-10013) Program of the National Research
Foundation (NRF) of Korea (T.H., N.P., and Y.-E.S.);
Portuguese Fundação para a Ciência e a Tecnologia projects
(PTDC/CTM/100468/2008 and REDE/1509/RME/2005) for
TEM work (M.-G.W. and N.P.); a Human Resources Development
of the Korea Institute of Energy Technology Evaluation and
Planning grant funded by the Korea government Ministry of
Trade, Industry and Energy (20124010203320) (K.K. and
D.-H.S.); and National Creative Initiative (2009-0081576), WCU
(R31-2009-000-10059), and Max Plank POSTECH/KOREA
Research Initiative (2011-0031558) programs though the NRF of
Korea (K.-T.K. and J.-H.P.). The Pohang Accelerator Laboratory is
supported by POSTECH and the Ministry of Science, ICT and
Materials and Methods
Figs. S1 to S22
Tables S1 and S2
3 January 2013; accepted 15 April 2013
The Cells and Circuitry for Itch
Responses in Mice
Santosh K. Mishra and Mark A. Hoon*
Itch is triggered by somatosensory neurons expressing the ion channel TRPV1 (transient receptor
potential cation channel subfamily V member 1), but the mechanisms underlying this nociceptive
response remain poorly understood. Here, we show that the neuropeptide natriuretic polypeptide b
(Nppb) is expressed in a subset of TRPV1 neurons and found that Nppb−/−mice selectively lose
almost all behavioral responses to itch-inducing agents. Nppb triggered potent scratching when
injected intrathecally in wild-type and Nppb−/−mice, showing that this neuropeptide evokes itch
when released from somatosensory neurons. Itch responses were blocked by toxin-mediated
ablation of Nppb-receptor–expressing cells, but a second neuropeptide, gastrin-releasing peptide,
still induced strong responses in the toxin-treated animals. Thus, our results define the primary
pruriceptive neurons, characterize Nppb as an itch-selective neuropeptide, and reveal the next
two stages of this dedicated neuronal pathway.
the ion channel TRPV1 (transient receptor po-
ruritic responses are triggered by somato-
agents acting through pathways involving
and the effector enzyme PLCb3 (3). Recently,
neurons was shown to result in a profound loss
of all itch responses (2) but also affects thermo-
sensation and pain (4). In contrast, a much more
lacking the gastrin-releasing peptide (GRP) re-
ceptor (5) and in animals in which the spinal
interneurons expressing this receptor had been
specifically targeted with a GRP-conjugated toxin
(GRP-saporin) (6). Thus, it was hypothesized that
GRP is the neurotransmitter that initiates a labeled
line for itch (5, 6).
Previously, we generated mice that had lost
all TRPV1-lineage neurons (7). These mice have
major pruritic deficits(Fig. 1A) as well as a com-
plete loss of thermosensory input (7), which is in
induced lesions (2,4).Toidentify candidate mol-
ecules that might mediate itch signaling, we used
a differential microarray–based screen that iden-
Amongthese,natriuretic polypeptideb (Nppb) is
root ganglia (DRG) neurons but is dramatically
decreased in sensory ganglia from TRPV1-DTA
animals (Fig. 1B). Double-label in situ hybrid-
expressing neurons contain TRPV1 (Fig. 1C) and
PLCb3 (Fig. 1D), which are critically required for
histamine-induced scratching in mice (2, 3). More-
over, double labeling (Fig. 1, E and F) shows al-
most complete overlap between the expression
of Nppb and two Mas-related G protein–coupled
receptors that have recently been shown to detect
specific pruritogens (8–10).
We generated Nppb−/−animals (Fig. 2A) and
showed that these mutants displayed no detect-
Molecular Genetics Unit, Laboratory of Sensory Biology, Na-
tional Institute of Dental and Craniofacial Research (NIDCR),
NIH, Building 49, Room 1A16, 49 Convent Drive, Bethesda,
MD 20892, USA.
