8274 • The Journal of Neuroscience, June 16, 2010 • 30(24):8274 – 8284

Behavioral/Systems/Cognitive

Oxytocin-Induced Analgesia and Scratching Are Mediated by
the Vasopressin-1A Receptor in the Mouse
Ara Schorscher-Petcu,1,2 Susana Sotocinal,3 Sorana Ciura,1 Anouk Dupré,4 Jennifer Ritchie,3 Robert E. Sorge,3Jacqueline N. Crawley,5 Shuang-Bao Hu,6 Katsuhiko Nishimori,7 Larry J. Young,8 Eliane Tribollet,4 Rémi Quirion,1,2
and Jeffrey S. Mogil3
1

Department of Neurology and Neurosurgery, 2Douglas Mental Health University Institute, and 3Department of Psychology and Alan Edwards Centre for
Research on Pain, McGill University, Montreal, Quebec H3A 1B1, Canada, 4Department of Basic Neurosciences, University Medical Center, CH-1211
Geneva, Switzerland, 5Laboratory of Behavioral Neuroscience and 6Division of Extramural Activities, National Institute of Mental Health, Bethesda,
Maryland 20892, 7Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Miyagi 981-8555, Japan, and8Center for Behavioral Neuroscience and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia 30302

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological
functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal
cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant
mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates.
Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in threedifferent assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory
nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced
analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated thatthe distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites
diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast,
quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal
root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal
root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

Introduction
Oxytocin (OXT) and arginine vasopressin (AVP) are two closely
related nonapeptides synthesized in the paraventricular and supraoptic nuclei of the hypothalamus (Swanson and Kuypers,
1980; Sofroniew, 1983). They are either transported to the posterior pituitary and secreted into the bloodstream, exerting a variety of hormonal effects, or released into the CNS where they act as
modulators of neuronal transmission (Leng and Ludwig, 2008;
Raggenbass, 2008). Recently, the actions of OXT and AVP in the
CNS have received increasing attention because of the documented role of these peptides in the regulation of complex socialand sexual behavior in mammals (Donaldson and Young, 2008;
Young et al., 2008), including humans (Baumgartner et al., 2008;
Petrovic et al., 2008; Walum et al., 2008). Concomitantly, a growing literature has demonstrated the analgesic effects of OXT and

Received March 29, 2010; revised April 26, 2010; accepted May 4, 2010.
This work was supported by the National Institutes of Health (J.S.M.), the Canadian Institutes for Health Research(R.Q.), and the Swiss National Foundation for Scientific Research (E.T.). We express our gratitude to Dr. Maurice
Manning for donating V1AR and OTR antagonists.
Correspondence should be addressed to Dr. Jeffrey S. Mogil, Department of Psychology, McGill University, 1205
Dr. Penfield Avenue, Montreal, QC H3A 1B1, Canada. E-mail: jeffrey.mogil@mcgill.ca.
DOI:10.1523/JNEUROSCI.1594-10.2010
Copyright © 2010 the authors 0270-6474/10/308274-11$15.00/0

AVP in both human and rodent species (Honda and Takano,
2009; Koshimizu and Tsujimoto, 2009). Indeed, OXT was reported to be analgesic in a variety of pain tests when administeredinto the brain (Ge et al., 2002; Wang et al., 2003; Gao and Yu,
2004), the spinal cord (Yu et al., 2003; Miranda-Cardenas et al.,2006; Condés-Lara et al., 2007), or systemically (Lundeberg et al.,
1994; Reeta et al., 2006). In the rat, oxytocin receptor (OTR)binding sites are abundant in laminae I and II of the dorsal horn
(Tribollet et al., 1989, 1997; Reiter et al., 1994; VéronneauLongueville et al., 1999; Liu et al., 2003), and it has been suggested
that OXT activates inhibitory spinal interneurons presynapticallyto induce analgesia (Robinson et al., 2002; Rojas-Piloni et al.,2007; Breton et al., 2008).
The central and peripheral effects of OXT are thought to be
mediated by its binding to a single isoform of the OTR (Gimpl
and Fahrenholz, 2001), which activates the phospholipase C␤(PLC␤) signal transduction pathway (Arnaudeau et al., 1994; Ku
et al., 1995; Phaneuf et al., 1995). Centrally, AVP exerts its effects
mainly through the vasopressin-1A receptor (V1AR), which isalso coupled to the PLC␤ pathway (Schöneberg et al., 1998;
Thibonnier et al., 1998; Birnbaumer, 2000). Importantly, both
OXT and AVP, as well as the OTR and V1AR, display a highdegree of sequence homology, and both peptides can therefore
activate both receptors (Chini and Manning, 2007).

Schorscher-Petcu et al. • Oxytocin Effects Are Mediated via the Vasopressin-1A Receptor

In the present studies, we used two lines of transgenic knockout (KO) mice lacking the OTR or the V1AR, respectively, toinvestigate the role of these receptors in pain. In a series of behavioral
and pharmacological experiments, baseline nociceptive sensitivity
and OXT-induced analgesia were compared between KO and wildtype (WT) littermates. The finding that OXT-induced analgesia isnormal in OTR KO mice but impaired in V1AR KO mice suggested
a V1AR-mediated effect of OXT, and this was confirmed by experiments using OTR- and V1AR-selective antagonists. To determine
the putative site of action of OXT, we mapped the autoradiographic
distribution of OTRs and V1ARs in mouse spinal cord and determined gene expression levels in dorsal root ganglia (DRGs) using
real-time quantitative reverse transcription (RT)-PCR as well as asingle-cell-based RT-PCR approach.

