RESEARCH ARTICLE
Hypoalgesia Induced by Rewa d
De alua ion in Ra s
Ana Ma ı
´a Jime
´nez-Ga cı
´a
1,4
, Leand o Ruı
´z-Ley a
1,4
, C uz Miguel Cenda
´n
1
,
Ca men To es
2
, Mau icio R. Papini
3
, Ignacio Mo o
´n
4
*
1Depa men o Pha macology, Biomedical Resea ch Cen e (CIBM) and Ins i u e o Neu oscience, Facul y
o Medicine, Uni e si y o G anada, Campus Ciencias de la Salud, 18016, G anada, Spain, 2Depa men o
Psychology, Uni e si y o Jae
´n, Campus Las Lagunillas, 23071, Jae
´n, Spain, 3Depa men o Psychology,
Texas Ch is ian Uni e si y, Fo Wo h, TX, 76129, Uni ed S a es o Ame ica, 4Depa men o
Psychobiology and Resea ch Cen e o Mind, B ain, and Beha io (CIMCYC), Uni e si y o G anada,
Facul y o Psychology, Campus Ca uja, 18071, G anada, Spain
*[email p o ec ed]
Abs ac
Reduced sensi i i y o physical pain (hypoalgesia) has been epo ed a e e en s in ol ing
ewa d de alua ion. Rewa d de alua ion was implemen ed in a consumma o y successi e
nega i e con as (cSNC) ask. Food-dep i ed Wis a a s had access o 32% suc ose du -
ing 16 sessions ollowed by access o 4% suc ose du ing 3 addi ional sessions. An
unshi ed con ol g oup had access o 4% suc ose h oughou he 19 sessions. Pain sensi-
i i y was measu ed using on F ey ilamen s (Expe imen 1) and Ha g ea es he mal s i-
muli (Expe imen 2) in p e aining baseline, 5 min, and 300 min a e ei he he i s (session
17) o second (session 18) de alua ion session in he cSNC si ua ion. Suc ose consump-
ion was lowe in downshi ed g oups ela i e o unshi ed g oups du ing pos shi sessions
— he cSNC e ec . Hypoalgesia was obse ed in downshi ed g oups ela i e o unshi ed
con ols when pain sensi i i y was assessed 5 min a e ei he he i s o second de alua-
ion session, ega dless o he pain sensi i i y es used. Bo h pain sensi i i y es s yielded
e idence o hypoalgesia 300 min a e he second downshi session, bu no 300 min a e
he i s de alua ion session. Whe eas hypoalgesia was p e iously shown only a e he
second de alua ion session, he e we epo e idence o hypoalgesia a e bo h he i s and
second de alua ion sessions using mechanical and he mal nocicep i e s imuli. Mo eo e ,
he hypoalgesia obse ed 300 min a e he second de alua ion session in bo h expe i-
men s p o ides unique e idence o he e ec s o ewa d loss on sensi i i y o physical pain
5 hou s a e he loss episode. The unde lying neu obeha io al mechanisms emain o be
iden i ied.
In oduc ion
Sensi i i y o physical pain is in luencedby a a ie y o emo ional s a es [1,2], including s ess
inducedby immobiliza ion[3] and ood dep i a ion [4]. The emo ional modula iono physical
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 1 / 15
a11111
OPEN ACCESS
Ci a ion: Jime
´nez-Ga cı
´a AM, Ruı
´z-Ley a L,
Cenda
´n CM, To es C, Papini MR, Mo o
´n I (2016)
Hypoalgesia Induced by Rewa d De alua ion in
Ra s. PLoS ONE 11(10): e0164331. doi:10.1371/
jou nal.pone.0164331
Edi o : Yuqing Li, Uni e si y o Flo ida, UNITED
STATES
Recei ed: Augus 13, 2016
Accep ed: Sep embe 25, 2016
Published: Oc obe 20, 2016
Copy igh : This is an open access a icle, ee o all
copy igh , and may be eely ep oduced,
dis ibu ed, ansmi ed, modi ied, buil upon, o
o he wise used by anyone o any law ul pu pose.
The wo k is made a ailable unde he C ea i e
Commons CC0 public domain dedica ion.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape .
