Visceral hyperalgesia caused by peptide YY deletion and Y2 receptor antagonism OPEN

Abstract

Altered levels of colonic peptide YY (PYY) have been reported in patients suffering from functional and inflammatory bowel disorders. While the involvement of neuropeptide Y (NPY) and Y receptors in the regulation of nociception is well established, the physiological role of PYY in somatic and visceral pain is poorly understood. In this work, the role of PYY in pain sensitivity was evaluated using PYY knockout (PYY(−/−)) mice and Y2 receptor ligands. PYY(−/−) mice were more sensitive to somatic thermal pain compared to wild type (WT) mice. Visceral pain was assessed by evaluating pain-related behaviors, mouse grimace scale (MGS) and referred hyperalgesia after intrarectal administration of allyl isothiocyanate (AITC, 1 or 2%) or its vehicle, peanut oil. The pain-related behaviors induced by AITC were significantly exaggerated by PYY deletion, whereas the MGS readout and the referred hyperalgesia were not significantly affected. The Y2 receptor antagonist, BII0246, increased pain-related behaviors in response to intrarectal AITC compared to vehicle treatment while the Y2 receptor agonist, PYY(3–36), did not have a significant effect. These results indicate that endogenous PYY has a hypoalgesic effect on somatic thermal and visceral chemical pain. The effect on visceral pain seems to be mediated by peripheral Y2 receptors.

Full text

1
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
www.nature.com/scientificreports
Visceral hyperalgesia caused by
peptide YY deletion and Y2 receptor
antagonism
Ahmed M. Hassan1,*, Piyush Jain1,*, Raphaela Mayerhofer1, Esther E. Fröhlich1, Aitak Farzi1,
Florian Reichmann1, Herbert Herzog2 & Peter Holzer1
Altered levels of colonic peptide YY (PYY) have been reported in patients suering from functional
and inammatory bowel disorders. While the involvement of neuropeptide Y (NPY) and Y receptors in
the regulation of nociception is well established, the physiological role of PYY in somatic and visceral
pain is poorly understood. In this work, the role of PYY in pain sensitivity was evaluated using PYY
knockout (PYY(−/−)) mice and Y2 receptor ligands. PYY(−/−) mice were more sensitive to somatic
thermal pain compared to wild type (WT) mice. Visceral pain was assessed by evaluating pain-related
behaviors, mouse grimace scale (MGS) and referred hyperalgesia after intrarectal administration of
allyl isothiocyanate (AITC, 1 or 2%) or its vehicle, peanut oil. The pain-related behaviors induced by
AITC were signicantly exaggerated by PYY deletion, whereas the MGS readout and the referred
hyperalgesia were not signicantly aected. The Y2 receptor antagonist, BII0246, increased pain-
related behaviors in response to intrarectal AITC compared to vehicle treatment while the Y2 receptor
agonist, PYY(3–36), did not have a signicant eect. These results indicate that endogenous PYY has a
hypoalgesic eect on somatic thermal and visceral chemical pain. The eect on visceral pain seems to be
mediated by peripheral Y2 receptors.
e gut hormone peptide YY (PYY) is a member of the neuropeptide Y (NPY) family which also includes pan-
creatic polypeptide. PYY release is regulated by food intake, the parasympathetic nervous system, inammatory
mediators, and other gut hormones including cholecystokinin, vasoactive intestinal polypeptide, gastrin, and
glucagon-like peptide-11–4. PYY is secreted by L-cells of the gastrointestinal tract (GIT) which release it in the
form of PYY(1–36). e amino acids tyrosine and proline are cleaved from the N-terminus of the polypeptide by
the dipeptidyl peptidase IV (DDP-IV) enzyme to produce PYY(3–36). is changes the pharmacological proper-
ties of the peptide. While PYY(1–36) binds to all Y receptor subtypes, PYY(3–36) has a higher anity to the Y2
subtype3. e two peptides have endocrine actions in the GIT where they act as brakes to inhibit gastric emptying,
intestinal motility, mouth to anus transit time, and electrolyte secretion in the intestine1,2.
In addition to regulation of GIT functions and food intake, the NPY system is involved in the regulation of
several physiological functions including stress coping, emotional-aective behavior, cognition, neurogenesis,
immunity, and nociception5. Knockout of Y1 receptors is associated with thermal, chemical, and mechanical
somatic hyperalgesia and exaggerated acetic acid- and MgSO4 -induced visceral pain5,6. Electrophysiological
studies conrm a role of spinal Y1 and Y2 receptors in controlling postsynaptic currents in pain signaling path-
ways7,8. NPY acting through Y1 receptors controls the production of several neuropeptides including substance P
and calcitonin gene-related peptide (CGRP), which subserve pronociceptive functions in the CNS9. In addition to
their eect on spinal pain pathways, Y receptor ligands aect visceral pain mediated by vagal aerent pathways, as
c-Fos expression in the nucleus tractus solitarii (NTS) following intragastric acid challenge is enhanced in Y2 and
Y4 receptor knockout mice10. Furthermore, Y1 receptors are involved in the descending control of pain signaling.
For example, NPY injection into the arcuate nucleus reduces somatic pain in rats, an eect that is blocked by Y1
but not Y2 receptor antagonists11. In spite of the well-established role of NPY in the regulation of pain sensitivity,
the role of the gut hormone PYY in nociception is poorly understood.
1Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology,
Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria. 2Neurobiology Research Program, Garvan
Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia. *These authors
contributed equally to this work. Correspondence and requests for materials should be addressed to P.H. (email:
peter.holzer@medunigraz.at)
Received: 13 September 2016
Accepted: 13 December 2016
Published: 20 January 2017
OPEN

www.nature.com/scientificreports/
2
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
Expression of colonic PYY is altered in patients suering from functional and inammatory bowel disorders.
e density of PYY-containing cells in the colon is lowered in patients with irritable bowel syndrome (IBS)12,13
and Crohn’s disease (CD)14,15. Similarly, patients suering from ulcerative colitis (UC) have lower rectal PYY
levels compared to healthy controls16. Given that IBS and inammatory bowel disease (IBD) are associated with
pain, we hypothesized that a deciency in PYY contributes to the hyperalgesia associated with these pathologies.
To examine a possible role of PYY in nociception, we rst investigated somatic and visceral pain percep-
tion in PYY knockout (PYY(−/−)) mice. Somatic sensitivity to thermal pain was tested with the plantar test17
while visceral sensitivity to chemical pain was quantied by the pain reactions to intrarectal allyl isothiocyanate
(AITC)18,19. e visceral pain readouts included spontaneous pain-related behaviors, locomotor activity, facial
pain expressions, and referred hyperalgesia18–20. Concomitantly, the expression of NPY, Y1, and Y2 receptor
mRNA in the spinal cord of wild type (WT) and PYY(−/−) mice was assessed. Since PYY(3–36), the main circu-
latory form of PYY, has a preferential anity to Y2 receptors3, the eects of the Y2 receptor agonist, PYY(3–36),
and the Y2 receptor antagonist, BIIE0246, on visceral pain sensitivity were also evaluated.
Results
PYY(−/−) mice are hypersensitive to thermal pain. Compared to WT mice, PYY(−/−) mice were more
sensitive to thermal pain as indicated by a signicantly shorter withdrawal latency in the plantar test (t19.5 = 3.3;
p = 0.004) (Fig.1).
PYY knockout exaggerates pain-related behaviors induced by intrarectal AITC. Following
intrarectal instillation of PO or AITC (2%), one-way ANOVA revealed statistically signicant dierences in
pain-related behaviors (F(3,24) = 4.6; p = 0.011) and time spent freezing (F(3,24) = 4.6; p = 0.003) while the dif-
ferences in the latency to rst pain-related behavior and time spent grooming were not statistically signicant
(Fig.2). Post-hoc testing revealed that pain-related behaviors (stretching, squashing, arching and licking of the
abdomen) were enhanced in PYY(−/−) mice that received AITC (PYY(−/−) + AITC group) compared to WT mice
that received PO (WT + PO group) and WT mice that received AITC (WT + AITC group) (Fig.2A). e time
spent freezing was prolonged in both the WT + AITC and the PYY(−/−) + AITC groups relative to the WT + PO
group (Fig.2B). None of the parameters recorded in the LabMaster system (horizontal activity, vertical activity,
traveling distance) during the rst 15 min aer intrarectal instillation of PO or AITC were signicantly dierent
between the experimental groups (Supplement;FigureS1).
PYY knockout does not alter AITC-induced facial pain expressions. Following intrarectal instil-
lation of PO or AITC (2%), one-way ANOVA revealed statistically signicant dierences in Δ MGS among the
experimental groups ((F(3,23) = 53.1; p < 0.001) (Fig.3). Post hoc testing revealed that the Δ MGS in the groups
which received AITC was signicantly higher than in the groups that received PO while other dierences between
the groups were statistically not signicant (Fig.3).
PYY knockout does not alter AITC-induced referred hyperalgesia. e mechanical pain threshold
(MPT) on the plantar surface of the hind paws was measured with von Frey laments in WT and PYY(−/−) mice
before and aer intrarectal treatment with PO or AITC (1%). e baseline value reects somatic sensitivity to
mechanical pain while the dierence between the pre- and post-treatment measurements was taken as index
of referred hyperalgesia which accompanies visceral pain. One-way ANOVA failed to disclose any statistically
signicant dierences in MPT among the experimental groups both under baseline conditions and aer intra-
rectal administration of PO and AITC (Fig.4A,B). A comparison of baseline and post-treatment recordings
Figure 1. Eect of PYY knockout (PYY(−/−)) on withdrawal latency in the plantar test. e data shown are
means + SEM, n = 11 per group; **p < 0.01.

www.nature.com/scientificreports/
3
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
with the paired t test revealed, however, that in the WT + PO group the MPT aer instillation of PO was signif-
icantly higher than at baseline. In contrast, Δ MPT was signicantly dierent among the experimental groups
(F(3,20) = 3.3; p = 0.041). Post-hoc testing showed that the Δ MPT was signicantly lower in the PYY(−/−) + AITC
compared to the WT + PO group but did not signicantly dier compared to the WT + AITC group (Fig.4C).
MPT measurements were also taken on the abdomen. e quantitative MPT and Δ MPT readouts over the
abdomen showed a similar but highly variable pattern, and no signicant dierences could be detected among the
experimental groups (Supplement,FigureS2).
