ORIGINAL RESEARCH
published: 17 Decembe 2018
doi: 10.3389/ nins.2018.00952
F on ie s in Neu oscience | www. on ie sin.o g 1Decembe 2018 | Volume 12 | A icle 952
Edi ed by:
Rubem C. A. Guedes,
Fede al Uni e si y o Pe nambuco,
B azil
Re iewed by:
Robe a Mon e azzo Cysnei os,
Mackenzie P esby e ian Uni e si y,
B azil
Na isa M. Jada ji,
Ca le on Uni e si y, Canada
*Co espondence:
Valen ina Eche e ia
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Neu oene ge ics, Nu i ion and B ain
Heal h,
a sec ion o he jou nal
F on ie s in Neu oscience
Recei ed: 24 July 2018
Accep ed: 30 No embe 2018
Published: 17 Decembe 2018
Ci a ion:
Mendoza C, Pe ez-U u ia N,
Al a ez-Rica es N, Ba e o GE,
Pé ez-O dás R, Ia ko A and
Eche e ia V (2018) Co inine Plus K ill
Oil Dec eased Dep essi e Beha io ,
and Inc eased As ocy es Su i al in
he Hippocampus o Mice Subjec ed
o Res ain S ess.
F on . Neu osci. 12:952.
doi: 10.3389/ nins.2018.00952
Co inine Plus K ill Oil Dec eased
Dep essi e Beha io , and Inc eased
As ocy es Su i al in he
Hippocampus o Mice Subjec ed o
Res ain S ess
C is hian Mendoza1, Nelson Pe ez-U u ia1, Na halie Al a ez-Rica es1,
Geo ge E. Ba e o2,3, Raquel Pé ez-O dás4, Alex Ia ko 1and Valen ina Eche e ia1,5*
1Uni e sidad San Sebas ián, Fac. Cs de la Salud, Concepción, Chile, 2Depa amen o de Nu ición y Bioquímica, Facul ad de
Ciencias, Pon i icia Uni e sidad Ja e iana, Bogo á, Colombia, 3Ins i u o de Ciencias Biomédicas, Uni e sidad Au ónoma de
Chile, San iago, Chile, 4Facul ad de Ciencias de la Ac i idad ísica y el depo e, Uni e sidad Pablo de Ola ide, Se illa, Spain,
5Resea ch & De elopmen Se ice, Bay Pines VA Heal hca e Sys em, Bay Pines, FL, Uni ed S a es
Res ain s ess (RS) is a condi ion a ec ing millions o people wo ldwide. The
in es iga ion o new he apies o alle ia e he consequences o p olonged RS is much
needed. Co inine, a nico ine-de i a i e, has shown o p e en he dec ease in ce eb al
synap ic densi y, wo king memo y de ici s, anxie y, and dep essi e-like beha io a e
p olonged es ain s ess (RS) in mice. Fu he mo e, pos - ea men wi h co inine
educed he ad e se e ec s o ch onic RS on as ocy e su i al and a chi ec u e. On
he o he hand, he nu i ional supplemen k ill oil (KO), has shown o be bene icial in
dec easing dep essi e-like beha io and oxida i e s ess. In his s udy, in he sea ch o
e ec i e p e en a i e ea men s o be used in people subjec ed o educed mobili y,
he e ec o co- ea men wi h co inine plus KO in mice subjec ed o p olonged RS was
in es iga ed. The esul s show ha co inine plus KO p e en ed he loss o as ocy es,
he appea ance o dep essi e-like beha io and cogni i e impai men induced by RS.
The use o he combina ion o co inine plus KO was mo e e ec i e han co inine alone in
p e en ing he dep essi e-like beha io in he es ained mice. The po en ial use o his
combina ion o alle ia e he psychological e ec s o educed mobili y is discussed.
Keywo ds: dep ession, co inine, anxie y, es ain s ess, k ill oil, as ocy es
INTRODUCTION
S ess is gene a ed when an indi idual is unable o cope wi h o e whelming physical o
psychological demands. Al hough, he ho monal and beha io al changes ha occu in esponse
o h ea ening s imuli is c ucial o su i al and can be bene icial in ec ui ing adap i e esponses
o cope wi h a s ess ul si ua ion, howe e , p olonged s ess can esul in maladap i e changes ha
may lead o men al illness, cogni i e and mo o de ici s (Yehuda e al., 1998; Hammen, 2003; Luine
e al., 2007).
In he clinical ealm, he emo ional and physical al e a ions induced by RS u he diminish
he quali y o li e o people wi h es ic ed mobili y as s ess/induced Neu oin lamma ion induce
Mendoza e al. Co inine Plus K ill and S ess
dep ession (Iwa a e al., 2013; Musca elli e al., 2017). A me a-
analysis including 354 s udies, 18,374 indi iduals e ealed ha
mo e han 70% o dep essed indi iduals showed signs o s ess
such as ele a ed co isol le els in plasma(S e le and Mille ,
2011).