Funding: This pape was pa ially suppo ed by
g an PSI-2013-44945-P om he Minis y o
Economy and Compe i i eness, Spain; he PhD
P og amme in Biomedicine and he Mas e ’s
p og am in Neu oscience and Pain, Uni e si y o
G anada, Spain. MRP’s pa icipa ion was
suppo ed by a Fulb igh US Schola Awa d. The
unde s had no ole in s udy design, da a collec ion
and analysis, decision o publish, o p epa a ion o
he manusc ip .
pain alsooccu sa e an expe iencein ol ing he de alua iono a la ge ewa d, as documen ed
below. In he p esen expe imen s, ewa d de alua ion was implemen ed in e ms o he con-
summa o y successi e nega i e con as (cSNC) ask [5]. In a ypical cSNC expe imen ,ani-
mals ecei e ee access o 5 min o a high- aluesuc ose solu ion ( ypically 32% suc ose)
du ing se e aldaily sessions, ollowed by se e alsessions o access o 4% suc ose. Pe o mance
du ing hese downshi sessions is compa ed o he consumma o y beha io o animals ha
ha e always ecei ed access o 4% suc ose (unshi ed con ols). The cSNC e ec in ol es a sup-
p ession o consumma o ybeha io in 32- o-4% suc ose animals ollowed by a eco e yo le -
els simila o hose o unshi ed con ols. The ini ial supp ession ( ypically obse eddu ing he
i s de alua ionsession) and he eco e y ha ollows ( ypicallys a ingdu ing he second
de alua ion session) a e dissociables ages o he cSNC e ec ,as ex ensi ely demons a ed by
Flahe y’s esea ch on cSNC [5]. Se e al sou ces o e idencesugges ha cSNC modula es and
is also modula ed by physical pain. In one expe imen [6], sensi i i y o physical pain was
assessedin e ms o he paw-wi hd awal la ency in he ho pla e a e ewa d de alua ion.The
esul s showed educed pain sensi i i y (i.e.,inc easedpaw-wi hd awal la ency) a e he sec-
ond 32- o-4% suc ose downshi session,bu no a e he i s one, ela i e o unshi edcon-
ols. Con e sely, a subcu aneous o malin injec ionin a hind paw be o e he i s and second
downshi sessions enhanced he consumma o y supp ession induced by ei he a 32- o-4% o a
16- o-4% suc ose downshi [7]. These s udies add o g owing e idencesugges ing ha si ua-
ions ha ac ually o po en ially in ol e issue damage sha e common unde lyingmechanisms
wi h si ua ions in ol ing ewa d de alua ion.Basedon ex ensi e e idence,Papini e al. [8] sug-
ges ed a connec ionbe weenphysical pain ( issue damage) and psychologicalpain ( ewa d
loss) ha in i es u he compa isons be ween hese wo se s o phenomena. Fo example, opi-
oid ligands known o modula e physical pain also modula e cSNC, ei he du ing he i s down-
shi session ([D-Pen2,D-Pen5]enkephalin, a selec i edel a- ecep o agonis ; [9]), du ing he
second downshi session (U50,488H, a selec i ekappa- ecep o agonis ; [10]), o du ingbo h
sessions (mo phine;[11]). Two opioid- ecep o an agonis s enhance he cSNC e ec ei he
selec i elydu ing he i s downshi session (nal indole, a selec i edel a ecep o an agonis )
o du ing bo h he i s and second downshi sessions (naloxone, a nonselec i eopioid ecep-
o an agonis ) [12]. Simila ly, lesions o b ain a eas in ol ed in pain p ocessing, such as he
an e io cingula e co ex, also a ec eco e y om ewa d downshi om he seconddown-
shi session onwa d [13].