Materials and Methods
Animals. All procedures were performed according to national guidelines
and approved by a McGill University animal use committee. OTR KO
mice and WT controls were obtained from Larry J. Young (Emory University, Atlanta, GA) (Takayanagi et al., 2005) and V1AR KO mice andWT controls were obtained from Jaqueline N. Crawley (National Institute of Mental Health, Bethesda, MD) (Hu et al., 2003). Both lines, bredcongenic on a C57BL/6 background for ⬎10 generations, were maintained as heterozygote (HET) ⫻ HET breeding colonies, and all offspring
was genotyped by custom PCR analysis of DNA isolated from tail tissue.
The nociceptive battery (see below) was performed on WT, HET, and KO
genotypes; other experiments compared only WT and KO mice.
For V1AR, the wild-type allele was identified by a 987 bp PCR productusing the following primers: 5⬘-CAGGAAATCTTCTGTAACATACTTGGTTTGG, 5⬘-CAAGATCCGCACAGTGAAGATGACC. The KO allele was identified by a 620 bp PCR product using the following primers:
5⬘-CAGGAAATCTTCTGTAACATACTTGGTTTGG, 5⬘-ACCCCTTCCCAGCCTCTGAGCCCAGAAGCGAAGG. Both reactions were run in
the same tube under the following conditions: 95°C for 4 min (95°C for 1
min, 60°C for 1 min, 72°C for 1 min) times 40 cycles, 72°C for 5 min.
For OTR, the wild-type allele was identified by a 630 bp PCR productusing the following primers: 5⬘-CTGGGGCTGAGTCTTGGAAG, 5⬘CTGGATACTCCAGTTGGGTGC. The KO allele was identified by 410
bp PCR product using the following primers: 5⬘-CTGGGGCTGAGTCTTGGAAG, 5⬘-GTTGGGAACAGGGGTGATTA. Both reactions wererun in the same tube under the following conditions: 95°C for 5 min(94°C for 30 s, 55°C for 30 s, 72°C for 1 min) times 35 cycles.
Nociceptive assays. Adult (6 –10 weeks of age) mice of both sexes(Mogil and Chanda, 2005) were used for behavioral testing and allowed
to habituate to the testing room for at least 1 h. Males and females were
tested separately. Moreover, mice were unable to see each other during
testing (Langford et al., 2006). For baseline nociceptive phenotyping,each mouse first underwent the whole series of acute thermal and mechanical assays, with 2 d between consecutive tests. We have determinedthat these acute assays are associated with no detectable carryover or
repeated-testing effects (Mogil et al., 2006). After the acute assays, mice
were tested in either the formalin, the zymosan, or the abdominal constriction (writhing) test and killed immediately thereafter.
Thermal nociception was assessed using three different assays, the tailwithdrawal, hot-plate, and paw withdrawal tests. (1) Tail withdrawal test:
Mice were lightly restrained in a cloth/cardboard “pocket” while thedistal half of their tail was dipped into a bath maintained at either 47 or
49°C. Latency to reflexively withdraw the tail from the water was measured three consecutive times (separated by 320 s) and averaged. (2)Hot-plate test: Mice were placed on a metal surface maintained at either
50 or 53°C (IITC model PE34MHC) within a 20-cm-high Plexiglas cylinder (15 cm diameter). The latency to either lick or shake the hindpaw
was measured as a nocifensive endpoint. (3) Paw withdrawal test: The
noxious stimulus was a high-intensity light beam (IITC; setting, 20%;⬇45 W) aimed at the plantar surface of the hindpaw of an inactive mouse(Callahan et al., 2008) placed within Plexiglas cubicles (9 ⫻ 5 ⫻ 5 cmhigh) atop a glass floor. For the assessment of baseline thermal sensitivity,

J. Neurosci., June 16, 2010 • 30(24):8274 – 8284 • 8275

the latency to lift the targeted hindpaw (i.e., paw withdrawal latency) was
measured six times (separated by 15 min) on each side.
Mechanical nociception was assessed using two different assays: thetail-clip and the von Frey filament test. (4) Tail-clip test: A modificationof the tail-pinch test was used (Tagaki et al., 1966). Each mouse wasplaced in a cloth/cardboard holder, and a binder clip (exerting ⬇700 g offorce) was applied to the tail ⬇1 cm from the base. The mouse wasimmediately removed from the holder, placed on a table top, and thelatency to lick, bite, grab, or bring the nose to within 1 cm of the clip was
measured with a stopwatch to the nearest 0.1 s, after which the clip wasimmediately removed. (5) von Frey test: Mice were placed within Plexiglas cubicles (9 ⫻ 5 ⫻ 5 cm high) on a metal mesh floor. Mechanicalsensitivity was measured by determining the median 50% hindpaw withdrawal threshold with calibrated von Frey nylon monofilaments (0.015–
2.0 g) using the up– down method (Chaplan et al., 1994). Reported values
represent the average of four distinct threshold evaluations, two on eachside.
Chemical nociception was assessed with the abdominal constriction(writhing) and the formalin test. (6) Writhing test: Mice were placed atopa glass floor within 20-cm-high Plexiglas cylinders (15 cm diameter) andhabituated for at least 30 min. Mice were briefly removed, injected intraperitoneally with 0.9% acetic acid in a 10 ml/kg volume, and returned
immediately to the cylinder. Each mouse was videotaped for the next 20
min by individual video cameras placed underneath the glass floor, andtheir behavior was later scored by a blinded observer using The Observer(Noldus). The presence or absence of “writhes” (lengthwise stretches of
the torso with a concomitant concave arching of the back) was sampled at
20 s intervals and is reported as the percentage of “positive” samples (i.e.,samples containing writhing behavior). (7) Formalin test: Mice wereplaced atop a glass floor within 20-cm-high Plexiglas cylinders (15 cm
diameter) and habituated for at least 30 min. Mice were briefly removed,
and 25 ␮l of a 2.5% formaldehyde solution was injected subcutaneously
into the plantar surface of the right hindpaw using a 50 ␮l microsyringewith a 30 gauge needle. After being returned to the cylinder, each mouse
was videotaped from below for the next 60 min, and the presence orabsence of licking/biting of the right hindpaw was sampled once perminute by a blinded observer (see above). The early/acute phase of thebiphasic formalin test was defined conservatively as the first 5 min ofobservation, and the late/tonic phase as the last 50 min of observation.
Inflammatory hypersensitivity was assessed using zymosan A from
Saccharomyces cerevisiae (Meller and Gebhart, 1997). (8) Zymosan thermal hypersensitivity: Paw withdrawal baseline latencies were assessed as
described above. The following day, 20 ␮l of a 2.5 mg/ml zymosan solution was injected into the right hindpaw and two postinjection latency
measures on each paw were taken every hour for 6 h.
Pharmacology. For pharmacological experiments with OXT or AVP,
four baseline measures were taken before and 30 min after injection oneither the radiant-heat paw withdrawal or the von Frey test. For thepaw withdrawal test, a cutoff latency of 30 s was used to prevent thepossibility of tissue injury. Reported values represent percentage analgesia produced by OXT or AVP and were calculated as follows:[(postdrug latency/threshold ⫺ baseline latency/threshold)/(cutoff
latency/threshold ⫺ baseline latency/threshold)] ⫻ 100. The same
protocol was used for hyperosmotic challenge experiments; instead ofa peptide injection, mice received a 10 ml/kg (intraperitoneal) injection of
hyperosmotic (1 M) or physiological (150 mM) saline. This protocol has beenshown to induce an average increase of 15.8 mOsm/kg in wild-type mice
(Ciura and Bourque, 2006), which in turn raises serum AVP levels from 2 to40 pg/ml (Sharif Naeini et al., 2006). Where used, OTR and V1AR antagonists were injected intraperitoneally, intracerebroventricularly, or intrathecally 10 min before intraperitoneal injection of OXT. Intracerebroventricular
injections were delivered using a 2.5 ␮l volume under light isoflurane/oxygen anesthesia according to the method of Laursen and Belknap (1986).
Intrathecal injections were delivered using a 5 ␮l volume based on themethod of Hylden and Wilcox (1980).
Compounds. OXT and AVP were both obtained from Sigma-Aldrich
and were dissolved in a 0.9% saline solution and injected intraperitoneally except where otherwise noted. d(CH2)[Tyr(Me) 2]AVP (Kruszynski
et al., 1980), a selective V1AR antagonist, and desGly-NH2-D(CH2)5[D-Schorscher-Petcu et al. • Oxytocin Effects Are Mediated via the Vasopressin-1A Receptor