AITC increases spinal NPY and Y1 receptor mRNA expression in PYY(−/−) but not WT mice. e
eect of intrarectal administration of PO and AITC (1%, 0.05 ml) on the expression of NPY as well as Y1 and
Y2 receptor mRNA in the lumbosacral spinal cord was examined in WT and PYY(−/−) mice. One-way ANOVA
revealed significant differences among the experimental groups with regard to NPY mRNA (F(3,18) = 3.3;
p = 0.045) (Fig.5A) and Y1 receptor mRNA (F(3,18) = 3.7; p = 0.032) expression (Fig.5B) while Y2 receptor mRNA
expression did not dier to a statistically signicant extent (Fig.5C). Post-hoc testing revealed that NPY mRNA
Figure 2. Eect of intrarectally administered AITC (2%, 0.1 ml) and PYY knockout (PYY(−/−)) on pain-related
behaviors (A), time spent freezing (B), latency to rst pain-related behavior (C), and time spent grooming (D).
e behavioral parameters were evaluated blindly from 15 min videos recorded immediately aer intrarectal
treatment. One-way ANOVA revealed statistically signicant dierences among the groups with regard to pain-
related behaviors (p < 0.05) and the time spent freezing (p < 0.01). e data shown are means + SEM, n = 6–8
per group; *p < 0.05, **p < 0.01 compared to WT + PO; #p < 0.05 compared to WT + AITC.

www.nature.com/scientificreports/
4
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
and Y1 mRNA expression in the PYY(−/−) + AITC group was signicantly higher than in the WT + PO group
(Fig.5A,B).
Y2 receptor antagonism increases pain-related behaviors induced by intrarectal AITC. In this
experiment, the eects of the Y2 receptor agonist PYY(3–36) (0.2 mg/kg) and the Y2 receptor antagonist BII0246
(0.03 mmol/kg) on visceral pain sensitivity were evaluated in conjunction with intrarectal administration of AITC
(2%) to C57BL/6 N mice. e results revealed that the Y2 receptor antagonist increased pain-related behaviors
induced by intrarectal AITC while the Y2 receptor agonist had no eect.
As depicted in Fig.6, one-way ANOVA with Welch’s correction disclosed statistically signicant dierences
among the groups with regard to pain-related behaviors (Welch’s F(6,21.5) = 9.4; p < 0.001), time spent freezing
(Welch’s F(6,19.1) = 26.1; p < 0.001), latency to rst pain-related behavior (Welch’s F(6,18.2) = 15.6; p < 0.001), and
time spent grooming (Welch’s F(6,18.7) = 10.9; p < 0.001).
Post-hoc testing failed to reveal any signicant dierences in pain-related behavior counts between the vehi-
cle + PO group and vehicle + AITC group. Signicantly higher pain-related behavior counts were recorded in the
BIIE0246 + AITC and BIIE0246 + PYY(3–36) + AITC groups compared to the vehicle + PO and BIIE0246 + PO
groups. e BIIE0246 + AITC group also exhibited signicantly higher pain-related behavior counts than the
vehicle + AITC group (Fig.6A). e time spent freezing was signicantly higher in all groups receiving AITC
compared to PO, but no signicant dierences were observed within the groups receiving PO or within the groups
receiving AITC (Fig.6B). Similarly, the time spent grooming and the latency to the rst pain-related behavior
were shorter in all groups receiving AITC than in the PO group, while no signicant dierences were observed
within groups receiving PO or within groups receiving AITC (Fig.6C,D). The parameters recorded by the
LabMaster system (horizontal activity, vertical activity, traveling distance) measured for 15 min aer intrarectal
injection of PO or AITC were not signicantly dierent among the experimental groups (Supplement,FigureS3).
Discussion
Although the role of Y receptors in nociception is well established9, relatively little is known about the specic
implication of PYY in pain regulation. In this work, we examined the eects of genetic PYY deletion and Y2
receptor ligands on somatic sensitivity to thermal and mechanical pain and visceral sensitivity to chemical pain.
To probe chemonociception in the rectum we used AITC which stimulates transient receptor potential ankyrin 1
(TRPA1) and transient receptor potential vanilloid 1 (TRPV1) ion channels expressed by nociceptive aerent
nerve bers21,22. On the somatic level, PYY(−/−) mice were found to be hypersensitive to thermal somatic pain. On
the visceral level, PYY knockout exaggerated pain-related behaviors in response to intrarectal AITC. In conrma-
tion of these results, the Y2 receptor antagonist BII0246 increased pain-related behaviors in response to intrarec-
tal AITC. ese ndings attest to an involvement of PYY in the regulation of somatic and visceral pain sensitivity.
To study visceral pain and hyperalgesia by a minimally invasive method that does not require surgery, we
took advantage of the models of Laird et al.18 and Langford et al.20 and the recording of locomotor activity19 to
gauge pain by a multitude of behavioral readouts considered to reect several dimensions of pain experience. By
recording several behavioral manifestations of visceral pain (stretching, squashing, arching, licking of the abdo-
men)18 we found that intrarectal AITC evoked pain-related behaviors only in PYY(−/−) mice but not in WT mice.
It appears as if PYY knockout enhances the susceptibility of the mice to respond to the stress and mechanical
Figure 3. Eect of intrarectally administered AITC (1%, 0.05 ml) and PYY knockout (PYY(−/−)) on the mouse
grimace scale (Δ MGS) expressed as action units. One-way ANOVA revealed statistically signicant dierences
among the groups (p < 0.001). e data shown are means + SEM, n = 6–7 per group; ***p < 0.001 compared to
WT + PO; 𝛿𝛿𝛿p < 0.001 compared to PYY(−/−) + PO.