The goalo heseexpe imen swas o look o e idenceo he modula iono physical pain by
ewa d de alua ion du ing he i s and second downshi sessions by using di e en echniques
o assess pain sensi i i y: he on F ey es and he Ha g ea es es . These es s es ima e pain
h esholds in e ms o paw-wi hd awal la ency applying a localizedmechanicalo he mals im-
ulus. Bo h a e ela i ely easy o apply, and ha e beenex ensi ely used wi h oden s [14,15,16,17]
and e en humans [18]. Addi ionally, hese expe imen s we e designed o p o ide e idenceo
he pos session ime cou se o he e ec by measu ingpain sensi i i y 5 and 300 min a e he
end o he ewa d de alua ionsession.The on F ey es consis s o ilamen s a yingin hick-
ness ha a e p essed agains he skino a hind paw. De ec iono he mechanicalp essu e esul s
in paw wi hd awal, hus p o iding an objec i emeasu e o mechanicalhypo- o hype algesia.
The Ha g ea es es es ima es pain sensi i i y also in e ms o paw wi hd awal, bu a e apply-
ing a localized he mal s imulus [16]. These echniques di e in he ype o nocicep i es imulus
used—mechanicalo he mal. Mo eo e , hese echniques s imula e only a small pa ch o skin
in a hind leg and, he e o e, a e p obably mo e sensi i e han o he echniques, such as he ho
pla e es p e iously used in a simila expe imen [6]. An addi ional ad an age o he on F ey
and Ha g ea es es s o si ua ions in ol ing epea ed es ing is ha he e is li le e idence ha
hey induce a e si e condi ioningin in ac animals [19]. By con as , jus a single exposu e
Hypoalgesia and Rewa d De alua ion
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 2 / 15
Compe ing In e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
using he ho pla e es has beenshown o induce a e si e condi ioning [20]. Thus, using he
on F ey es (Expe imen 1) and Ha g ea es es (Expe imen 2) would end o minimizea pos-
sible in e ac ion be ween ewa d de alua ion and a e si e condi ioning.Based on p e ious
esul s [6], a hypoalgesice ec was expec eda e he seconddownshi session, bu no a e
he i s downshi session, a leas when es ing occu ed5 min a e he session. Tes ing 300
min a e bo h sessions was expec ed o yield li le o no e idenceo changes in pain sensi i i y.
While he e is no a ailable e idencedesc ibing he pos session ime cou se o pain sensi i i y
e ec sa e ewa d de alua ion,pos ainingd ug manipula ions in he cSNC ask ypically
ailed o show e ec sa e a 180-min in e al [21,22].
Ma e ials and Me hod
Subjec s
In Expe imen 1, he subjec swe e 41 male Wis a a s pu chased om Ha lan Labo a o ies
(Ba celona, Spain). In Expe imen 2, he subjec swe e 40 male Wis a a s pu chased om
Cha les Ri e s (Les Oncins, F ance). A pa o i uscon amina ion in a s om Ha lan Labo a-
o ies explains he swi ch o endo s; howe e , qua an ine (one week) and habi ua ion o he
labo a o y we e done as usual. Ra s we e app oxima ely 75 days old a he beginningo each
expe imen .The mean (±SEM) ad lib weigh o all he a s was 281.2 (±2.1) g. Ra s we e housed
indi iduallyin polyca bona e cages wi h ad lib wa e , in a oom wi h cons an empe a u e
(24°C) and humidi y (50–60%). Animals we e housed unde a 12:12 h cycle o ligh : da kness
(ligh son a 08:00 h) and ooddep i ed o 82–85% o hei ad lib weigh s h oughou he
expe imen .Dep i a ion le els we e main ained by p o iding a chow a leas 5–50 min a e
he end o all beha io al es ing. In Expe imen 1, a chow was om Ha lan, Mucedola, I aly;
in Expe imen 2, a chow om En igo ( o me ly Ha lan), Ba celona, Spain. Wa e was con in-
uously a ailable in he home cage ( hese a s we e ne e wa e dep i ed).These animals pa ic-
ipa ed in a p e ious expe imen in an ins umen al successi e nega i e con as (iSNC) ask in
a unway si ua ion, wi h solid oodpelle sas ewa d. These animals also had pos session access
o ei he 2% e hanol o wa e . Assignmen o he newcondi ionswas ma ched as a as possible
o p io expe ience.In Expe imen 1, he numbe s o a s wi h downshi ed/unshi edp io
expe iencewe e 6/4, 4/6, 6/5, and 6/4, espec i ely, o G oups 32/17, 4/17, 32/18, and 4/18. In
Expe imen 2, he equi alen numbe s we e 6/4, 6/4, 4/6, and 4/6, espec i ely, o G oups 32/
17, 4/17, 32/18, and 4/18. As o p io e hanol/wa e exposu e, he numbe s o hesame
g oups we e 7/3, 7/3, 8/3, and 8/2 in Expe imen 1, and 5/5, 5/5, 5/5, and 5/5 in Expe imen 2.