8276 • J. Neurosci., June 16, 2010 • 30(24):8274 – 8284

Table 1. Phenotyping of WT, HET, and OTR KO mice on a battery of nociceptive assays
OTR
Assay
ThermalTail withdrawal
Hot plate
Paw withdrawal
Mechanical
Tail clip
von Frey
ChemicalWrithing
Formalin (early)
Formalin (late)
Inflammatory
Zymosan

V1AR

Intensity

Units

na

WTb

HET

KO

p valuec

na

WTb

HET

KO

p valuec

49°C
47°C
53°C
50°C
20%
15%

s
s
s
s
s
s

16
16
16
16
16
16

2.7 ⫾ 0.2
4.1 ⫾ 0.1
15.1 ⫾ 1.0
29.1 ⫾ 2.9
9.5 ⫾ 0.6
15.1 ⫾ 0.8

2.5 ⫾ 0.1
4.1 ⫾ 0.1
15.2 ⫾ 1.4
27.8 ⫾ 3.2
10.5 ⫾ 0.5
15.9 ⫾ 0.8

2.6 ⫾ 0.1
4.1 ⫾ 0.1
14.3 ⫾ 1.0
26.1 ⫾ 2.0
11.5 ⫾ 0.7
7.1 ⫾ 1.0

0.74
0.93
0.82
0.75
0.08
0.26

13–18
13–18
13–18
13–18
14 –1614 –16

2.1 ⫾ 0.1
3.9 ⫾ 0.1
14.7 ⫾ 1.1
27.6 ⫾ 1.3
7.4 ⫾ 0.3
12.3 ⫾ 0.8

2.4 ⫾ 0.1
3.9 ⫾ 0.1
14.3 ⫾ 1.0
28.1 ⫾ 1.7
8.0 ⫾ 0.4
12.2 ⫾ 0.5

2.1 ⫾ 0.1
3.7 ⫾ 0.1
17.4 ⫾ 1.0
23.3 ⫾ 3.0
8.4 ⫾ 0.4
12.5 ⫾ 0.5

0.08
0.64
0.08
0.21
0.14
0.88

⬇700 g0.015–2.0 g

s
g

20
20

2.1 ⫾ 0.5
1.1 ⫾ 0.1

4.7 ⫾ 1.4
1.1 ⫾ 0.1

2.8 ⫾ 1.1
1.0 ⫾ 0.1

0.14
0.54

7– 8
6–8

2.2 ⫾ 0.7
1.1 ⫾ 0.1

1.6 ⫾ 0.5
0.9 ⫾ 0.1

2.4 ⫾ 0.3
1.2 ⫾ 0.1

0.52
0.08

0.9%
2.5%
2.5%

%d
%
%

14 –1516
16

23.6 ⫾ 2.4
45.0 ⫾ 6.4
18.4 ⫾ 2.8

25.2 ⫾ 3.0
37.5 ⫾ 6.0
21.7 ⫾ 2.3

18.6 ⫾ 3.2
33.8 ⫾ 6.5
19.4 ⫾ 2.4

0.28
0.45
0.64

10 –12
9 –17
9 –17

27.8 ⫾ 4.4
55.6 ⫾ 8.7
24.6 ⫾ 2.9

31.7 ⫾ 3.8
52.0 ⫾ 4.9
30.8 ⫾ 2.8

26.1 ⫾ 4.5
55.0 ⫾ 5.6
35.2 ⫾ 1.8

0.62
0.90
0.04*

2.5 ␮g%e

1667.4 ⫾ 1.8

n.d.

68.7 ⫾ 2.0

0.62

12

61.1 ⫾ 4.6

n.d.

54.3 ⫾ 3.3

0.24

n.d., Not determined.
aSample size per genotype.
b
All values represent mean ⫾ SEM.
c
From a one-way ANOVA of genotype.
d
Percentage of positive samples featuring writhing or licking/biting behavior.
e
Percentage of maximum possible hyperalgesia using areas over the time–response curve (Mogil et al., 2006).
*p ⬍ 0.05.

Tyr 2,Thr 4]OVT (Manning et al., 1995), a selective OTR antagonist, wereboth kindly donated by Dr. Maurice Manning (University of Toledo,
Toledo, OH).
Scratching. Mice were placed atop a glass floor within 20-cm-highPlexiglas cylinders (15 cm diameter) and allowed to habituate for 30 min.
Then, they were removed, lightly anesthetized with isoflurane/oxygen,and given an intracerebroventricular injection of OXT or AVP using a
volume of 2.5 ␮l. Mice were immediately returned to their test cylinders
and videotaped by individual video cameras from below for the next 30
min. Blinded experimenters using The Observer scored the cumulative
duration of vigorous scratching of the flanks with the contralateralhindpaws.
Receptor binding study. Three male and three female adult mice of eachof the OTR KO, the V1AR KO, and the C57BL/6 (WT) genotypes were
killed, and their spinal cords and brains were rapidly dissected and frozen
in isopentane at ⫺80°C. The lumbar section of the spinal cord (L4 –L6)
was cut with a freezing microtome in six series of coronal sections, 14 ␮m
thick, and mounted on chrome-alum-gelatin-coated microscope slides.
Two brains of each genotype were also cut in coronal sections of equal
thickness at the level of the lateral septum and the ventromedial hypothalamus. These sections served as a control for OTR and V1AR binding.
All slides were stored at ⫺80°C until the day of the experiment. The
binding procedure was performed as previously described (Tribollet etal., 1997). Before incubation, sections were lightly fixed by dipping theslides for 5 min in a solution of 0.2% paraformaldehyde in 0.1 M PBS, pH
7.4, and then rinsed by two 5 min washes in 50 mM Tris-HCl buffer, pH
7.4, supplemented with 0.1% bovine serum albumin. OXT binding sites
were labeled with the radioiodinated OTR antagonist D(CH2)5[Tyr(Me) 2,Thr 4,Tyr 9-NH2]OVT ( 125I-OTA) at 0.02 nM (Elands et al., 1988).
AVP binding sites were labeled with the radioiodinated V1AR antagonist
HO-Phaa 1-D-Tyr(Me) 2-Phe 3-Gln 4-Asn 5-Arg 6-Pro 7-Arg 8-NH2 ( 125IVPA) at 0.02 nM (Barberis et al., 1995). The specific activity of radioligands was 2200 Ci/mmol. Their binding properties in mouse spinal andbrain sections were assessed in preliminary experiments by using selective agonist compounds as competitors (Chini and Manning, 2007). Results indicated that 125I-VPA and 125I-OTA have the same receptor
selectivity as in the rat (i.e., label selectively V1AR and OTR, respectively,
when used at the proper concentration) (results not shown). Nonspecificbinding was determined in the presence of 2 ␮M OXT or AVP. Binding
was performed in a humid chamber, at room temperature, by covering
each slide with 400 ␮l of the incubation medium (50 mM Tris-HCl,