S ess esponse when engaged o ex ended du a ions o
ac i a ed by a auma ic e en ; i is linked o he dys egula ion
o he Hypo halamus-pi ui a y-ad enal (HPA) axis (Baue
e al., 2001). This dys egula ion esul s in al e ed le els o
glucoco icoid ho mones and neu o ansmi e s in he b ain
and may lead o psychological dep ession (Hayase, 2011). Fo
example, since he HPA axis has bidi ec ional ela ionships
wi h he se o onine gic sys em i s dys egula ion can esul
in dec eased le els o se o onin leading o dep ession and
i i abili y. Also, neu ons om he amygdala (AMY), which
a e esponsi e o he co ico opin- eleasing ho mone (CRH),
p ojec o he aphe nuclei, he main se o onin sou ce o he
o eb ain. In addi ion, dys egula ion o he HPA axis co ela es
wi h mo phological al e a ions o he b ain such as he educ ion
o hippocampal olume obse ed in indi iduals wi h majo
dep essi e diso de (MDD) and pos auma ic s ess diso de
(PTSD) (Sheline, 2000; Bonne e al., 2001; Czeh e al., 2001;
Schmi z e al., 2002; Villa eal e al., 2002; D e e s e al., 2008;
Felmingham e al., 2009; Ap el e al., 2011; Filipo ic e al., 2011;
Gonul e al., 2011; Teiche e al., 2012; Admon e al., 2013;
Ahmed-Lei ao e al., 2016).
Ch onic s ess has many o he ha m ul e ec s on
he b ain including neu oin lamma ion, oxida i e s ess,
mic ogliosis, educed neu ogenesis, and diminished numbe s
and a chi ec u al complexi y o neu ons. Ch onic s ess also
e okes synap ic al e a ions including spine numbe and shape
(Kassem e al., 2013; Benne and Lagopoulos, 2014; Scha man
and MacLusky, 2014). In oden s, es ain s ess (RS) a ec s
spa ial memo y (Bowman e al., 2002; Kleen e al., 2006)
and long- e m po en ia ion, a cellula model o lea ning and
memo y p ocesses, in he hippocampus (Pa lides e al., 2002).
Such e ec s ha e been associa ed wi h he e ac ion o apical
dend i es, as well as he loss o synapses in he CA1, CA3, and
den a e gy us (DG) sub- egions o he hippocampus (McEwen
e al., 1997; Maga inos e al., 2011). These cellula changes and
b ain connec i i y, con ibu e o he de elopmen o PTSD in
people subjec ed o auma and su e ing wi h educed mobili y.
As a esul , se e al neu ological symp oms appea such as
wo king memo y loss, impulsi i y, agg essi e beha io , and
dep ession in auma ized indi iduals who de eloped PTSD
(Ta e and Be na dini, 2003; Reagan e al., 2008; Con ad and
Bimon e-Nelson, 2010; Luine, 2016; Mo ei a e al., 2016).
Abb e ia ions: AMPA, α-amino-3-hyd oxy-5-me hyl-4-isoxazolep opionic acid;
ANOVA, Analysis o a iance; CRH, co ico ophin eleasing ho mone; FS, Fo ced
swimming; GFAP, Glial ib illa y acidic p o ein; HPA, Hypo halamus-pi ui a y-
ad enal; IR, Immuno eac i i y; KO, K ill oil; MD, Majo dep ession; MAOIs,
Monoamine oxidase inhibi o s; MGV, Mean g ay alues; NOR, No el objec
ecogni ion; OF, Open ield; PBS, Phospha e bu e ed saline; PFC, P e on al
co ex; PTSD, Pos auma ic s ess diso de ; ROI, Region o in e es ; SSRIs,
Region se o onin- eup ake inhibi o s; SNRIs, Se o onin-no epineph ine eup ake
inhibi o s; TBS, T is-bu e ed saline; TBST, TBS wi h 0.1% Tween 20.
Nume ous s udies ha e emphasized he ole o as ocy es
in media ing b ain homeos asis by suppo ing he blood-b ain
ba ie unc ion, sus aining neu onal ene gy me abolism (S oba
and Ande son, 2013), and neu o ansmission (Schousboe e al.,
1992). Fu he mo e, as ocy es modula e synap ic plas ici y
p ocesses including synap ogenesis, neu ogenesis, and lea ning
and memo y (Honsek e al., 2012; Be na dinelli e al.,
2014; Haydon and Nede gaa d, 2014). Fo hese easons,
as ocy es a e conside ed use ul he apeu ic a ge s o se e al
neu ological diso de s (Ga zon e al., 2016; Gonzalez-Gi aldo
e al., 2017).
As ocy es ha e been classi ied acco ding o hei cellula
mo phologies (cell body size, and numbe , leng h, hickness,
di ec ion, and leng h o p ocesses) and loca ion, in wo main
sub ypes, p o oplasmic o ib ous (So oniew and Vin e s, 2010).
P o oplasmic as ocy es a e ound in he g ay ma e and p esen
nume ous s em b anches ha o igina e om se e al b anching
p ocesses in a egula sphe e-like dis ibu ion. Fib ous as ocy es
ha e elonga ed p ocesses and a e p esen in he whi e ma e
(So oniew and Vin e s, 2010). I has been p oposed ha he
mo phology o as ocy es can be a good indica o o hei
unc ions (de Filippis, 2011). Recen ly, Choi e al. s udied he
molecula and mo phological changes o as ocy es induced by
ea condi ioning; he esul s showed a signi ican dec ease o
he immuno eac i i y (IR) o he glial ib illa y acidic p o ein
(GFAP) in he hippocampus o ea condi ioned oden s (Sau
e al., 2016). Ou eam ound a simila educ ion in he
numbe o GFAP+as ocy es in he CA1, CA3, and DG o
he hippocampus a e p olonged es ain s ess (RS) in mice
(Pe ez-U u ia e al., 2017). P e ious s udies indica ed ha in he
hippocampal o ma ion pos ea men wi h co inine no malized
he numbe o GFAP+as ocy es as ocy e and GFAP IR, in
all hippocampal egions being he e ec especially signi ican in
he DG.