www.nature.com/scientificreports/
5
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
irritation associated with the instillation procedure with pain-related behaviors. is phenotype is consistent with
impaired stress coping and enhanced depression-like behavior seen in PYY(−/−) mice23. e moderate behavioral
response of the WT mice to intrarectal AITC was untypical when compared with the more pronounced response
observed in C57BL/6 mice19,24. Although the reason for this discrepancy is not understood, we think of a strain
eect, given that the genetic background of the WT animals is dierent from that of C57BL/6 mice.
e MGS described by Langford et al.20 is a novel method to assess the pain experience in response to sev-
eral noxious stimuli. In addition, recording of the MGS does not interfere with the evaluation of pain-related
behaviors or referred hyperalgesia. On the basis of our study we contend that a combination of several behavioral
methods to assess visceral pain, including facial pain expression, a variety of pain-related behaviors and referred
hyperalgesia, addresses several dimensions of nociception and has the potential to overcome strain-related dif-
ferences in the nociceptive responses to noxious stimuli. Although, for instance, the WT mice used here did not
show the typical pain-related behaviors described by Laird et al.18 in response to intrarectal AITC, they exhibited
a very prominent MGS response: the Δ MGS was more than 20 fold larger in response to AITC than aer admin-
istration of PO. On the other hand, the eect of PYY knockout on MGS was not statistically signicant.
A particular dimension of the visceral pain experience manifests itself in the phenomenon of referred
pain. We used a decrease in the MPT of the hindpaws as an index of referred hyperalgesia and found that the
Figure 4. Mechanical pain threshold (MPT) of the plantar surface of the hindpaws in WT and PYY knockout
(PYY(−/−)) mice before (baseline, A) and aer (post-treatment, B) intrarectal administration of PO or AITC
(1%, 0.05 ml) and the dierence between the two measurements (Δ MPT, C). MPT was assessed with von Frey
hairs, and the values represent the average of measurements in both hind paws. With regard to Δ MPT, one-
way ANOVA revealed statistically signicant dierences among the groups (p < 0.05). e data shown are
means + or − SEM, n = 5–7 per group; *p < 0.05 compared to WT + PO.
Figure 5. Eect of intrarectal AITC (1%) on the relative expression of NPY (A), Y1 receptor (B), and Y2
receptor (C) mRNA in the lumbosacral spinal cord. Tissue was collected 1 h aer intrarectal administration
of PO or AITC. Relative mRNA expression was assessed with real time PCR. One-way ANOVA revealed
statistically signicant dierences among the groups with regard to NPY and Y1 receptor but not Y2 receptor
mRNA expression. e data shown are means + SEM, n = 5–6 per group; *p < 0.05 compared to WT + PO.

www.nature.com/scientificreports/
6
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
AITC-induced reduction of the MPT was most pronounced in PYY(−/−) mice. It must not be overlooked, how-
ever, that the AITC-induced Δ MPT in PYY(−/−) mice was only nominally, but not signicantly, dierent from the
AITC-induced Δ MPT in WT mice. e absence of a signicant AITC-evoked referred hyperalgesia in WT mice
is consistent with the relative resistance of this genotype towards pain-related behaviors induced by AITC. It is
worth noting, however, that in WT but not PYY(−/−) mice administration of the vehicle (PO) increased the MPT
of the hindpaws. is rise of the MPT may be interpreted as stress-induced analgesia which is known to be strain
dependent25,26. If so, the MPT changes in the dierent experimental groups may represent the composite result of
stress-induced analgesia and referred hyperalgesia evoked by intrarectal irritation.
Since PYY(3–36), the circulatory form of PYY, has a higher anity for Y2 receptors than PYY itself5, we exam-
ined the eects of this Y2 receptor agonist (0.2 mg/kg) and the Y2 receptor antagonist BIIE0246 (0.03 mmol/kg)
Figure 6. Eects of subcutaneously injected BIIE0246 (0.03 mmol/kg) and intraperitoneally injected PYY
(3–36) (0.2 mg/kg) on pain evoked by intrarectally administered PO or AITC (2%, 0.05 ml) in C57BL/6 N
mice. Shown are pain-related behaviors (A), time spent freezing (B), latency to rst pain-related behavior
(C), and time spent grooming (D). e behavioral parameters were evaluated blindly from 15-min videos
recorded immediately aer intrarectal treatment. One-way ANOVA with Welch’s correction revealed signicant
dierences among the groups. e data shown are means + SEM, n = 6 for PO groups and 9–12 for AITC
groups; *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle + PO, #p < 0.05 compared to vehicle + AITC.