The expe imen alp o ocols we e app o ed by he Uni e si y o G anada Resea ch E hics
Commi ee.
Appa a us
All he beha io al p ocedu es (cSNC and pain sensi i i y measu es) we e conduc edin he
same lab oom. The cSNC p ocedu e was iden ical o bo h expe imen s. cSNC aining was
ca iedou in ou boxes made o clea Plexiglas and measu ing30 x 30 x 15 cm (L x H x D).
A g adua ed cylinde (in 0.01-ml uni s) con aining he suc ose solu ion was in oduced o he
insidein he cen e o one o he la e al walls o he box.The amoun o suc osesolu ion con-
sumedby each animal in each session (in millili e s)was ob ainedby sub ac ing he amoun
o suc ose solu ion eco deda e he session om he amoun p o ided be o e he session.
Animals licked he solu ions om a me allic sippe ube p o uding 3 cm inside he box.
Suc ose solu ions we e p epa ed weigh /weigh by mixing 32 g o comme cial suga o e e y
68 g o dis illedwa e (32% suc ose)and 4 g o suga o e e y96 g o dis illedwa e (4%
suc ose).
Hypoalgesia and Rewa d De alua ion
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 3 / 15
The on F ey and Ha g ea es es s we e conduc ed in wo boxes, also made o clea Plexi-
glas, and measu ing20 x 20 x 24 cm (L x H x D). Fo he on F ey es , he loo was made o
aluminum ba s. Fo he Ha g ea es es he loo was made o glass. Pain sensi i i ywas de e -
mined by measu ing he paw-wi hd awal esponse o a punc a e mechanical o he mal s imu-
la ion o one o he hindpaws. In he on F ey es , s imula ion in ol ed applying one o a
ange o 9 on F ey ilamen s (Touch-Tes Senso yE alua o s, No h Coas Medical, CA,
USA) anging om 0.4 o 10 g (3.92–98.1mN) o a hind paw. Fo he Ha g ea es es , adian
hea (42–43°C h ough he glass loo , o a maximum o 13 s) was applied by a plan a es
appa a us (Ugo Basile,Come io, I aly). Tempe a u e was kep cons an h oughou he
expe imen .
P ocedu e
To he au ho s’ knowledge, his is he i s expe imen explo ing he e ec so ewa d downshi
in he cSNC si ua ion on physical pain assessedwi h he on F ey ilamen sand he Ha g ea es
he mal es . The p o ocols implemen ed he e combine es ing pa ame e s usedsepa a ely in
p io expe imen sin ol ing cSNC, on F ey es ing,and Ha g ea es es ing.
cSNC aininglas ed 19 daily sessions: 16 p eshi sessions ollowed by 3 pos shi sessions.
Fou g oups we e included:32/17 (n= 10), 4/17 (n= 10), 32/18 (n= 11 in Expe imen 1,
n= 10 in Expe imen 2), and 4/18 (n= 10). In g oup labels, he i s numbe e e s o he
suc ose concen a ion adminis e ed du ing p eshi sessions (32 o 4% suc ose;all animals had
access o 4% suc osedu ingpos shi sessions),whe eas he secondnumbe e e s o he ses-
sion when he on F ey es o Ha g ea es es was adminis e ed, ei he a e he i s pos shi
session (17) o he second pos shi session (18).
Each day, he animal ack was mo ed in o he expe imen al oom, a s we e allowed 15 min
in hei home cage o se led,and hen hey we e placedin he con as box. The con as ses-
sion las ed 5 min om he i s con ac wi h he sippe ube. Session du a ion o each box was
measu ed manually wi h digi al clocks (Digi al Ons a 100). Animals we e un in squads o 4
and he o de o squads a iedac oss days. A he end o all sessions, e e yday, con as boxes
we e wipedwi h a we pape owel and eceswe e emo ed whenp esen .