*Corresponding author. E-mail: email@example.com
24 MAY 2013VOL 340
Bouts of scratching
Fig. 1. Nppb is always coexpressed with itch-related signaling mol-
scratch responses after intradermal injection of pruritic agents; histamine
(30 mg); PAR2 agonist SLIGRL-NH2 (100 mg); and compound 48/80 (100 mg).
Itch-inducing substances were injected intradermally into the shoulder of
mice, and numbers of scratching bouts were assessed over 30 min; data are
mean T SEM (n ≥ 7 animals) normalized to wild-type litter controls. (B) ISH of
sections through DRG illustrating loss of Nppb-expression in TRPV1-DTA an-
imals. Quantitation of Nppb-expressing neurons revealed that 7 T 0.6% of
NeuN-positive C4 DRG neurons express the neuropeptide in wild-type mice
(n = 3 animals). (C) Double-label ISH of DRG demonstrates that Nppb
(green) and TRPV1 (red) are coexpressed in the same sensory neurons; only a
subset of TRPV1-expressing neurons contain Nppb. (D) All Nppb-positive neu-
Double-label ISH also shows that Nppb-positive neurons (green) all express
MrgprA receptors (including MrgprA3, the receptor for chloroquine; red), with
more than 70% of MrgprA-expressing neurons also containing the neuro-
peptide. (F) Double-label immunostaining demonstrates complete overlap
between expression of MrgprC11 (red), the receptor for the pruritogen SLIGL-
of neurons expressing Nppb is available in fig. S2.
Normalized response (%)
Bouts of scatching
Bouts of scatching
Fig. 2. GenerationandcharacterizationofNppb−/−
mice. (A) Schematic representation showing the dis-
ruption of the Nppb gene by insertion of a splice
acceptor-lacZ cassette into the second exon used to
generate Nppb−/−mice. (B) ISH of sections through
DRG reveals that Nppb expression is lost in Nppb−/−
animals. (C) Nppb−/−mice retained normal reactions to
thermal, touch, and proprioceptive stimuli, but (D) ex-
hibit greatly attenuated responses to a range of pruritic
normalized to wild-type litter controls. Significant
differences between genotypes were determined by
using Student’s t test; *P < 0.001. (E) Intrathecal
injection of Nppb (5mg in 10ml) into the lumbar 4-5
segment of control and Nppb−/−mice induced re-
peated bouts of scratching; injection of GRP (1nM in
10ml) also triggered itch responses in both mutant and
5 animals); no significant differences in response be-
tween genotypes were found (Student’s t test). Typical
behavioral responses of control and Nppb−/−mice to
histamine, Nppb, and the nociceptive neuropeptide substance P are illustrated in movies S1 to S3.
VOL 340 24 MAY 2013
able expression of Nppb (Fig. 2B). The mice
were healthy, had normal numbers of nocicep-
tive, touch, and proprioceptive neurons, and the
distribution and number of dorsal horn inter-
S1). Nppb−/−mice retained normal responses to
thermal, nociceptive, touch, and proprioceptive
stimulation when tested with standard assays
(Fig. 2C). We performed intradermal injections
and recorded numbers of scratching bouts for
substances that directly activate pruriceptors and
with compound 48/80 that causes itch via an
indirect route (11). All of these agents (table S2)
reliably triggered multiple bouts of scratching in
control animals (Fig. 2D, gray bars), but Nppb−/−
mice were almost completely insensitive to the
full range of pruritic substances tested (Fig. 2D,
How does Nppb induce this stereotyped
scratch response? We reasoned that because this
peptide is prominently expressed in somatosen-
sory neurons, the most plausible explanation for
its role would be if it acted as a specific itch-
related neuromodulator (or neurotransmitter) in
the spinal cord. Indeed, intrathecal injection of
Nppb induced profound scratching behavior in
wild-type animals (Fig. 2E, gray bar), demon-
strating that spinal-Nppb is sufficient to induce
itch even without activation of the peripheral neu-
rons that express it. Intrathecal injection of Nppb
type (Fig. 2E, red bar). Loss of Nppb in sensory
afferents was thus responsible for the pruricep-
tive deficits seen in mutantmice. Therefore, we
suggest that Nppb-expression delineates the sub-
set of somatosensory neurons that detect pruritic
neurons is necessary for the itch response.