In his s udy, ou goal was o assess he e ec o co-
ea men wi h KO plus co inine on GFAP+IR in he DG,
and dep essi e-like beha io and wo king memo y in a mouse
model o p olonged RS. The esul s highligh he ad an age o
using co inine plus a na u al an ioxidan nu ien o diminish
s ess-de i ed dep essi e beha io , cogni i e impai men ,
and as ocy es abno mali ies in he hippocampus a e
p olonged RS.
MATERIALS AND METHODS
Animals
Two-mon h-old male C57BL/6J mice (ob ained om he
Uni e si y o Chile), weighing 25-30 g, we e main ained on a 12-
h (h) ligh /da k cycle (ligh on a 07:00 h) wi h ad libi um access
o ood and wa e and main ained a a con olled empe a u e (25
±1◦C). Upon a i al, mice we e g oup housed and acclima ed
o 7 days be o e beha io al es ing. Expe imen s we e comple ed
du ing he ligh pe iod o he ci cadian cycle. Animal handling
and ca e we e pe o med in compliance wi h he Guide o he
ca e and use o labo a o y animals adop ed by he Na ional
Ins i u e o Heal h (USA), acco ding o a p o ocol app o ed
F on ie s in Neu oscience | www. on ie sin.o g 2Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
by he e hical commi ee o he Uni e sidad San Sebas ian,
Chile.
D ug P epa a ion
Co inine ((5S)-1-me hyl-5-(3-py idyl) py olidine-2-one)
(Sigma-Ald ich Co po a ion, S . Louis, MO, USA) was p epa ed
by dissol ing he powde ed compound in s e ile phospha e
bu e ed saline (PBS). KO capsules (300 mg) we e pu chased
om Walg eens (Supe ba, USA). So gels con ain 300 mg KO
(90 mg omega-3 a y acids, 50 mg eicosapen aenoic acid, 24 mg
docosahexaenoic acid, 130 mg phospholipids). Manu ac u e s
p o ided no in o ma ion abou he As axan hin con en in he
so gels.
Expe imen al G oups and D ug T ea men s
Ini ially, he mice we e acclima ed o 1 week and subjec ed o
handling o educe s ess. Then, mice we e andomly di ided in o
i e g oups (n=8 mice/condi ion) and ea ed ia ga age wi h
0.05 ml o ehicle (PBS, pH 7.4), Co inine (Co ), KO o Co plus
KO dissol ed in 0.05 ml o ehicle as de ailed: (1) con ol non-
es ained mice ea ed wi h ehicle; (2) es ained mice ea ed
wi h ehicle; (3) es ained co inine- ea ed mice (5 mg/kg in
ehicle); (4) es ained KO- ea ed mice (143 mg/kg in ehicle);
(5) es ained mice ea ed wi h co inine (5 mg/kg) plus KO (143
mg/kg, in ehicle). Mice we e ea ed ia a he same ime o
he day, 30 min (min) be o e es ain and con inuously un il
eu hanasia. A e 21 days (d) in o ea men s, mice we e es ed
o beha io in he o de om less s ess ul es o mo e s ess ul
as indica ed in Figu e 1, wi h 24 o es ing be ween es s o pe mi
adequa e es ing o mice and o p e en in e e ence be ween
es . A e he end o expe imen s, mice we e eu hanized using
ce ical disloca ion by well- ained pe sonnel.
Beha io al P ocedu es
Res ain S ess
RS is a eliable me hod o model ch onic s ess ha mimics
he e ec s o es ain s ess on b ain unc ions and beha io in
oden s (Pa e and Gla in, 1986; Jaggi e al., 2011). Mice we e
gen ly in oduced in o 50-ml conical anspa en plas ic ubes
(Co ning Inc.) con aining non-p o uding pe o a ions in bo h
ends and in he walls o pe mi en ila ion. Mice we e kep inside
hese ubes, allowing sligh mo emen s, o 6 h a day o 21 d a
oom empe a u e (RT). A e he daily es ain ime, mice we e
e u ned o hei home cages and pe mi ed o mo e eely o he
es o he day. Following he 3 weeks o RS, mice we e es ed o
locomo o ac i i y and dep essi e-like beha io .
Open Field Tes (OF)
The open ield (OF) es (Belzung and G iebel, 2001) was
conduc ed as p e iously desc ibed wi h mino modi ica ions
(No c oss e al., 2008) o iden i y changes in locomo o ac i i y
in esponse o s ess and d ug ea men s. Mice we e indi idually
placed in a co ne and pe mi ed o eely explo e an open squa e
a ena (40 ×40 ×35 cm) o 10 min (Figu e 2A). To al dis ance
a eled, speed and ime spen in he cen e zone we e measu ed
unde mode a e ligh ing using he ideo acking so wa e (ANY-
Maze, S oel ing Co.).
Fo ced Swim Tes
The o ced swim (FS) is a b oadly used ask o assess dep essi e-
like beha io in oden s (Dalla e al., 2010). The FS is pe o med
in oducing each mouse in he su ace o a anspa en and
inescapable cylinde wo- hi ds illed wi h wa e a 26 ±1◦C
(Figu e 3A). Mice engage in pe iods o in ense mo emen
ollowed o inc easing pe iods o immobili y. The immobili y
ime is conside ed an exp ession o dep essi e-like beha io .