In Expe imen 1, Von F ey es ing was conduc ed 5 and 300 min a e session 17 o G oups
32/17 and 4/17, and a e session 18 o G oups 32/18 and 4/18. Animals ecei edone addi-
ional pos shi session (19) o de e minewhe he on F ey es ing a ec ed consumma o y
beha io on he ollowing session.To minimizeno el y e ec swi h he on F ey boxes, ani-
mals we e amilia izedbe o e he c i ical es sessions acco ding o he ollowing schedule.
Baselinemeasu emen s we e conduc ed wo days be o e he s a o iSNC aining,du inga
p e ious phase no epo ed he e (see Subjec s o a desc ip ion o p e ious expe ience).In
addi ion, a s we e exposed o he on F ey boxes o 5 min pe session,5 min a e each o
eigh p eshi sessions du ing he cu en expe imen .Du ing hese 8 box-exposu e sessions no
measu emen s we e aken wi h on F ey ilamen s.
In e e y es , each ilamen was applied h ee imes o 2–3 s, sepa a ed by 5-s in e als
using he up-down pa adigm[23]. Tes ing s a ed wi h he 2-g (19.6 mN) on F ey ilamen
(i.e., he middle o he ange). The ilamen was manually p essed agains he paw’s plan a
su ace wi h su icien o ce o cause a dep ession in he skin. The paw chosen o s imula-
ion was always he same o a gi en animal and i was coun e balanced o igh and le
hind paw wi hin each g oup (5/5 o h ee g oups and 6/5 o G oup 32/18). In each
consecu i e es , i he e was no esponse o he ilamen , a s onge s imulus was hen
selec ed;i he e was a posi i e esponse, a weake one was hen used.The esponse o he
Hypoalgesia and Rewa d De alua ion
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 4 / 15
ilamen was conside ed posi i e when immedia e wi hd awal o shaking o he paw was
obse ed.Obse e swe e blind wi h espec o he con as assignmen o he subjec (i.e.,
32% s. 4%).
In Expe imen 2, he mal pain sensi i i y was assessedwi h a echnique desc ibedin [24]
wi h sligh modi ica ions.Ha g ea es es s we e conduc ed 5 and 300 min a e session 17 o
G oups 32/17 and 4/17, and a e session 18 o G oups 32/18 and 4/18. Animals ecei edone
addi ional pos shi session (19) o de e minewhe he Ha g ea es es ing a ec edconsumma-
o y beha io on he ollowing session.To minimizeno el y e ec swi h he Ha g ea es es
boxes, animals we e amilia izedbe o e he c i ical es sessions acco ding o he ollowing
schedule.Baselinemeasu emen s we e conduc ed wo days be o e he s a o iSNC aining
(see Subjec s).In addi ion, a s we e exposed o he Ha g ea es es boxes o 5 min pe ses-
sion, 5 min a e each o eigh p eshi sessions du ing Expe imen 2. A ideo came a (Sony
Handycam HD) was placed o e he Ha g ea es es boxes. Du ing hese sessions, no ideo
eco dings o pain measu emen s we e aken.
Pain sensi i i y was assessed o each animal a e session 17 in G oups 32/17 and 4/17, o
a e session 18 in G oups 32/18 and 4/18. The Ha g ea es es s a ed wi h 5 min o habi ua-
ion o he boxes. Du ing his pe iod, oile pape was placed on he loo o collec u ine and
eces. A e 5 min, he oile pape was emo ed and a beamo adian hea was ocused o he
plan a su aceo a hindpaws wi h a plan a es appa a us, un il he a made a wi hd awal
esponse. Paw wi hd awal in e up ed he ligh e lec ed om he paw on o a pho ocell and
au oma ically u ned o he ligh and he ime . The la ency o he wi hd awal esponse (as an
indi ec measu e o he hea -induced pain h eshold) was hus eco ded au oma ically. The
in ensi y o he ligh was adjus ed a he s a o Expe imen 2 such ha a e age baseline
la ency was abou 13 s. This in ensi y was ne e changed. Each a was es ed wice al e na ely
on each hind paw. All he la encies eco ded we e a e aged o ob ain a single la ency measu e
pe animal. Typically, a measu emen was de i ed om ou eco dings, wo om each hind
paw. A leas 1 min was allowed be weenconsecu i e measu emen s in he same paw. A cu -o
la ency ime o 30 s was used o a oid skin damage and minimize pain. A Ha g ea es es usu-
ally las ed 15 min.