0.025% bacitracin, 5 mM MgCl2, 0.1% bovine serum albumin) containing 125I-VPA or 125I-OTA, either alone or in the presence of unlabeled
peptide. Incubation lasted 1 h at room temperature under gentle agitation and was followed by two 5 min washes in ice-cold incubation medium and a quick rinse in distilled water. The slides were air-dried,apposed to Kodak Biomax MR Films for 14 d, and developed in Kodak
D19 for 5 min. Both radioligands and films were obtained from
PerkinElmer.
Quantitative gene expression analysis. Five adult male and five adultfemale C57BL/6 mice (The Jackson Laboratory) were killed, and their
DRGs were rapidly transferred into RNAlater (Ambion). Total RNA
was isolated from these tissues with the TRIzol method according to
the manufacturer’s instructions (Invitrogen) and reverse transcribedalong with standards of known concentrations (Ambion). Amplification of Oxtr (assay ID, Mm01182684_m1) and Avpr1a (assay ID,
Mm00444092_m1) cDNA was performed using an ABI 7000 with TaqMan
probes and primers as described in the manufacturer’s protocol (Applied
Biosystems). Unknown samples were amplified in quadruplicate andstandards in triplicate. Relative quantification was made following thestandard curve method. Results were averaged and normalized by dividing mean values of the Oxtr or Avpr1a gene by mean values of Gapdh, theendogenous reference gene.
Single-cell gene expression analysis. Three adult male and three adultfemale C57BL/6 mice (The Jackson Laboratory) were killed, and their
DRGs were rapidly transferred into PIPES buffer supplemented with 10
mM glucose.
DRGs were cleaned of conjunctive tissue with fine forceps and transferred into oxygenated PIPES to which 0.5 mg/ml proteases X and XIV(Sigma-Aldrich) had been added. After 30 min of enzymatic digestion,
DRGs were rinsed for 30 min in oxygenated PIPES, and then triturated
and plated on Petri dishes. Cell harvesting was performed under a phasic
microscope (Olympus IX71) equipped with an electrode manipulator.
Glass electrodes with polished ends were filled with 1.5 ␮l of PIPES
containing 10% RNase inhibitor and used to aspire individual cells. For
subsequent determination of size, a picture of each cell was taken at 40⫻
magnification. ImageJ software was used to measure cell diameter. Immediately after harvesting, the content of each cell was expelled into anindividual 200 ␮l PCR tube. DNase treatment and first-strand cDNA
synthesis were performed using the Superscript III Reverse Transcriptase
kit (Invitrogen). Avpr1a and Gapdh transcripts were amplified with
nested multiplex PCR in 74 individual cell samples as previously de-Schorscher-Petcu et al. • Oxytocin Effects Are Mediated via the Vasopressin-1A Receptor

Figure 1. OXT-inducedanalgesiaintheradiant-heatpawwithdrawaltest(a,b)andvonFreytest(c) of mechanical nociception is V1AR dependent. Systemically administered OXT dose-dependently
producesanalgesiainWTandOTRKO(a),butnotinV1ARKOmice(b)(n⫽7–16/group).A4mg/kg
dose of OXT produces analgesia in WT but not V1AR KO mice (c) (n ⫽ 8/group). *p ⬍ 0.05, **p ⬍0.01, ***p ⬍ 0.001, compared with corresponding saline group. Error bars indicate SEM.

J. Neurosci., June 16, 2010 • 30(24):8274 – 8284 • 8277

scribed by Hoyda et al. (2007) using a QIAGEN Multiplex PCR kit(QIAGEN). Water blanks and samples of the dissociation buffer wereused as negative controls. Whole DRG samples were positive controls.
Primers were designed based on Ensembl database cDNA sequences using
Primer3 software (Rozen and Skaletsky, 2000). The following primers wereused for first-round amplification: Avpr1a, 5⬘-CCTACATGCTGGTGGTGATG-3⬘ (forward) and 5⬘-TCTTCACTGTGCGGATCTTG-3⬘ (reverse); Gapdh, 5⬘-AACTTTGGCATTGTGGAAGG-3⬘ (forward) and5⬘-CCCTGTTGCTGTAGCCGTAT-3⬘ (reverse). For second-round(nested) amplification, the forward and reverse primer sequences were as
follows: Avpr1a, 5⬘-GGGCTGAGTTTCGTTCTGAG-3⬘ and 5⬘-GAAATGCTCTTCACGCTGCT-3⬘, respectively; Gapdh, 5⬘-ACCCAGAAGACTGTGGATGG-3⬘ and 5⬘-AGGAGACAACCTGGTCCTCA-3⬘, respectively. Final amplification products had a predicted size of 338 bp (Avpr1a)and 301 bp (Gapdh) and were separated by electrophoresis on a cyanine
dye-stained (SYBR Gold; Invitrogen) 2% agarose gel.
Statistical analyses. Statistical analysis was performed using Prism(GraphPad). Criterion significance level was set at p ⬍ 0.05 for all tests,
and post hoc analyses were only performed when appropriate. Variance isillustrated in figures as the SEM.
Data from the behavioral phenotyping were analyzed using one-way
ANOVA. Behavioral data from pharmacological experiments were analyzed using two-way ANOVA with genotype and dose as between-subject
factors. Dunnett’s case-comparison post hoc analysis was used for pairwise comparisons between vehicle and different doses of OXT or AVP withineach genotype; where only one dose was used, significance was established byStudent’s t test. FlashCalc 31.5 software (M. Ossipov, University of Arizona,
Tucson, AZ) was used to calculate half-maximal effective dose (ED50) values
and associated 95% confidence intervals— using the method of Tallarida
and Murray (1981)—for the analgesic efficacy of OXT and AVP in the paw
withdrawal test. Oxtr and Avpr1a quantitative gene expression were compared using Student’s t test (two-tailed).