Immobili y ime is de ined as no longe exhibi ing any escape
beha io , mo ionless o mo ing only o keep loa ing. Immobili y
ime du ing a 5-min ial was eco ded and quan i ied by wo
in es iga o s blind o he ea men g oups.
No el Objec Recogni ion (NOR)
This ask e alua es ecogni ion memo y, and i is based on he
na u al p e e ence o oden s o no el objec s when exposed o
new and p e iously encoun e ed objec s (de B uin and Pouze ,
2006). Du ing he ask, a o ed explo a ion o he no el objec
p o ides a measu e o ecogni ion memo y. A e a habi ua ion
s ep in a squa e a ena (40 ×40 ×35 cm), each mouse was
placed in he same a ena bu con aining wo iden ical anspa en
objec s loca ed equidis an o each o he ( amilia iza ion phase)
and led o explo e he objec s o 5 min (Figu e 4A). Then, mice
we e e u ned o hei cages and pe mi ed o es o 30 min.
A e es ing, mice we e placed back in he a ena con aining one
o he amilia objec s and a new objec (Figu e 4B). The ime
explo ing he wo objec s was eco ded o 5 min. Explo a o y
beha io was eco ded and he ime o explo a ion o each objec
was no malized o animal ac i i y by calcula ing he explo a ion
index (EI) ha co esponds o he ime spen by he mouse
explo ing one o he equal objec s o he new objec / o al ime
spen explo ing bo h objec s ×100%. The beha io al eco ding
and analysis we e pe o med using he (ANY-Maze, S oel ing
Co.).
Suc ose P e e ence Tes
The suc ose p e e ence es was used o assess anhedonia, an
indica o o dep essi e-like beha io . The suc ose p e e ence es
was pe o med as p e iously desc ibed (Se cho e al., 2016)
wi h some modi ica ions. The mice we e habi ua ed gi en wo
iden ical bo les wi h ap wa e in hei cage. Then, mice we e
gi en ee access o bo h, one wi h 2% suc ose solu ion and
ano he wi h ap wa e o 3 days. The posi ion o bo les was
swi ched daily o a oid place p e e ence in d inking beha io .
Bo le weigh s we e aken e e y day o de e mine solu ion in ake.
The p e e ence o suc ose was calcula ed as a pe cen age o he
olume o suc ose in ake o e he o al olume o luid in ake and
a e aged o e he 3 days o he es .
Mo phological Analyses o As ocy es in
he Den a e Gy us
B ain Tissue P epa a ion
Fo he p o ein analyses, mice we e eu hanized, and b ains
emo ed. The le hemisphe e o b ains was dissec ed ou o
collec he egions o in e es and quickly ozen o la e
biochemical analyses. Fo he immunohis ochemis y (IHC) and
luo escen IHC (F-IHC) analysis, he igh hemisphe e o each
F on ie s in Neu oscience | www. on ie sin.o g 3Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
FIGURE 1 | Expe imen al design. Mice we e subjec ed o es ain s ess 6 h/day o 21 days and co- ea ed wi h PBS, k ill oil (KO), Co inine (Co ) o Co plus KO.
A e es ain and unde con inuing ea men s, mice we e es ed o locomo o unc ion, ecogni ion memo y using he no el objec ecogni ion es (NOR) and
dep essi e-like beha io using he o ced swim (FS) es and he suga p e e ence es (SPT).
FIGURE 2 | Co- ea men wi h co inine and k ill oil does no a ec locomo o ac i i y in mice. A e p olonged es ain s ess (RS) and co- ea men wi h ehicle (PBS),
co inine (Co , 5 mg/kg), k ill oil (KO, 143 mg/kg) o (Co plus KO), mice we e es ed o locomo o ac i i y in he open ield es o 25 min. The esul s show he e ec o
RS and ea men s on. Time in he cen e zone (A) and o al dis ance a elled (B).
mouse b ain was placed in 4% pa a o maldehyde in PBS pH 7.4
a 4◦C o 24 h. The issues we e embedded in 2% aga ose molds
o ib a ome sec ioning. The egion o in e es was loca ed using
he Paxinos A las as a e e ence (F anklin and Paxinos, 1997), and
se ial sec ions o 20 µm (n≥2/mouse) we e collec ed using he
Vib a ome Leica VT1000S and placed on posi i ely cha ged slides
(Bioca e Medical, Conco d, CA).
Immuno luo escence and Con ocal Mic oscopy
Fo he F-IHC, samples we e washed h ee imes o 7 min wi h
T is-bu e ed saline (TBS), pH 7.8. The p ima y an ibody an i-
GFAP (1:50, BioSB) was dilu ed in diluen bu e , con aining
TBS supplemen ed wi h 1% bo ine se um albumin (BSA) and
0.2% T i on X-100 and incuba ed wi h he issue sec ions
o e nigh (ON) a 4◦C. A e h ee washes wi h TBS o
10 min, sec ions we e incuba ed wi h he seconda y an ibody,
Cy2-conjuga ed abbi an i-mouse IgG (1:200, Jackson Immuno
Resea ch, Pennsyl ania, USA) dilu ed in TBS con aining 1% BSA
o 2 h a RT. The samples we e coun e s ained wi h Hoechs
(1:1,000) and moun ed wi h luo escence moun ing medium
(P olong, In i ogen). Con ocal z-s acks we e acqui ed using an
LSM 780 con ocal mic oscope (Zeiss, Obe kochen, Ge many), z-
s acks we e no malized o main ain a consis en signal in ensi y
h ough he dep h o he sample, con ocal z-s ack image se ies
we e supe posed in maximum in ensi y p ojec ions by ImageJ
(Na ional Ins i u e o Heal h, Be hesda, MA, USA) o he
measu emen s.