S a is ics
In he cSNC si ua ion, he dependen a iable was he o al amoun o suc ose consumedin
each session (in millili e s).In Expe imen 1, pain sensi i i y in he on F ey es was exp essed
as a mechanical h eshold p oducing a esponse in 50% o he ials.This paw wi hd awal
h eshold alue was calcula ed using he ollowing o mula:
50% h eshold ðgÞ ¼ 10ðX þkdÞ=10;000
whe e X
is he alue (in log uni s) o he inal on F ey ilamen used,κis he abula alue (see
Appendix in [23]) o he pa e n o posi i e/nega i e esponses, and δis he mean di e ence
(in log uni s) be weens imuli. In Expe imen 2, pain sensi i i y in he Ha g ea es es was
exp essed in e ms o he mean paw-wi hd awal la ency (in seconds)o e measu emen s aken
om each paw. All da a analyses we e compu ed wi h he IBM SPSS S a is ics 21 package. The
speci ic analysis o a iance used is desc ibedin he Resul s sec ion.In e ac ions we e analyzed
wi h pai wiseLSD es s de i ed om he main analysis. No mali y was assessed wi h he Kol-
mogo o -Smi no es whene e a signi ican e ec was de ec ed in downshi sessions and in
he on F ey and Ha g ea es es s o minimizeType I e o . The alpha alue was se a he 0.05
le el in all s a is ical analyses.
Hypoalgesia and Rewa d De alua ion
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 5 / 15

Resul s
Expe imen 1
A Con as (32% s. 4% suc ose) x on F ey (session 17 s. 18) x Session (1–16) analysis o p e-
shi consumma o y pe o mance indica ed ha 32% g oups consumed signi ican lymo e
suc ose han 4% g oups, F(1, 37) = 28.65, p<0.001, and also he e was a signi ican inc ease in
consump ion ac oss sessions, F(15, 555) = 87.15, p<0.001. Suc ose consump ion du ing he
las p eshi session (16) and he h ee pos shi sessions (17–19) is p esen ed in Fig 1. Con-
sump ion was highe in g oups exposed o 32% suc ose han o 4% suc oseon session 16, he
las p eshi session,F(1, 37) = 20.80, p<0.001, bu he e was no di e encebe weeng oups
assigned o on F ey es ing on session 17 o 18, o in e ac ion be ween hese wo ac o s,
Fs<1.91, ps>0.17. An analysis o pos shi sessions da a indica ed ha he e was a weak cSNC
e ec las inga singlesession.This analysis yieldeda signi ican in e ac ionbe weencon as
and pos shi session,F(2, 74) = 5.68, p<0.006, and LSD pai wisecompa isons con i med ha
downshi ed g oups consumed signi ican ly less suc ose on session 17 han unshi edcon ols,
F(1, 37) = 4.48, p<0.05. The di e enceswe e no signi ican o pos shi sessions 18 and 19.
The e was no e idenceo de ia ions om no mali y on sessions 16–19 (s a is ics: <0.18,
ps>0.09). Thus, consumma o y beha io showed no e idenceo a downshi e ec on session
18, be o e he on F ey es was adminis e edin G oups 32/18 and 4/18. Addi ionally, he e
was no e idence ha on F ey es ing in one day a ec edconsumma o y beha io in he con-
as box he ollowing day.
Fig 1. Mean (±SEM) suc ose consump ion (ml) du ing he las p eshi session (16) and ei he session 17 o 18
(Pos ) depending on he g oups. 32: animals exposed o ewa d de alua ion om 32% o 4% suc ose du ing pos shi
sessions. 4: animals exposed o an unshi ed ewa d condi ion, always ecei ing access o 4% suc ose h oughou he
expe imen . The as e isk e lec s a signi ican di e ence be ween bo h downshi ed g oups s. bo h unshi ed con ols (see
ex o de ails).