Results

Nociceptive phenotype of OTR KO and V1AR KO mice
Table 1 shows that nociceptive sensitivity of OTR KO mice is
identical with that of their WT and HET
littermates in a battery of eight nociceptive assays, including tests for thermal,
mechanical, and chemical/inflammatory
nociception. No significant main effects
of genotype were detected in any assay. In
four assays (53°C hot-plate, 49°C tailwithdrawal, 15% paw withdrawal, and vonFrey tests), significant main effects of sex
were observed—in every case with females
more sensitive than males, as has been previously reported (Mogil et al., 2006)— but
no significant genotype by sex interactions
were observed, so data were pooled fromboth sexes for presentation.
Table 1 further shows that V1AR WT,
HET, and KO mice also generally responded similarly to each other. Significant main effects of genotype wereobtained only in one assay, the late phaseof the formalin test, in which V1AR KOs
displayed significantly increased sensitivity compared with WT mice (p ⬍0.05). In four assays (49 and 47°C tailwithdrawal, 15 and 20% paw withdrawal tests), significant main effects of
Figure 2. OXT-induced analgesia in the formalin test is V1AR dependent. A dose of 4 mg/kg OXT reduces licking behavior in WT sex were observed, again with females
and OTR KO mice in both the early (a) and late (b) phases (n ⫽ 6 – 8/group). In V1AR KO mice, the analgesic effect of OXT is being more sensitive.
The power to detect genotype differabolished in both the early (c) and late (d) phases (n ⫽ 6 – 8/group). *p ⬍ 0.05, **p ⬍ 0.01, compared with corresponding salinegroup. Error bars indicate SEM.
ences in both mutant lines was compar-

8278 • J. Neurosci., June 16, 2010 • 30(24):8274 – 8284

Schorscher-Petcu et al. • Oxytocin Effects Are Mediated via the Vasopressin-1A Receptor

atively high, since sample sizes used herein are large comparedwith standard practices [Mogil et al. (2005), their Fig. 5].
Oxytocin- and vasopressin-induced analgesia in OTR KO andV1AR KO mice
OXT injected systemically dose-dependently increased paw withdrawal latencies in WT and OTR KO mice in the paw withdrawal
test of thermal nociception (Fig. 1a). Two-way ANOVA confirmed a main effect of oxytocin dose (F(3,73) ⫽ 23.4; p ⬍ 0.001).
According to Dunnett’s case comparison post hoc tests, effective
doses of OXT were 2 and 8 mg/kg for both genotypes (p ⬍ 0.01).
A dose of 8 mg/kg OXT produced ⬃60% of maximum possible
analgesia (with the arbitrary cutoff latency chosen); a higher dose
(16 mg/kg) (data not shown) was no more effective.
Although still present in V1AR WT mice, OXT-induced analgesia was completely absent in V1AR KO mice (Fig. 1b). Two-way
ANOVA revealed a main effect of oxytocin dose (F(3,79) ⫽ 3.6;
p ⬍ 0.05) and of genotype (F(1,79) ⫽ 78.1; p ⬍ 0.001), as well as asignificant dose by genotype interaction (F(3,79) ⫽ 10.6; p ⬍0.001). Follow-up one-way ANOVA and Dunnett’s post hoc testing on data from V1AR WT mice revealed that OXT effectively
induced analgesia at doses of 0.1, 2, and 8 mg/kg (p ⬍ 0.01)
compared with saline.
The V1AR-dependent analgesic effect of systemic OXT was
confirmed using the von Frey test of mechanical sensitivity. Usinga dose of 4 mg/kg, we observed a significant analgesia (i.e., increase in paw withdrawal threshold) in V1AR WT mice compared with saline or zero (one-way Student’s t(14) ⫽ 2.3, p ⬍ 0.05,
or t(7) ⫽ 2.6, p ⬍ 0.05, respectively) that was absent in their KO
littermates (Fig. 1c).
Systemic OXT was similarly analgesic in the formalin test of
inflammatory nociception. In OTR KO and WT mice, there was amain effect of oxytocin on nocifensive (licking/biting) behavior
in both the early (Fig. 2a) (F(1,24) ⫽ 5.4; p ⬍ 0.05) and late phase(Fig. 2b) (F(1,24) ⫽ 23.7; p ⬍ 0.01) of the test. In contrast, although still reducing the pain behavior in V1AR WT mice, no
OXT-induced analgesia was seen in V1AR KOs in either the early(Fig. 2c) or the late (Fig. 2d) phase of the formalin test. In the earlyphase, only the main effect of genotype was significant (F(1,24) ⫽15.4; p ⬍ 0.001), although the genotype by oxytocin interaction
approached significance (F(1,24) ⫽ 2.5; p ⫽ 0.13). In the latephase, significant main effects of oxytocin (F(1,24) ⫽ 12.9; p ⬍0.01) and genotype (F(1,24) ⫽ 5.5; p ⬍ 0.05), as well as a significant interaction (F(1,24) ⫽ 4.5; p ⬍ 0.05) were observed. In
WTs only, OXT significantly reduced licking behavior in thelate phase (p ⬍ 0.01).
Systemically administered AVP also induced analgesia in WT,
but not in V1AR KO mice (Fig. 3a). A two-way ANOVA revealed
a significant main effect of genotype (F(1,49) ⫽ 14.2; p ⬍ 0.001)
and a significant dose by genotype interaction (F(3,49) ⫽ 2.9; p ⬍0.05). AVP was found to be more potent than OXT, since 50%
analgesia was obtained with a dose of 0.5 mg/kg already ( p ⬍0.05 compared with saline). Again, higher AVP doses demonstrated no greater efficacy (data not shown). All these conclusions are also supported by estimated ED50 values and potency
ratios (Table 2).
Systemic hyperosmotic challenge, a known trigger of endogenous AVP release (Robertson et al., 1976), also elicited analgesia
in the paw withdrawal test in WT mice at 1 M [one-way t18 ⫽ 3.1,
p ⬍ 0.01, compared with 150 mM (physiological saline)], but notin V1AR KO mice (Fig. 3b).