Mo phome ic Analysis and Cell Coun ing
In each image, egions o in e es (ROI) we e de ined on he
den a e gy us using ee-hand d awing. Fo each ROI, he mean
g ay alues (MGV), ep esen ing he a ea immuno eac i e o
GFAP, we e measu ed. To measu e GFAP immunos aining in
he DG, maximum luo escence in ensi y p ojec ions o con ocal
z-s acks acqui ed om sagi al b ain sec ions we e con e ed
in o 8-bi g eyscale images wi h 256 scales (pixel in ensi y 0
co esponding o no signal and 255 o maximal signal) by Image
J so wa e. To calcula e he a ea ac ion o GFAP+IR, bina y
images he immuno eac i e a ea o he h esholded images was
di ided by he o al o he ROI. Fo he GFAP+cell coun ing, cell
F on ie s in Neu oscience | www. on ie sin.o g 4Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
FIGURE 3 | Co- ea men wi h co inine and k ill oil does no a ec locomo o ac i i y in mice. A e p olonged es ain s ess (RS) o 10 min and co- ea men wi h
ehicle (PBS), co inine (Co , 5 mg/kg), k ill oil (KO, 143 mg/kg) o (Co plus KO). The esul s show ha RS did no signi ican ly a ec locomo o ac i i y in he s essed
mice. (A) To al dis ance a eled. (B) Mean speed (me e s/seconds) (C) Time in he cen e zone. *P<0.05, **P<0.01.
FIGURE 4 | Co- ea men wi h co inine plus k ill oil p e en ed he es ain s ess-induced dep essi e-like beha io in mice. A e h ee-week es ain and
co- ea men wi h ehicle (PBS), co inine (Co , 5 mg/kg) o k ill oil (KO, 143 mg/kg), mice we e es ed o dep essi e-like beha io in he o ced swim es s (5 min).
(A) o he suc ose p e e ence es s (B). *P<0.05, **P<0.01, ***P<0.001.
o be coun ed mus ha e a leas hal o he cell nucleus isible on
he edge o he ROI and cells o be included in he analysis mus
be no adhe en o blood essels.
S a is ical Analysis
To analyze g oup and ea men e ec s di e ences in he means
be ween g oups one-way analysis o a iance (ANOVA), and
pos hoc Dunne ’s es was pe o med using he G aphPad
P ism so wa e o assess di e ence signi icance be ween g oups.
Di e ences we e conside ed signi ican wi h P<0.05.
RESULTS
E ec o K ill oil and Co inine on
Locomo o Ac i i y
To de e mine he changes in locomo o ac i i y induced by co-
ea men s du ing RS, mice we e es ed using he OF es o
25 min. The esul s show ha s essed mice showed highe ime
spen in he cen e zone han non-s essed con ols in he i s
10 min o he es s, bu he di e ences diminished a e his
ime be ween g oups. The ime spen in he cen e zone by he
di e en g oups was signi ican ly di e en in he i s 5 min o
F on ie s in Neu oscience | www. on ie sin.o g 5Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
he OF es [F(4, 63) =6.42, P<0.0003; Figu e 2A]. A he
con a y, he dis ance a eled show ma ked di e ences be ween
non-s essed mice and all g oups o s essed mice, independen
o ea men s (Figu e 2B). Conside ing hese esul s we p oceed
o analyze he changes in locomo o ac i i y in he OF du ing
he i s 10 min o es ing. One-way ANOVA analysis e ealed
signi ican di e ences in he dis ance a eled in he i s 10 min
[F(7, 67) =16, P<0.0001; Figu e 3A] and speed [F(7, 76) =8.91,
P<0.0001; Figu e 3B]. The e we e no s a is ically signi ican
di e ences in dis ance a eled o speed be ween non-s essed
mice ea ed wi h ehicle, co inine o KO. Howe e , hey showed
signi ican ly lowe dis ances han non-s essed mice ea ed wi h
KO plus co (P<0.05), and es ained mice ea ed wi h ehicle,
co inine, KO o KO plus Co inine (P<0.0001).
The inc ease in dis ance a eled was accompanied by a
signi ican inc ease in speed in he mice subjec ed o RS when
compa ed o non-s essed ehicle- ea ed mice (& RS, P<0.05;
& RS plus Co P<0.05; & RS, KO, P<0.01; & RS, KO plus
Co , P<0.05; Figu e 3B). Also, he ime spen in he cen e
zone du ing he i s 10 min o es ing was analyzed, when anxie y
beha io was mo e e iden . The esul s showed ha in he i s
10 min he e was a non-signi ican inc ease in ime spen in he
cen e zone be ween con ol non-s essed mice and he mice
subjec ed o RS [F(7, 67) =2.11, P<0.053] (Figu e 3C). Howe e ,
hese di e ences we e signi ican be ween ea men g oups a e
5 min o es ing [F(4, 67) =9.17, P<0.0001; (Figu e 2A) wi h
signi ican di e ences be ween non-s essed mice and s essed-
mice independen o ea men s (& RS, P<0.0001; & RS plus Co
P<0.001; & RS, KO, P<0.001; & RS, KO plus Co , P<0.0001).