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PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 6 / 15
Fig 2 shows he mean mechanicalpain h esholds in g oups es ed on baseline session, and
a e session 17 ( op) o 18 (bo om). Whe eas he baselinemeasu emen was ob ained a he
same pos session ime o all animals, he pos shi measu emen s we e ob aineda e he con-
as session. Thus, hese h ee alues (baseline,5 min, and 300 min) a e sepa a ed by di e en
ime in e al.Because he ele an compa isons a e be weendownshi ed and unshi edg oups
es ed equally on any gi en day, excep o hei p io his o y, hese esul s we e analyzed sepa-
a ely wi h one-way designs.
Baseline measu emen s did no di e be weendownshi ed and unshi ed g oups o bo h
es day condi ions, Fs<1. Two main ou comes a e obse edin Fig 2. Fi s , in g oups es ed
a e he i s downshi session (32/17, 4/17), pain h esholds inc ease a e 5 min, bu hey
dec easeda e 300 min o ma ch he alues o unshi edcon ols. This was con i meds a is i-
cally. Rela i e o unshi edcon ols, downshi ed animals exhibi ed hypoalgesia5 min a e ses-
sion 17, F(1, 18) = 8.61, p<0.01, bu no 300 min a e ha session, F<1. Second,in g oups
es ed a e he second downshi ial, pain h esholds also inc easedin downshi edanimals
ela i e o unshi ed con ols a e 5 min, F(1, 19) = 14.28, p<0.002, bu , unexpec edly, his
g oup di e ence emained signi ican e en 5 hou s a e he end o he seconddownshi ses-
sion, F(1, 19) = 13.58, p<0.003. No e idenceo de ia ions om no mali y was de ec ed on any
o he signi ican e ec sshown in Fig 2 (s a is ics: <0.23, ps>0.18).
The esul s epo edabo e we e no dependen on g oup di e encesin eedingmo i a-
ion, as assessed in e ms o bodyweigh . The mean (±SEM) weigh s ac oss sessions 1–19
we e 239.6 (5.5), 240.0 (5.6), 232.4 (3.2), and 231.6 (4.1) g o G oups 32/17, 32/18, 4/17, and
4/18, espec i ely. A Con as x on F ey analysis de ec edno e ec s,all Fs<1. A simila
analysis on he mean weigh s du ing pos shi sessions 17–19 also de ec edno e ec s,all
Fs<1.
Expe imen 2
The esul s o he cSNC ask we e analyzed wi h Con as (32% s. 4% suc ose)x Ha g ea es
es (session 17 s. 18) x Session analyses. Da a om ou indi idualsessions, one om each
g oup, we e los due o echnical di icul y;in hese cases, he g oup a e age was subs i u ed
o he missing alue. P eshi consumma o y pe o mance(sessions 1–16) showed ha a s
consumed signi ican lymo e 32% suc ose han 4% suc ose,as shown by signi ican e ec s o
con as , F(1, 36) = 84.33, p<0.001, and o he con as by session in e ac ion,F(15, 540) =
5.94, p<0.001. The e was also a signi ican inc ease in consump ion ac oss p eshi sessions,
F(15,540) = 73.22, p<0.001. O he e ec swe e no signi ican .Fig 3 shows he pe o mance
du ing he las p eshi session, session 16; an analysis o jus his session indica ed a signi i-
can con as e ec ,F(1, 36) = 11.85, p<0.002. Fig 3 also shows he esul s o he h ee pos -
shi sessions, sessions 17–19. A simila analysis yielded a signi ican con as by session
in e ac ion,F(2, 72) = 9.64, p<0.001. In addi ion, he e we e signi ican main e ec s o con-
as and Ha g ea es es , Fs(1, 36) >4.74, ps<0.04. The sou ce o he con as by session
in e ac ionwas a signi ican lylowe suc ose consump ion o downshi edg oups compa ed
o unshi ed con ols on sessions 17 and 18, as indica ed by LSD pai wisecompa isons,
Fs(1, 36) >11.61, ps>0.003. Ac oss all sessions o aining, he e we e no signi ican in e ac-
ions in ol ing he Ha g ea es es ac o , which indica ed ha g oup assignmen s we e no
biased.Howe e , du ing pos shi sessions, he g oups es ed o physical pain a e session
17 consumed signi ican lymo e suc ose han he g oups es ed a e session 18. No ice ha
since he iple in e ac ion was nonsigni ican ,F<1, he sizeo he cSNC e ec was simila
in bo h se s o downshi ed-unshi edg oups. The e was no e idenceo de ia ions om no -
mali y on sessions 16–19 (s a is ics: <0.24, ps>0.12).