Figure 3. Exogenous and endogenous AVP analgesia is V1AR dependent. AVP injected systemically is analgesic in WT, but not in V1AR KO mice (a) (n ⫽ 6 –9/group). The graph in b
shows the effect of osmotic stress, known to release endogenous AVP. Intraperitoneal injectionof 1 M but not 150 mM (physiological) saline produces analgesia in WT but not V1AR KO mice(n ⫽ 6 –10/group). *p ⬍ 0.05, compared with corresponding saline group; **p ⬍ 0.01,
compared with corresponding 150 mM (physiological saline) group or other genotype. Error bars
indicate SEM.
Table 2. Half-maximal effective doses (ED50 values) and associated 95% confidence
intervals (CIs) for systemic OXT- and AVP-induced analgesia in the paw withdrawal
test
Analgesic/genotype
Oxytocin-induced analgesia
OTR WTOTR KOV1AR WTV1AR KO
Vasopressin-induced analgesia
V1AR WTV1AR KO

ED50

95% CI

3.3
14.3
6.4
n.c.

1.6 –7.0
1.3–158
1.8 –22.4
n.c.

0.5
n.c.

0.1–2.0
n.c.

All values are in milligrams per kilogram. Note that these ED50 estimates are extrapolations because of submaximal
analgesia, and their absolute values should be treated with caution. n.c., Not calculable.

Oxytocin and vasopressin-1A receptor antagonists
To further address the hypothesis that the analgesic effects of
OXT are actually mediated by the V1AR, OTR- or V1ARselective antagonists were administered 10 min before injectionof 4 mg/kg OXT. When given systemically (Fig. 4a), only the
V1AR antagonist could dose-dependently prevent OXT-induced
analgesia. One-way ANOVA confirmed a main effect of antagonist
treatment (F(3,28) ⫽ 17.1; p ⬍ 0.001). Likewise, a supraspinally administered V1AR antagonist (Fig. 4b) prevented OXT-induced analgesia completely and dose-dependently (F(5,45) ⫽ 5.3; p ⬍ 0.001).
Interestingly, in this compartment, a higher dose of the OTR
antagonist displayed a partial inhibitory effect on OXT-induced
analgesia. The same results were obtained using spinal injectionsof OTR and V1AR antagonists (Fig. 4c). Again, OXT-induced
analgesia was fully and dose-dependently prevented by the V1AR
antagonist (F(4,40) ⫽ 16.9; p ⬍ 0.001), whereas the OTR antago-

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J. Neurosci., June 16, 2010 • 30(24):8274 – 8284 • 8279

Figure 5. OXT (100 ng) administered intracerebroventricularly produces a robust and longlasting scratching response in WT and OTR KO (a), but not in V1AR KO mice (b) (n ⫽ 7– 8/group). AVP (25 ng, i.c.v.) also elicits scratching in WT, but not in V1AR KO mice (c) (n ⫽7– 8/group). ***p ⬍ 0.001, compared with saline vehicle. Error bars indicate SEM.

Figure 4. A selective V1AR antagonist (VPA), but not a selective OTR antagonist (OTA),
prevents OXT-induced analgesia in the radiant-heat paw withdrawal test (n ⫽ 6 – 8/dose/
drug/route). When injected systemically (a), 5 mg/kg VPA fully inhibits, whereas OTA does notaffect OXT-induced analgesia. Centrally (intracerebroventricularly) administered (b), a 500 ng
dose of VPA is sufficient to block OXT-induced analgesia, whereas up to 2.5 ␮g of OTA only
partly attenuated the effect of OXT. At the spinal level (intrathecal) (c), both antagonists display
some efficacy in inhibiting OXT-induced analgesia, but only the VPA is fully effective, at both 100
and 500 ng. *p ⬍ 0.05, **p ⬍ 0.01, compared with saline group. Error bars indicate SEM.

nist could only partially inhibit the effect of OXT, even when usedat higher doses.
Oxytocin- and vasopressin-induced scratching
It has been well documented that both OXT and AVP elicit strong
scratching and grooming behaviors in rats (Van Wimersma
Greidanus et al., 1990) and mice (Meisenberg and Simmons,
1982) when injected into the brain or spinal cord (Thurston et al.,1992). This vigorous response strongly interfered with nociceptive testing in every assay tried, and thus OXT and AVP wereadministered systemically in analgesia studies. Scratching induced by 100 ng of OXT acutely injected into the third ventricle
was present in both WT and OTR KO mice (Fig. 5a) (main effectof oxytocin only: F(1,27) ⫽ 210.6, p ⬍ 0.001), but absent in V1AR
KOs (Fig. 5b). In the latter data set, in addition to significant main
effects of oxytocin (F(1,27) ⫽ 274.9; p ⬍ 0.001) and genotype(F(1,27) ⫽ 252.1; p ⬍ 0.001), there was also a significant interaction (F(1,27) ⫽ 279.6; p ⬍ 0.001). Scratching behavior occurred inbouts beginning immediately after injection, sometimes even before the animal had fully recovered from isoflurane anesthesia,
and lasted for ⬃20 min. Within a 30 min observation period, thecumulative amount of scratching bouts totaled ⬃7 min.
Figure 5c shows that, in WT mice only, intracerebroventricular AVP elicited a similar duration of scratching at a dose of 25 ng( p ⬍ 0.001 compared with saline). This response was absent in
V1AR KO mice. The main effects of vasopressin (F(1,27) ⫽ 153.2)
and of genotype (F(1,27) ⫽ 138.8), as well as the vasopressin bygenotype interaction (F(1,27) ⫽ 144.0) were all highly significant(all p ⬍ 0.001).

Schorscher-Petcu et al. • Oxytocin Effects Are Mediated via the Vasopressin-1A Receptor

8280 • J. Neurosci., June 16, 2010 • 30(24):8274 – 8284

Spinal OTR and V1AR distribution
Figures 6 and 7 illustrate the distributionof OXT and AVP receptor binding sites,
respectively, in mouse spinal cord at thelevel of the lumbar enlargement (L4 –L6).
OXT binding sites are restricted to the superficial layers of the dorsal horn (Fig. 6a).
This densely labeled band corresponds tolaminae I and II of the dorsal horn, and isabsent in sections from OTR KO mice
(Fig. 6d). In contrast, Figure 7a shows that
AVP binding sites are diffusely present
and weakly labeled in all laminae of the
spinal cord, with only a slight increase oflabeling intensity in the dorsal horn. As
expected, AVP binding sites are absent insections from V1AR KO mice (Fig. 7c).
Tissue was obtained from both male andfemale mice, and no obvious sex difference in spinal distribution of OXT or AVP
binding sites was observed (data notshown).
The specificity of the radioligands used
was controlled for by competition with
unlabeled ligand (Figs. 6b, 7b), as well as
by processing of brain sections of WT animals. Figures 6e and 7e show that 125IOTA binding sites are enriched in theventromedial hypothalamus (arrow),whereas 125I-VPA binds to the lateral septum (arrow), both regions where abundant expression of OTR and V1AR,
respectively, has been well documented(Barberis and Tribollet, 1996). Binding ofradioligands was successfully competedby unlabeled OXT or AVP, respectively(Figs. 6f, 7f ).