E ec o K ill Oil and Co inine on
Dep essi e-like Beha io
To in es iga e he po en ial an i-dep essan e ec s o co inine,
KO and KO plus co inine when adminis e ed du ing s ess, a
he las day o RS mice we e es ed o dep essi e-like beha io
using he o ced swim es and suc ose p e e ence es (Figu e 4).
A wo-way ANOVA showed a signi ican impac o p olonged RS
on he le el o dep essi e-like beha io , exp essed as a gene al
inc ease in he immobili y ime du ing he FS es by he
es ained mice when compa ed o con ol mice [F(1,38) =15.35,
P=0.0004]. Also, his analysis e ealed a signi ican e ec o
ea men s on dep essi e-like beha io [F(3,38) =5.23, P=0.004].
Mul iple compa ison es s showed ha mice subjec ed o RS
and co- ea ed wi h Co (P<0.05), KO (P<0.05) o KO
plus co inine (P<0.01) showed lowe le els o immobili y han
ehicle- ea ed es ained mice (Figu e 4A).
To co obo a e he an idep essan e ec o ea men s, a
suc ose p e e ence es s we e pe o med. Also, he suc ose
p e e ence es e ealed signi ican di e ences be ween
ea men s. One-way ANOVA e ealed a signi ican e ec o
ch onic s ess in suc ose p e e ence [F(4, 28) =8.65, P=0.0001].
Mul iple compa ison es s showed signi ican dec ease in suc ose
p e e ence in he ehicle- ea ed es ained mice when compa ed
o ehicle- ea ed non-s essed mice. Howe e , ea men s
showed an idep essan e ec s and he s essed mice ea ed
wi h co inine (P<0.001), KO (P<0.001) o co inine plus KO
(P<0.05) showed signi ican ly lowe le els o anhedonia and
highe suc ose consump ion han he es ained mice ea ed
wi h ehicle (Figu e 4B).
E ec o K ill Oil and Co inine on
Recogni ion Memo y
To assess whe he co- ea men wi h co inine du ing RS
in luenced ecogni ion memo y, mice we e es ed in he
no el objec ecogni ion NOR es . Non-signi ican di e ences
we e ound be ween non-s essed and es ained mice in he
amilia iza ion s ep o he ask, and all mice explo ed each
o he wo objec s almos 50% o he ime, no showing a
p e e ence o any o hem (Figu e 5A). Howe e , when mice
we e exposed o one old objec and a new objec in he a ena, one-
way ANOVA analysis e ealed signi ican di e ences be ween
g oups on ecogni ion memo y [F(4,48) =4.29, P=0.0049].
Mul iple compa ison es s showed a signi ican di e ence in
he ime spen wi h he new objec be ween he ehicle- ea ed
non- es ained mice and he ehicle- ea ed es ained mice
(P<0.05). Howe e , es ained mice ea ed wi h co inine
plus KO showed no di e ences in ecogni ion wo king memo y
e lec ed as a simila le el o p e e ence o he new objec han
ehicle- ea ed non- es ained mice (P>0.05) (Figu e 5B).
Analysis o Changes in Mo phology and
Cell Viabili y o GFAP+As ocy es
Cell Coun ing
To assess changes induced by RS and ea men s on as ocy e
a chi ec u e and numbe s. The numbe o GFAP+cells, GFAP IR
and a ea ac ion we e in es iga ed in he DG, one o he egion
mo e a ec ed in hose pa ame e s by RS (Pe ez-U u ia e al.,
2017).
Mul iple compa ison es s e ealed no signi ican e ec s o
ea men s in GFAP IR in he non-s essed mice, bu signi ican
di e ences in he es ained mice. One-way ANOVA analysis o
GFAP+cells in he DG showed a signi ican e ec o ea men s
on he numbe o GFAP+cells in he DG [F(7, 46) =4.88,
P=0.0004]. GFAP+cell densi ies in he DG we e signi ican ly
educed in he ehicle- ea ed es ained mice when compa ed
o ehicle- ea ed non-s essed mice (P<0.001). T ea men wi h
KO alone did no induced changes in he numbe o GFAP+cell
in he KO- ea ed es ained mice, when compa ed o ehicle-
ea ed es ained mice. Howe e , a subs an ial inc ease o
GFAP+IR cells was obse ed in he co inine- ea ed es ained
mice o KO plus co inine, when compa ed o ehicle- ea ed
es ained mice (P<0.05) (Figu es 6A,B).
Mean G ay Value
One-way ANOVA analysis o mean GFAP IR in he GFAP+cells
in he DG showed a signi ican e ec o ea men s on GFAP
IR in ensi y in he DG [F(7, 33) =5.10, P=0.0005]. Mul iple
compa ison es s e ealed no signi ican e ec s o ea men s
on he mean GFAP IR in he non-s essed mice. Howe e ,
a signi ican dec ease in he mean IR in ensi y was ound in
he ehicle- ea ed es ained mice g oup when compa ed o
ehicle- ea ed non- es ained mice (P<0,05). Nonsigni ican
changes in GFAP IR we e ound in he KO- ea ed es ained
mice when compa ed o he ehicle- ea ed es ained mice
(P>0.05). On he o he hand, a signi ican inc ease o IR
F on ie s in Neu oscience | www. on ie sin.o g 6Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
FIGURE 5 | Co- ea men wi h co inine dec eased he es ain s ess-induced de ici in ecogni ion memo y. A e es ain and co- ea men wi h ehicle (PBS),
co inine (Co , 5 mg/kg) k ill oil (KO, 143 mg/kg) o Co plus KO, mice we e es ed o locomo o ac i i y in he open ield es and nex day mice we e es ed o
ecogni ion memo y in he no el objec ecogni ion es (NOR). (A) Familia iza ion: mice we e indi idually exposed o wo iden ical objec s. (B), No el objec ecogni ion
s ep: a e 30 min o es , mice we e exposed o one o he old objec s and a new objec . Ch onic es ain s ess impai ed no el objec ecogni ion. Co- ea men wi h
KO plus Co p ese ed ecogni ion memo y abili ies in he s essed mice o le els non-signi ican ly di e en om con ol non-s essed mice. **P<0.01.