www.nature.com/scientificreports/
7
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
on pain-related behaviors induced by AITC (2%) in C57BL/6 N mice. e experiments revealed that Y2 recep-
tor antagonism led to visceral hyperalgesia in response to intrarectal AITC, while Y2 receptor agonism had no
eect. Specically, PYY(3–36) failed to attenuate pain-related behaviors and to attenuate the eect of BII0246
to exaggerate AITC-induced pain-related behaviors. e inecacy of PYY(3–36) could be due to pharmacody-
namic reasons, pharmacokinetic factors and/or particular experimental conditions. First, the simplest explana-
tion would be to assume that dosing of PYY(3–36) was inadequate. It should be noted, however, that the dose
used here (0.2 mg/kg) is considered to be a high dose, given that doses as low as 0.01 mg/kg are sucient to
enhance c-Fos expression in the brain and to induce several behavioral changes in mice27,28. Second, the failure of
PYY(3–36) to antagonize the hyperalgesic eect of BII0246 may be due to dierent pharmacokinetic proles of
the two compounds, PYY(3–36) being unable to achieve eective concentrations at the site where BII0246 exerts
its hyperalgesic action. ird, it should not go unnoticed that a failure of PYY(3–36) to induce expected pharma-
cological eects is a common observation in the literature29. e inconsistent ecacy of PYY(3–36) in vivo has
been attributed to the stress in handling the animals, its poor pharmacokinetic prole in rodents, or concomitant
activation of Y1 and Y5 receptors, as PYY(3–36) is not totally receptor-selective30,31.
e ndings of this study that PYY knockout and Y2 receptor antagonism enhance particular aspects of
AITC-evoked visceral pain behavior raise the question as to the site in the pain pathway where PYY and Y2
receptors operate. Y1 and Y2 receptors are widely expressed in the primary somatic and visceral pain pathways,
including the skin and colon, the inferior ganglion of the vagus nerve, the dorsal root ganglia, and the dorsal horn
of the spinal cord9,32–35. An implication of the NPY system in AITC-evoked rectal pain can be deduced from the
increased expression of NPY and Y1 receptor mRNA in the spinal cord of PYY(−/−) mice. e functional impli-
cation of these ndings is not clear. However, given that NPY and Y1 receptor signaling in the spinal cord have
an analgesic function8, it could be contended that the increase in spinal NPY and Y1 receptor mRNA expression
in PYY(−/−) mice reects a counterregulatory mechanism to balance the hyperalgesic state in this genotype. Due
to the widespread distribution of multiple Y receptors in the peripheral and central pain pathways it is dicult
to hypothesize on the sites where PYY could inuence visceral and somatic pain, given that PYY can cross the
blood brain barrier36. e observation that both somatic and visceral pain sensitivity was increased in PYY(−/−)
mice is compatible with both a peripheral and central site of action. In contrast, BII0246 acts primarily by block-
ing peripheral Y2 receptors37, which suggests that peripheral Y2 receptors play a role in controlling visceral pain
signaling. is contention is in keeping with the expression of Y2 receptors by vagal and spinal primary sensory
neurons in rodents and rabbits9,32,38.
In conclusion, PYY knockout and Y2 receptor antagonism have a hyperalgesic eect in mice, exaggerating
particular behavioral aspects of both somatic and visceral pain. Circumstantial evidence indicates that the eect
on visceral pain is mediated by peripheral Y2 receptors. Our experimental observations in mice may have a trans-
lational value in view of the deciency of PYY reported to occur in the colon of IBS and IBD patients12–15. e
results of the current work suggest that the decreased PYY content in the lower GIT of IBS and IBD patients may
contribute to the visceral pain associated with these pathologies.
Methods
Experimental animals. PYY(−/−) mice were generated by removing the entire coding sequence including
the initiation start as reported previously39. e genetic background of the knockout as well as WT mice was a 1:1
mixture of C57Bl/6 and129/SvJ. e PYY(−/−) mice and the age-matched WT mice were 2–4 months old when
used in the plantar test and in the assessment of spontaneous pain-related behaviors and 3–9 months old when
MGS and referred pain were assessed in parallel. ree dierent lots of mice were used for the plantar test, the
assessment of spontaneous pain-related behaviors, and the combined assessment of MGS and referred pain. e
animals subjected to the MGS and referred pain measurements were also used to assess the expression of NPY, Y1
receptor and Y2 receptor expression in the spinal cord. e total number of WT and PYY(−/−) mice employed in
this study amounted to 77 male mice.
e eects of BII0246 and PYY(3–36) on pain behavior were evaluated in a total of 59 male C57BL/6 N mice
obtained from Charles River (Sulzfeld, Germany). e C57BL/6 N mice arrived in the institutional animal house
at 8 weeks of age and were housed 2–3 per cage. ey were habituated for 2 weeks before they were subjected to
the experiments.
Ethical statement. All experiments were approved by an ethical committee at the Federal Ministry of
Science, Research, and Economy of the Republic of Austria (BMWFW-66.010/0054-WF/II/3b/2014) and con-
ducted according to the Directive of the European Communities Council of 24 November 1986 (86/609/EEC)
and the Directive of the European Parliament and of the Council of 22 September 2010 (2010/63/EU).
Intrarectal administration. Chemically induced nociception in the colon was induced by intrarectal AITC
administered through a Teon feeding cannula (length 38.1 mm, gauge 20) (Scanbur, Karlslunde, Denmark).
Vaseline (Rösch & Handel, Vienna, Austria) was applied to the perianal region and the tip of the cannula to
reduce any discomfort induced by the insertion of the cannula. AITC (1% (v/v)) in a volume of 0.05 ml in the
MGS and referred pain experiment, 2% in a volume of 0.1 ml in assessment of pain related behavior in WT and
PYY(−/−) mice and 2% in a volume of 0.05 ml in assessment of pain related behaviors in C57BL/6 N mice or its
vehicle, PO, was injected intrarectally through the feeding cannula18,19,21. e concentration (1%) and volume
(0.05 ml) of AITC used in the MGS and referred pain experiment was chosen to avoid any ceiling eect as noted
with higher dosing in pilot trials.