Figure 6. Autoradiographic localization of specific 125I-OTA binding sites in mouse brain and spinal cord. In WT (a) and V1AR KO
mice (c), OTR binding is dense in all superficial laminae of the lumbar spinal cord. OTR binding is absent in OTR KO mice (d). In WT
mice, OTR binding is dense in the ventromedial hypothalamus (e, arrow). OTR binding is displaced by coincubation of radioligand
with 2 ␮M cold OXT (b, f ). Scale bars: d, 500 ␮m; f, 1500 ␮m.

Figure 7. Autoradiographic localization of specific 125I-VPA binding sites in mouse brain and spinal cord. In WT (a) and OTR KO
mice (d), V1AR binding is diffuse in all regions of the lumbar spinal cord. V1AR binding is absent in V1AR KO mice (c). In WT mice,
V1AR binding is dense in the lateral septum (e, arrow). V1AR binding is displaced by 2 ␮M cold AVP (b, f ). Scale bars: d, 500 ␮m;
f, 1500 ␮m.

Oxtr and Avpr1a gene expressionin DRGIn the final experiment, Oxtr and Avpr1a
gene expression levels were measured in DRG samples of WT
mice. Oxtr mRNA was only barely expressed in mouse DRG(0.4 ⫾ 0.2 arbitrary units compared with Gapdh), whereas
Avpr1a mRNA expression levels were ⬇10-fold higher (4.3 ⫾ 0.1
arbitrary units compared with Gapdh; p ⬍ 0.001 by Student’s ttest). To further characterize Avpr1a expression at the cellularlevel in the DRG, a single-cell RT-PCR approach was adopted.
Figure 8a shows a scatterplot illustrating that, of the total of 74
sampled DRG neurons, 25 (33.8%) expressed Avpr1a. Interestingly, Avpr1a-positive neurons were predominantly of small (⬍20␮m) or medium (20 –30 ␮m) diameter. In contrast, Avpr1a mRNAwas detected in only two (2.8%) large-diameter neurons (⬎30 ␮m).
The PCR products obtained from nine DRG cells, representative ofthe large-, medium-, and small-size populations of DRG neurons,
are illustrated in Figure 8b, and representative large- and smalldiameter cells are pictured in Figure 8, c and d.

Discussion

neuropathic pain states were not assessed here, and thus it remains possible that the OTR and/or the V1AR play as yet undetermined roles in the mediation of mechanical or thermal
hypersensitivity in chronic neuropathic pain. The major finding of this study is that systemically administered OXT produced robust, dose-dependent analgesia in OTR KO mice, but
not in V1AR KO mice, in three distinct nociceptive assays.
Likewise, the analgesic effects of OXT could be fully prevented
by a V1AR-selective antagonist, but not by an OTR-selective
antagonist. Scratching, a behavior elicited by centrally administered OXT and AVP, was also absent in V1AR KO mice, but
intact in OTR KO mice, generalizing our findings beyond nociception itself. Finally, receptor binding and gene transcription experiments demonstrated that, whereas the OTR
predominates in pain-relevant regions of the mouse lumbar
spinal cord, DRGs mostly express the V1AR. Thus, it appears
that OXT acts via the V1AR to modulate pain and scratching
behaviors.

Using a battery of assays of acute and tonic pain sensitivity, we
systematically investigated mutant mice lacking the OTR or the
V1AR. Mice of both KO lines displayed nociceptive phenotypes
mostly similar to their WT and HET littermates. We note that

Pain sensitivity is normal in OTR KO and V1AR KO mice
A number of studies have reported on the analgesic actions ofexogenously administered neurohypophyseal hormones against

Schorscher-Petcu et al. • Oxytocin Effects Are Mediated via the Vasopressin-1A Receptor

Figure 8. Avpr1a gene expression in individual DRG cells. a, Scatterplot illustrating the
proportion of Avpr1a mRNA-expressing DRG neurons of different size. b, Gel showing nine
single DRG cells positive or negative for Avpr1a. c, d, Examples of large-diameter (c) and smalldiameter (d) dissociated DRG neurons. Scale bar, 15 ␮m.

different pain modalities (Lundeberg et al., 1994; Ge et al., 2002;
Yu et al., 2003; Gao and Yu, 2004; Koshimizu and Tsujimoto,2009), but little is known about the role of endogenous OXT and
AVP in pain processing.
The anatomical distribution of OXT and OTRs in rat strongly
supports such a role. OTRs are enriched in pain-relevant laminae
I and II of the dorsal horn (Reiter et al., 1994; Tribollet et al., 1997;
Véronneau-Longueville et al., 1999) and are a likely target ofspinal OXTergic projections from the paraventricular nucleus of
the hypothalamus (Swanson and Kuypers, 1980; Puder andPapka, 2001; Condés-Lara et al., 2007). A few functional studies
have linked paraventricular activity to analgesia, which was mimicked by OXT and prevented by OTR antagonists (Condés-Laraet al., 2006; Miranda-Cardenas et al., 2006; Rojas-Piloni et al.,2007). Here, we used mice deficient in OTRs or V1ARs to investigate the physiological contribution of these neurohypophyseal
peptides to pain control and found that the nociceptive phenotype of both KO lines was virtually identical with WT control
animals. Although this may indicate that endogenous OXT and
AVP do not significantly contribute to (non-neuropathic) pain
processing, we cannot exclude the possibility of compensatory
mechanisms in the mutant mice.
Systemic oxytocin elicits analgesia through the V1AR
As mentioned above, anatomical data point toward the oxytocinergic system as a strategically located player in spinal pain
processing. A number of groups have studied the cellularmechanisms by which spinal OTRs contribute to analgesia (Joet al., 1998; Condés-Lara et al., 2003; Rojas-Piloni et al., 2007).
Recently, it has been suggested that OXT could act on glutama-J. Neurosci., June 16, 2010 • 30(24):8274 – 8284 • 8281