Because Nppb is responsible for transmitting
its receptor Npra (12) should be expressed at the
site of afferent fiber synaptic connections in the
spinal dorsal horn and mark the secondary neu-
rons in the itch-response circuit. Therefore, we
used ISH to localize Npra expression in the dor-
sal horn and found thatit is indeedexpressedin a
limited subset of neurons (most likely interneu-
rons), primarily in the outer layer—lamina I (Fig.
3A, left)—corresponding to the terminal field of
TRPV1-expressing sensory neurons (13).
To examine whether the Npra neurons in
the spinal cord function in the itch circuit and
whether they are selectively required for pruritogen-
induced scratching (rather than other somatosensory
responses), we used a targeted-toxin cell-ablation
strategy (14). We injected a Nppb-saporin con-
jugate intrathecally into wild-type mice so as to
target their Npra-expressing cells and assessed
the effectiveness, specificity, and behavioral conse-
quences of administering this toxin. Approximately
70% of Npra-positive neurons were eliminated
after administration of toxin (Fig. 3A and fig. S3).
This targeting was highly selective, with neither
cells expressing the GRP-receptor nor other dorsal
horn interneurons affected by Nppb-saporin treat-
ment (Fig. 3A and fig. S3). Toxin-injected mice
displayed normal responses to thermal, touch, and
painful stimulation (Fig. 3B). However, we ob-
served a dramatic reduction in scratching evoked
by histamine (Fig. 3C), indicating that these neu-
rons are required for itch responses but not for
other somatosensory pathways.
of itch behavior? The GRP-receptor has been
(5, 6), with the suggestion that GRP might be the
primary neurotransmitter for itch. However, this
view has also been questioned (11, 15). We were
unable to detect more than trace quantities of
GRP expression in the DRG using a sensitive
quantitative polymerase chain reaction (PCR) as-
say (Fig. 4A). Similarly, somatosensory neurons
negative for enhanced green fluorescent protein
(EGFP) expression (fig. S4A). In contrast, ISH
for GRP and analysis of EGFP expression in the
GRP-reporter mice revealed that this neuropep-
tide is present in a population of neurons in the
dorsal horn (Fig. 4B and fig. S4A), as reported
previously (15, 16). Therefore, we concluded that
GRP cannot act at the level of pruriception but
must function downstream of Nppb and so ap-
plied three complementary functional strategies
to substantiate this hypothesis and dissect the
First, we demonstrated that GRP-induced
scratching was unaltered either by knocking
out Nppb (Fig. 2E) or by the ablation of Npra-
expressing neurons (Fig. 4D). Second, we
showed that pharmacological inhibition of the
GRP-receptor not only attenuated behavioral re-
sponses to the pruritic agent histamine or GRP
injection (fig. S4B) but also inhibited scratching
after intrathecal administration of Nppb (Fig. 4E).
Last, we tested mice with selective ablation of
significantly reduced itch responses to Nppb (Fig.
4E). These data place GRP downstream of Nppb
in the itch response circuit and suggest that the
secondary pruriceptors are targets for one neu-
ropeptide, Nppb, and in turn signal through a sec-
ond peptide, GRP. Just as this model predicts, all
Npra-expressing neurons in the dorsal horn con-
tain GRP (Fig. 4F), and Nppb-saporin treatment
cells (Fig. 4B, right).
Our results molecularly characterize the first
(Fig. 4G), demonstrate that Nppb marks the pri-
is both necessary and sufficient for transmission
of Npra receptor neu-
rons in the spinal cord
impairs pruriception. (A)
Expression of Npra in the
dorsal spinal cord was as-
mal mice (top), a subset of
interneurons in the outer
layer express Npra; howev-
er, after intrathecaladmin-
istration of Nppb-saporin
(bottom), few Npra neu-
rons remained. In contrast,
prioceptive stimuli, but (C)
exhibit greatly attenuated
responses to intradermal
mg in 10 ml) or intrathecal
administration of Nppb.
controls T SEM (n ≥ 5
ences between genotypes
were determined by using
Student’s t test; *P < 0.01.