FIGURE 6 | Analysis o he e ec o co inine plus k ill oil on as ocy es in he den a e gy us o he hippocampus. A igu e ep esen ing he changes in cell GFAP+cells
numbe s and mo phology in he den a e gy us egion o he hippocampus in male mice subjec ed o no o es ain s ess (R. S ess) (A). G aph depic ing he
changes in he numbe o GFAP+IR cells (B); main g ay alues (MGV) (C); a ea o GFAP IR in he DG o ehicle- ea ed non- e ained mice o es ained (RS) mice,
co inine (Co , 5 mg/kg) o Co plus KO (143 mg/kg) (Co +KO) (D). *P<0.05, **P<0.01, ***P<0.001.
F on ie s in Neu oscience | www. on ie sin.o g 7Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
in ensi y was ound in he co inine- ea ed es ained mice when
we e compa ed o ehicle- ea ed es ained mice (P<0.01).
Also, he e was a signi ican inc ease in GFAP IR in he KO
plus co inine- ea ed es ained mice when compa ed o ehicle-
ea ed es ained mice (P<0.01; Figu e 6C).
A ea F ac ion
The analysis o he pe cen a ea ac ion occupied by GFAP+
cells e ealed signi ican e ec s o ea men s in he DG
[F(7, 34) =17.28, P<0.0001]. Mul iple compa ison analysis
showed ha ehicle- ea ed es ained mice show a signi ican
dec ease o he GFAP+IR a ea in compa ison o non-s essed
ehicle- ea ed mice (P<0,001). Ne e heless, a signi ican
inc ease in he GFAP+IR ac ion a ea was ound in he
co inine and KO plus co inine- ea ed es ained mice when
compa ed o ehicle- ea ed es ained mice (P<0.001) in he
DG (Figu e 6D).
DISCUSSION
Ch onic immobiliza ion o educed mobili y s ess can esul
om obesi y, pa alysis induced by ascula e en s such as s oke,
spinal co d inju y, ad anced age, and many neu odegene a i e
condi ions such as a h osis, and a axia. These e en s esul in
dep ession and cogni i e impai men in he a ec ed indi iduals.
RS is a b oadly used model o s ess-induced dep essi e-like
beha io (Buyni sky and Mos o sky, 2009). P olonged RS esul s
in mo phological changes in he b ain such as e ac ion o
p ocesses in hippocampal neu ons and as ocy es (Maga inos
e al., 1997; McEwen e al., 1997), neu oin lamma ion (Baue
e al., 2001; de And ade e al., 2012; Tymen e al., 2013), and
cogni i e de ici s (Tho sell e al., 2000; Abidin e al., 2004; Con ad
e al., 2004; Che ian e al., 2009; Mika e al., 2012) and dep essi e-
like beha io in oden s (Buyni sky and Mos o sky, 2009; Chiba
e al., 2012). I has been shown ha co inine adminis e ed
be o e and a e RS, educes he dep essi e-like beha io , synap ic
de ici s, as ocy e al e a ions, and cogni i e impai men in mice
(G izzell e al., 2014; G izzell and Eche e ia, 2015; Pe ez-U u ia
e al., 2017). In his s udy, i was in es iga ed he e ec o co-
ea men wi h co inine combined wi h KO, du ing ch onic RS,
on he de elopmen o dep essi e-like beha io and cogni i e
impai men induced by ch onic s ess in mice. RS p o oked
a dec ease in ecogni ion memo y abili ies and dep essi e-like
beha io in he mice, howe e , co inine plus KO p e en ed hese
beha io al changes. These esul s showed a syne gis ic bene icial
e ec o bo h co inine and KO in p ese ing mood s abili y and
cogni i e abili ies unde condi ions o ch onic RS.
A he neu ochemical le el, ch onic s ess induces a de ici
in glu ama e gic neu o ansmission by mechanisms in ol ing
a dec ease o NMDA (N-Me hyl-D-aspa a e) and AMPA
(α-amino-3-hyd oxy-5-me hyl-4-isoxazolep opionic acid)
ecep o s in he pos synap ic si e in he p e on al co ex and
he hippocampus, wo b ain egions ha a e undamen al
o media ing decla a i e and wo king memo y abili ies. This
educ ion in he numbe o synap ic glu ama e ecep o s induces
a dec ease in he ac i i y o b ain ne wo ks con olling s ess
beha io including he p e on al co ex-amygdala-hippocampus
pa hways. Some e idence sugges s ha loss o glu ama e
FIGURE 7 | Diag am ep esen ing he e ec o co inine and k ill oil p e en ing
he e ec s o ch onic s ess on as ocy e and neu onal unc ion and beha io .