tergic interneurons located in lamina II of the dorsal horn, which
in turn activate a larger population of inhibitory GABAergic interneurons to reduce the net activity of spinal nociceptive neurons (Breton et al., 2008). Contrary to what would be expectedbased on this information, we found that OXT analgesia was fully
intact in OTR KO mice, but entirely absent in V1AR KOs.
Similarly, when endogenous AVP release was precipitated by a
hyperosmotic stressor, OTR KO, but not V1AR KO mice developed analgesia, suggesting that under physiological conditions as well the V1AR is necessary for mediating the analgesic
effects of increased circulating neurohypophyseal hormones.
Together, these results strongly suggest that, at least systemically, OXT acts via V1ARs, rather than OTRs to produce its
analgesic effects. This hypothesis was confirmed by studies
using V1AR- and OTR-selective antagonists. It is interesting
to note that, when administered systemically, only the V1AR
antagonist could prevent OXT-induced analgesia, whereas
when injected intracerebroventricularly or intrathecally, theOTR antagonist also displayed a partial inhibitory effect on
OXT-induced analgesia. This observation suggests that, centrally, and in particular at the spinal cord level, both receptors
contribute to the analgesic properties of OXT, whereas peripherally, the V1AR is solely responsible for OXT-induced
analgesia.
In the brain, OXT contributes to the regulation of numerous
behaviors and physiological functions (Gimpl and Fahrenholz,
2001; Kiss and Mikkelsen, 2005; Nishimori et al., 2008). We tried
to investigate the receptor basis of OXT modulation of anxietyand depression-like behaviors— using the elevated plus mazeand Porsolt forced-swimming tasks, respectively— but we failed
to obtain any reliable evidence of dose-dependent OXT effects in
WT mice using various routes of administration (data notshown). Interestingly, however, one central effect of OXT,
scratching, was also absent in V1AR KOs, while remaining intact
in OTR KOs. In light of the growing number of studies on neurohypophyseal hormones as key regulators of pair bonding andaffiliative behaviors (Young and Wang, 2004; Storm and Tecott,2005; Lim and Young, 2006; Caldwell et al., 2008; Donaldson andYoung, 2008; Young et al., 2008), our results should be considered before making any definite conclusions as to the receptor
type mediating effects of these peptides.
Oxytocin binding sites in superficial layers of the dorsal horn
On the basis of the high sequence homology of both peptides andtheir receptors, an action of OXT via the V1AR is highly plausible(Chini and Manning, 2007). In contrast, it is difficult to reconcilea V1AR-mediated analgesic effect of OXT with anatomical evidence demonstrating that OTRs are enriched in the superficial
layers of the dorsal horn, whereas V1ARs are only weakly expressed in the adult spinal cord (Tribollet et al., 1997). We would
note that the vast majority of the anatomical literature is from rat,
whereas our pharmacological experiments were performed inmice. Although two studies have systematically compared OTR
and V1AR distribution in mouse CNS, one of them focused on
forebrain structures (Insel et al., 1991), whereas the other examined spinal distribution only as caudally as the trigeminal nucleus
caudalis (Tribollet et al., 2002). One putative explanation for the
V1AR dependency of OXT-induced analgesia would be a species
difference in receptor distribution. Indeed, whereas OXTergicand AVPergic projections are evolutionary well conserved,
marked species differences in brain receptor distribution patterns
have been reported (Tribollet et al., 1988, 1992; Dubois-Dauphin
et al., 1996; Yoshida et al., 2009).

8282 • J. Neurosci., June 16, 2010 • 30(24):8274 – 8284

To investigate whether the spinal distribution of OTRs andV1ARs in mice is distinct from that seen in the rat, an autoradiographic study on lumbar spinal cord sections was performed.
Results clearly show that, globally, murine OTR and V1AR distribution is similar to that seen in rat, with OTRs, but not V1ARs,clustering in superficial layers of the dorsal horn. Although aslightly higher density of AVP binding sites in dorsal versus ventral parts of the spinal cord was observed, the intensity of dorsal
V1AR labeling remains weak and is not a likely explanation for
the above-reported V1AR-dependent OXT analgesia.
Avpr1a mRNA is expressed in dorsal root ganglia
One possible limitation of the present study is that OXT wasgiven intraperitoneally in analgesia experiments, rather than directly into the brain or spinal cord. The rationale for the peripheral route of administration was to circumvent the characteristic
and vigorous scratching response elicited by both intracerebroventricular and intrathecal OXT injections (Meisenberg andSimmons, 1982; Van Wimersma Greidanus et al., 1990; Thurston
et al., 1992) to maintain the animal in a state allowing for accurate
algesiometric testing (Wilson and Mogil, 2001). Since OXT and
AVP are low-molecular-weight peptides, it is highly likely thatthey can cross the blood– brain barrier. Indeed, Jin et al. (2007)
have reported that subcutaneously injected OXT can affect centrally mediated behaviors. Alternatively, intraperitoneally injected OXT could act peripherally. In the peripheral nervous
system, nerve endings innervating the skin originate in primarysensory neurons located in the DRG. In fact, these cell bodies
represent the first locus of nociceptive processing, since they relay
incoming pain signals to supraspinally projecting second-order
sensory neurons of the dorsal horn (Morris et al., 2004). Our gene
expression studies showed that Avpr1a mRNA levels greatly exceed those of Oxtr in the mouse DRG. This is in contrast to thedorsal horn, where OTR expression greatly exceeds V1AR expression. Single-cell gene expression experiments revealed that
⬃34% of DRG neurons are positive for Avpr1a mRNA. Based on
their size (⬍30 ␮m), these neurons probably belong to the population of small- and medium-sized unmyelinated nociceptors.
Thus far, the role of V1ARs in the DRG has received minimal
attention, but Hu et al. (2004) reported a hyperpolarizing effectof AVP in DRG neurons. Thus, it is tempting to speculate thatperipherally administered AVP and OXT both produce analgesia
through V1AR-mediated inhibition of DRG neurons. Additional
experiments will be needed to explore this hypothesis. In particular, it would be useful to conduct behavioral pain tests after AVP
or OXT administration to discrete regions of the CNS via microinjection or viral-mediated delivery.
Conclusions
In addition to demonstrating that analgesia induced by systemic
OXT is in fact mediated by the V1AR, we identify V1ARs expressed in DRGs as a potential target for this effect. Moreover, thescratching behavior provoked by intracerebroventricular OXT isalso mediated by the V1AR. These findings invite reflection on
the receptor substrates and cellular mechanisms of other effects
commonly attributed to OXTergic signaling.

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