Drug treatment. PYY(3–36) lyophilized powder (Sigma-Aldrich, Vienna, Austria) was dissolved in saline
(0.9% NaCl) containing 1% bovine serum albumin. Aliquots were stored at − 70 °C in polypropylene tubes and

www.nature.com/scientificreports/
8
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
thawed on the day of experiment. e peptide was injected intraperitoneally at the dose of 0.2 mg/kg (volume:
0.05 ml/10 g body weight). is dose of PYY(3–36) is sucient to induce several behavioral changes in mice27.
BIIE0246 (Tocris, Bristol, UK) was dissolved in 30% polyethylene glycol 200 in distilled water, prepared freshly
on the day of the experiment and injected subcutaneously (SC) at the dose of 0.03 mmol/kg (volume: 0.05 ml/10 g
body weight). is dose of BIIE0246 was used previously to evaluate the role of Y2 receptors in vagal aerent
signaling10. To assess the eects of the Y2 receptor agonist PYY(3–36) and the Y2 receptor antagonist BIIE0246,
7 groups of mice were used. e time line of the experiments was such that, rst, the mice received a SC injection
of BIIE0246 or its vehicle, 5 min later IP PYY(3–36) or its vehicle, and aer a further 5 min PO or 2% AITC intra-
rectally. e 7 experimental groups were as follows:
(1) SC vehicle, IP vehicle, PO intrarectally (vehicle + PO)
(2) SC vehicle, IP vehicle, 2% AITC intrarectally (vehicle + AITC)
(3) SC vehicle, IP PYY(3–36), PO intrarectally (PYY(3–36) + PO)
(4) SC vehicle, IP PYY(3–36), 2% AITC intrarectally (PYY(3–36) + AITC)
(5) SC BIIE0246, IP vehicle, PO intrarectally (BIIE0246 + PO)
(6) SC BIIE0246, IP vehicle, 2% AITC intrarectally (BIIE0246 + AITC)
(7) SC BIIE0246, IP PYY(3–36), 2% AITC intrarectally (BIIE0246 + PYY(3–36) + AITC)
After intrarectal administration of 2% AITC, pain-related behaviors and locomotion were assessed as
described below.
Assessment of pain-related behaviors. Aer intrarectal administration of AITC or vehicle, the behav-
ior of mice was video-recorded in the LabMaster system (TSE Systems, Bad Homburg, Germany) for 15 min. A
blinded trained investigator used the event marker module of the Videomot 2 tracking soware (TSE Systems) to
evaluate the video-recordings with regard to the following pain-related behaviors18,19,24: (a) stretching of the trunk
and squashing (pressing the abdomen towards the oor of the cage and stretching of the body), (b) licking of the
lower part of the abdomen, and (c) arching of the trunk.
e time spent freezing and grooming was also calculated from the video-recordings. Horizontal and vertical
locomotor activity was measured simultaneously with the LabMaster system19. Details about the LabMaster sys-
tem are available in the supplement.
The mouse grimace scale (MGS). Facial pain expression was evaluated with the MGS as described
previously20,40 with a slight modification that allowed for the simultaneous assessment of referred hyper-
algesia. Mice were kept on a wire mesh (Ugo Basile) in a homemade plexiglass box (9 cm × 5 cm × 5 cm;
length × height × width). All sides of the box except one (9 cm × 5 cm) were covered with white paper. Mice
were le to habituate in the box for 30 min. ereaer a 20-min video recording under baseline conditions was
taken with a Canon Legira HF R406 video camera. Aer taking the video, MPT was measured as described
later. Aerwards the mice received 1% AITC or the vehicle (PO) intrarectally as described above, followed by
another 20-min video recording for the assessment of the MGS and a second measurement of MPT. e MGS was
assessed as described in the supplement. e Δ MGS value was calculated by subtracting the baseline MGS from
post-treatment MGS and used as an index of visceral pain. One hour aer intrarectal treatment the mice were
sacriced by decapitation aer they had been deeply anesthetized with pentobarbital (150 mg/kg IP). e lum-
bosacral spinal cords were collected, shock frozen in liquid nitrogen and stored at − 70 °C until RNA extraction.
Assessment of mechanical pain threshold (MPT) and referred pain. Referred pain18,24 was evalu-
ated in parallel with the MGS assessment and determined by two measurements of MPT over the plantar surface
of the hind paws and abdomen. e rst measurement of MPT was made under baseline conditions aer the
baseline MGS video had been taken. e second measurement of MPT was taken 20 min aer intrarectal admin-
istration of PO or 1% AITC when the video-recording of the post-treatment MGS had been completed. e MPT
was evaluated with von Frey laments (Bioseb, Vitrolles, France) using the simplied up-down method (SUDO
method)41 as described in the supplement. e Δ MPT value was calculated by subtracting the baseline MPT from
the post-treatment MPT. A reduction of Δ MPT was considered as an index of referred pain.
Real time PCR. NPY, Y1 receptor and Y2 receptor mRNA expression in the lumbosacral spinal cord
was assessed by real-time PCR. RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden,
Germany). Reverse-transcription was performed with the High Capacity cDNA Reverse Transcription kit as
described by the manufacturer (Applied Biosystems, Foster City, CA, USA). e PCR conditions and the primer
sequences are described in the supplement. GAPDH and PGK were used as reference genes. Quantitative values
of mRNA relative to control were calculated with the 2−ΔΔCT method42.