Hypoalgesia and Rewa d De alua ion
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 7 / 15
Fig 2. Th eshold o ce (g) o paw wi hd awal in he on F ey es du ing baseline sessions and ei he 5 o 300
min a e sessions 17 ( op) o 18 (bo om), depending on he g oups. 32: 32- o-4% suc ose downshi . 4: unshi ed
con ols always exposed o 4% suc ose. The as e isks e lec a signi ican di e ence be ween he co esponding
downshi ed s. unshi ed g oups (see ex o de ails).
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PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 8 / 15
Fig 4 shows he paw-wi hd awal la ency in he Ha g ea es es o downshi ed and
unshi edg oups es ed a e cSNC session17 ( op) o session 18 (bo om).As in Expe imen
1, measu emen s we e sepa a ed by di e en imein e alsand, he e o e, he esul s we e ana-
lyzed sepa a ely o baseline,5 min, and 300 min es s. Becauseo echnical di icul ies,da a
om wo animals in G oup 32/17, in he 5-min Ha g ea es es , we e los , he e o e lea ingan
n= 8 o his measu e;howe e , hoseanimalsdid p oduce da a o baselineand 300-min es s,
hus lea ing an n= 10 o bo h o hese measu es. In addi ion,one o he wo measu emen s in
ei he he igh o le paw was los in 9 animals om G oups 32/17 and 4/17, in he 5-min es ;
in all hese cases, he secondmeasu e aken om he co espondingpaw was usedin place o
he a e age ( he a e age o wo measu es was used when bo h we e a ailable).
No di e enceswe e obse edin e ms o baselinela encies ei he a e session 17 o 18, Fs
(1, 18) <1.17, ps>0.29. Pain sensi i i y inc eased in downshi ed g oups ela i e o unshi ed
con ols, as measu ed5 min a e session 17 o 18. Thus, la encies we e longe in G oup 32/17
han 4/17, F(1, 16) = 6.43, p<0.03, and in G oup 32/18 han 4/18, F(1, 18) = 38.26, p<0.001.
As in Expe imen 1, he Ha g ea es es yielded di e en e ec sa he 300-min in e al
dependingon he session. A e he i s downshi e en , la encies in G oups 32/17 and 4/17
had becomenondi e en ial,F<1. Howe e , a e he seconddownshi e en , paw-wi h-
d awal la ency was s ill longe in G oup 32/18 han in G oup 4/18, F(1, 18) = 20.43, p<0.001.
None o he signi ican e ec sshown in Fig 4 we e basedon samples ha de ia ed om no -
mali y (s a is ics:<0.26, ps>0.05).
The esul s epo edabo e we e no dependen on g oup di e encesin eedingmo i a ion,
as assessed in e ms o bodyweigh . The mean (±SEM) weigh s ac oss sessions 1–18 (weigh s
Fig 3. Mean (±SEM) suc ose consump ion (ml) du ing he las p eshi session (16) and pos shi sessions (17–19).
32: animals exposed o a 32- o-4% suc ose downshi du ing pos shi sessions. 4: animals exposed o an unshi ed ewa d
condi ion, ecei ing access o 4% suc ose h oughou he expe imen . The as e isks e lec signi ican di e ences be ween
downshi ed s. unshi ed g oups (see ex o de ails).
doi:10.1371/jou nal.pone.0164331.g003
Hypoalgesia and Rewa d De alua ion
PLOS ONE | DOI:10.1371/jou nal.pone.0164331 Oc obe 20, 2016 9 / 15