Normalizeed response (%)
Bouts of scratching
Bouts of scratching
24 MAY 2013 VOL 340
of peripheral signals that induce stereotypic itch
responses. Unlike previously characterized re-
ceptors (8, 9) and signaling molecules (1–3) that
affect the detection of particular itch-inducing
range of pruritogens (compounds classed as in-
ducing histamine- and nonhistamine-related itch)
(table S2). Our data also refine the role GRP and
way by placing them at later stages than had been
hypothesized previously (5, 6). Many questions
about itch remain unanswered, including its close
relationship to sensing pain (17) and its slow
human and nonhuman primate studies that
different central pathways mediate histamine and
nonhistamine itch (18–21).Because Nppb is crit-
ically required for pruriception in mice, future
Nppb-expressing somatosensory neurons togeth-
er with circuit tracing may reveal whether these
cells directly trigger a spinal scratch reflex, are
selective detectors for the sensation of itch,or are
in fact more broadly tuned nociceptors. Such ex-
periments will help reveal the central mechanism
(or mechanisms) for itch, explain the interactions
between pruriception and other somatosensory
signals, and ultimately provide a powerful stim-
ulus for the rational design of novel therapies to
alleviate chronic itch.
References and Notes
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6. Y. G. Sun et al., Science 325, 1531 (2009).
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Acknowledgments: We are grateful to N. Ryba for
encouragement and helpful advice and for valuable
suggestions. The Nppb−/−mice used for this research
project were generated from embryonic stem cells obtained
from the National Center for Research Resources
(NCRR)–NIH–supported KOMP repository (www.komp.org)
and engineered by the Wellcome Trust Sanger Institute
and the Mouse Biology Program (www.mousebiology.org).
We thank NIDCR Gene Targeting Facility for help
generating Nppb mutant mice. The mouse strain STOCK
Tg(GRP-EGFP) was obtained from the Mutant Mouse Regional
Resource Center (MMRRC), a NCRR-NIH–funded strain
repository, and was donated to the MMRRC by the National
Institute of Neurological Disorders and Stroke–funded
GENSAT BAC transgenic project. This research was
supported by the intramural research program of the
Materials and Methods
Figs. S1 to S4
Tables S1 and S2
Movies S1 to S3
7 December 2012; accepted 13 March 2013
Untreated GRP injection
Bouts of scatching
Dorsal horn of spinal cord Skin
Fig. 4. GRPactsdirectlydownstreamofNppbintherodentpruriceptive
circuit.(A) Quantitative PCR was used to quantitate expression of GRP and
Nppb relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in
the DRG and spinal cord. GRP is robustly expressed in the spinal cord (at a
level comparable with Tac1) but is almost undetectable in theDRG.Nppb is
prominently expressed in DRG but is not present in the spinal cord. Data
represent mean T SEM for triplicate cDNA preparations each analyzed in two
separate PCR reactions. (B) ISH was used to analyze GRP expression in the
expressing interneurons are lost on ablation of Npra-expressing cells. (C) A
significant number of GRP neurons was eliminated after Nppb-saporin (Nppb-
sap) treatment. GRP-saporin (GRP-sap), which targets GRP-receptor neurons,
has no effect on the number of GRP interneurons; data represent mean T SEM
(n ≥ 4 animals). Significant differences between groups were determined by
using Student’s t test; *P < 0.001. (D) Ablation of Npra neurons had no effect
on intrathecally administered GRP-induced scratch responses. (E) Scratching
induced by means of lumbar injection of Nppb was strongly attenuated by
pretreatment with a selective GRP antagonist or by the ablation of GRPR-
6 animals); *P < 0.001 (Student’s t test). (F) Double-label immunohistochem-
istry was used to localize interneurons expressing Npra (green) and GRP-
driven EGFP (red) in sections through the dorsal horn of Tg(GRP-EGFP) mice.
(Left) Typical individual staining patterns. (Right) Merged Npra and GRP
dorsal horn is dotted. (G) Model of the first three stages of the pruriceptive
circuit, with the critical neuropeptide used at each stage indicated.
VOL 34024 MAY 2013