Statistical analysis. SPSS 22 and SigmaPlot 13 were used for statistical analysis and graphic presentation of
the results. e data were analyzed with two-sample t-test, paired t-test, or one-way ANOVA, as appropriate. Post
hoc pairwise comparisons with Tukey’s test were made when one-way ANOVA revealed signicant dierences
among the groups. Log transformation was considered whenever needed to meet one-way ANOVA assumptions.
When log transformation was not sucient to meet the equal variability assumption of one-way ANOVA as
tested by Levene’s test, the Welch correction and post-hoc Games-Howell test were used. Dierences between
groups in t test, one-way ANOVA, and post-hoc tests were considered signicant if p ≤ 0.05. All data are pre-
sented as means + SEM, n referring to the number of mice in each group.

www.nature.com/scientificreports/
9
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
References
1. Ballantyne, G. H. Peptide YY(1–36) and peptide YY(3–36): Part I. Distribution, release and actions. Obes. Surg. 16, 651–658 (2006).
2. Cox, H. M. Peptide YY: a neuroendocrine neighbor of note. Peptides 28, 345–351 (2007).
3. Neary, M. T. & Batterham, . L. Peptide YY: food for thought. Physiol. Behav. 97, 616–619 (2009).
4. Cosun, T. et al. Activation of prostaglandin E receptor 4 triggers secretion of gut hormone peptides GLP-1, GLP-2, and PYY.
Endocrinology 154, 45–53 (2013).
5. Holzer, P., eichmann, F. & Farzi, A. Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut–brain axis. Neuropeptides
(2012).
6. Naveilhan, P. et al. educed antinociception and plasma extravasation in mice lacing a neuropeptide Y receptor. Nature 409,
513–517 (2001).
7. Moran, T. D., Colmers, W. F. & Smith, P. A. Opioid-lie actions of neuropeptide Y in rat substantia gelatinosa: Y1 suppression of
inhibition and Y2 suppression of excitation. J. Neurophysiol. 92, 3266–3275 (2004).
8. Smith, P. A., Moran, T. D., Abdulla, F., Tumber, . . & Taylor, B. . Spinal mechanisms of NPY analgesia. Peptides 28, 464–474
(2007).
9. Brumovsy, P., Shi, T. S., Landry, M., Villar, M. J. & Hofelt, T. Neuropeptide tyrosine and pain. Trends Pharmacol. Sci. 28, 93–102
(2007).
10. Wultsch, T. et al. Endogenous neuropeptide Y depresses the aerent signaling of gastric acid challenge to the mouse brainstem via
neuropeptide Y type Y2 and Y4 receptors. Neuroscience 136, 1097–1107 (2005).
11. Li, J. J., Zhou, X. & Yu, L. C. Involvement of neuropeptide Y and Y1 receptor in antinociception in the arcuate nucleus of
hypothalamus, an immunohistochemical and pharmacological study in intact rats and rats with inammation. Pain 118, 232–242
(2005).
12. El-Salhy, M., Gundersen, D., Hatleba, J. G., Gilja, O. H. & Hausen, T. Abnormal rectal endocrine cells in patients with irritable
bowel syndrome. egul. Pept. 188, 60–65 (2014).
13. El-Salhy, M., Hatleba, J. G., Gilja, O. H. & Hausen, T. Densities of rectal peptide YY and somatostatin cells as biomarers for the
diagnosis of irritable bowel syndrome. Peptides 67, 12–19 (2015).
14. El-Salhy, M., Danielsson, A., Stenling, . & Grimelius, L. Colonic endocrine cells in inammatory bowel disease. J. Intern. Med. 242,
413–419 (1997).
15. El-Salhy, M., Suhr, O. & Danielsson, A. Peptide YY in gastrointestinal disorders. Peptides 23, 397–402 (2002).
16. Tari, A. et al. Peptide YY abnormalities in patients with ulcerative colitis. Jpn. J. Med. 27, 49–55 (1988).
17. Allen, J. W. & Yash, T. L. Assessment of acute thermal nociception in laboratory animals. Methods Mol. Med. 99, 11–24 (2004).
18. Laird, J. M., Martinez-Caro, L., Garcia-Nicas, E. & Cervero, F. A new model of visceral pain and referred hyperalgesia in the mouse.
Pain 92, 335–342 (2001).
19. Jain, P. et al. Behavioral and molecular processing of visceral pain in the brain of mice: impact of colitis and psychological stress.
Front. Behav. Neurosci. 9, 177 (2015).
20. Langford, D. J. et al. Coding of facial expressions of pain in the laboratory mouse. Nat. Methods 7, 447–449 (2010).
21. Mitrovic, M., Shahbazian, A., Boc, E., Pabst, M. A. & Holzer, P. Chemonociceptive signalling from the colon is enhanced by mild
colitis and bloced by inhibition of transient receptor potential anyrin 1 channels. Br. J. Pharmacol. 160, 1430–1442 (2010).
22. Everaerts, W. et al. e capsaicin receptor TPV1 is a crucial mediator of the noxious eects of mustard oil. Curr. Biol. 21, 316–321
(2011).
23. Painsipp, E. et al. Neuropeptide Y and peptide YY protect from weight loss caused by Bacille Calmette-Guerin in mice. Br. J.
Pharmacol. 170, 1014–1026 (2013).
24. Eijelamp, N. et al. Increased visceral sensitivity to capsaicin aer DSS-induced colitis in mice: spinal cord c-Fos expression and
behavior. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G749–57 (2007).
25. Mosowitz, A. S., Terman, G. W. & Liebesind, J. C. Stress-induced analgesia in the mouse: strain comparisons. Pain 23, 67–72
(1985).
26. Przewloca, B. et al. e dierence in stress-induced analgesia in C57BL/6 and DBA/2 mice: a search for biochemical correlates. Pol.
J. Pharmacol. Pharm. 40, 497–506 (1988).
27. Stadlbauer, U., Langhans, W. & Meyer, U. Administration of the Y2 receptor agonist PYY3-36 in mice induces multiple behavioral
changes relevant to schizophrenia. Neuropsychopharmacology 38, 2446–2455 (2013).
28. Stadlbauer, U., Weber, E., Langhans, W. & Meyer, U. e Y2 receptor agonist PYY(3-36) increases the behavioural response to
novelty and acute dopaminergic drug challenge in mice. Int. J. Neuropsychopharmacol. 17, 407–419 (2014).
29. Tschop, M. et al. Physiology: does gut hormone PYY3-36 decrease food intae in rodents? Nature 430, 1 p following 165; discussion
2 p following 165 (2004).
30. Batterham, . et al. Physiology: does gut hormone PYY3-36 decrease food intae in rodents? (reply). Nature 430 (2004).
31. DeCarr, L. B. et al. A long-acting selective neuropeptide Y2 receptor PEGylated peptide agonist reduces food intae in mice. Bioorg.
Med. Chem. Lett. 17, 1916–1919 (2007).
32. Ghilardi, J. ., Allen, C. J., Vigna, S. ., McVey, D. C. & Mantyh, P. W. Cholecystoinin and neuropeptide Y receptors on single rabbit
vagal aerent ganglion neurons: site of prejunctional modulation of visceral sensory neurons. Brain es. 633, 33–40 (1994).
33. Zhang, X. et al. Expression and regulation of the neuropeptide Y Y2 receptor in sensory and autonomic ganglia. Proc. Natl. Acad. Sci.
USA 94, 729–734 (1997).
34. Goumain, M., Voisin, T., Lorinet, A. M. & Laburthe, M. Identication and distribution of mNA encoding the Y1, Y2, Y4, and Y5
receptors for peptides of the PP-fold family in the rat intestine and colon. Biochem. Biophys. es. Commun. 247, 52–56 (1998).
35. Landry, M., Holmberg, ., Zhang, X. & Hofelt, T. Eect of axotomy on expression of NPY, galanin, and NPY Y1 and Y2 receptors
in dorsal root ganglia and the superior cervical ganglion studied with double-labeling in situ hybridization and
immunohistochemistry. Exp. Neurol. 162, 361–384 (2000).
36. Nonaa, N., Shioda, S., Nieho, M. L. & Bans, W. A. Characterization of blood-brain barrier permeability to PYY3-36 in the
mouse. J. Pharmacol. Exp. er. 306, 948–953 (2003).
37. Brothers, S. P. et al. Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput
screening. Mol. Pharmacol. 77, 46–57 (2010).
38. Brumovsy, P. et al. Neuropeptide Y2 receptor protein is present in peptidergic and nonpeptidergic primary sensory neurons of the
mouse. J. Comp. Neurol. 489, 328–348 (2005).
39. Boey, D. et al. Peptide YY ablation in mice leads to the development of hyperinsulinaemia and obesity. Diabetologia 49, 1360–1370
(2006).
40. Leach, M. C. et al. e assessment of post-vasectomy pain in mice using behaviour and the Mouse Grimace Scale. PLoS One 7,
e35656 (2012).
41. Bonin, . P., Bories, C. & De oninc, Y. A simplied up-down method (SUDO) for measuring mechanical nociception in rodents
using von Frey laments. Mol. Pain 10, 26-8069-10-26 (2014).
42. Schmittgen, T. D. & Liva, . J. Analyzing real-time PC data by the comparative CT method. Nature protocols 3, 1101–1108 (2008).

www.nature.com/scientificreports/
10
Scientific RepoRts | 7:40968 | DOI: 10.1038/srep40968
Acknowledgements
is work was supported by the Austrian Science Fund (FWF grants P23097-B18, P25912-B23 and W1241-B18).
e authors thank Margit Eichholzer for running the PCR assays, and Jerey Mogil and Susana Sotocinal, McGill
University, Montreal, Canada, for their guidance and help in setting up the mouse grimace scale recordings.
Author Contributions
A.M.H., P.J. and P.H. designed the experiments and wrote the manuscript. A.M.H. and P.J. acquired the behavioral
data and performed the statistical analysis. H.H. generated the PYY(−/−) mice. R.M. and E.E.F. collected the spinal
cords for PCR analysis. F.R. contributed to the validation and analysis of the PCR assay and A.F. contributed to
the analysis of the LabMaster data. All authors revised the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Hassan, A. M. et al. Visceral hyperalgesia caused by peptide YY deletion and Y2
receptor antagonism. Sci. Rep. 7, 40968; doi: 10.1038/srep40968 (2017).
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
© e Author(s) 2017