Co inine plus KO may coun e ac he neu oin lamma o y and oxida i e
p ocesses igge ed by ch onic s ess in he b ain. This e ec may p e en he
educ ion in as ocy e a ec ing hei ole suppo ing he neu onal plas ici y
p ocesses ha a e equi ed o wo king memo y and mood s abili y a ec ed
by ch onic s ess.
ecep o s in neu ons o he p e on al co ex a e epea ed s ess
in a s, i is due o inc eased ubiqui in–p o easome-dependen
deg ada ion o hese ecep o s (Joels e al., 2004; Yuen e al.,
2012). Fu he mo e, p e ious s udies using oden models o
ch onic s ess ound a educed p oli e a ion o glial p ogeni o
cells, and a dec ease o GFAP+cells in se e al b ain egions,
including he hippocampus and p e on al co ex in a s. In
a s, glucoco icoids can diminish he exp ession o GFAP in he
PFC, esul ing in >20% educ ion in GFAP exp ession ha was
accompanied by a dec ease o he GFAP mRNA le els (Zschocke
e al., 2005). Also, ch onic RS inhibi s he glu ama e up ake by
as ocy es enhancing exci o oxici y and long- e m dep ession
(Yang e al., 2005). Fu he mo e, some e idence shows ha a s
exposed o ea ly-li e s ess ha e a dec ease in as ocy es le els in
he on al co ex in adul hood, indica ing a long- e m e ec o
s ess on glial cells de elopmen (Le en opoulos e al., 2007). I is
easonable o p opose ha a de ici in as ocy e’s unc ion plays
a c ucial ole in he highe suscep ibili y o PTSD obse ed in
pe sons wi h a p e ious his o y o child abuse.
We ha e p e iously ound a p o ec i e e ec o in anasal
co inine adminis e ed agains RS-induced as ocy es dec ease. In
his s udy, we ound ha co- ea men o mice wi h o al co inine
plus KO p e en ed he dec ease in he numbe and complexi y
F on ie s in Neu oscience | www. on ie sin.o g 8Decembe 2018 | Volume 12 | A icle 952
Mendoza e al. Co inine Plus K ill and S ess
o as ocy es in he DG o mice subjec ed o RS. In his s udy, we
obse ed a bene icial e ec o co inine and co inine plus KO bu
no KO alone in p ese ing he numbe and a bo complexi y o
as ocy es unde condi ions o RS.
We ha e p e iously shown ha , in he absence o s ess, long-
e m co inine ea men o up o 8 mon hs did no induce
signi ican di e ences in senso y-mo o abili ies o anxie y in
mice (Zei lin e al., 2012). Alike hese esul s, no signi ican
changes in locomo o ac i i y in he mice ea ed wi h co inine,
KO o co inine plus KO and subjec ed o RS we e ound.
Thus, he supe io e ec o he combina ion o co inine plus
KO inc easing he escape-o ien ed beha io in he FS es ,
canno be explained by an inc ease in locomo o ac i i y induced
by he mix. The open ield es is a good es o assess
locomo o ac i i y. Howe e , he in e p e a ion o he ime spen
in he cen e zone as a measu e o anxie y has p o en o be
misleading and some imes con adic o y (Belzung, 2001a,b; P u
and Belzung, 2003). Fo example, i has been shown ha many
clinically e ec i e non-benzodiazepine anxioly ics, excep 5-
HT1A agonis s, hese anxioly ic d ugs exhibi ex emely a iable
e ec s in he open ield es s. Alike o he anxioly ic d ugs
co inine, KO o co inine plus KO did no diminish he inc eased
locomo o ac i i y o ime spen in he cen e zone du ing he
i s 10 min o he es , ha is conside ed a measu e o anxie y
beha io a e p olonged es ain s ess.
I is appealing ha compa able esul s we e ob ained in he
beha io al pa ame e s es ed, wi h a mo e signi ican e ec o he
mix co inine plus KO han he indi idual componen s in he mix.
The connec ion be ween changes in as ocy es and dep essi e-
like beha io has been epo ed be o e. Fo example, a p e ious
s udy epo ed ha he dec ease in as ocy es numbe s in he
on al co ex induced by L-alpha-aminoadipic acid p o oked
dep essi e-like beha io in oden s (Lee e al., 2013). This
e idence demons a ed ha as oglia abla ion in he PFC is
su icien o p omp dep essi e-like beha io s compa able o he
one induced by ch onic RS. This da a s ongly sugges s ha
he loss o as oglia may be a key ac o con ibu ing o he
de elopmen o long-las ing dep ession (Lee e al., 2013).
The e ec o co inine in he mix p e en ing he e ec o
s ess on mood can be he esul o he ac ion o co inine
as an an i-in lamma o y compound inhibi ing mic ogliosis and
neu oin lamma ion as well as p omo ing neu onal and as ocy e
su i al h oughou he ac i a ion o p o-su i al cell signaling
pa hways.
Inc eased le els o as ocy es may p o ide neu ons wi h
mo e ene gy subs a es, glu ama e p ecu so s, and neu o ophic
ac o s. Also, as ocy es can dec ease he oxic e ec o
he abno mal inc ease in glu ama e elease induced by
co icos e oids a he p esynap ic le el, by up aking he
glu ama e om he synap ic space. On he o he hand, KO
componen s such as omega-3 and as axan hin can p e en
oxida i e s ess and diminish he dele e ious e ec s o s ess
and Neu oin lamma ion on b ain unc ion (Ba os e al., 2014;
Polo ow e al., 2015;Figu e 7).
A mo e de ailed s udy o he e ec on co inine and KO on
as ocy e unc ion is gua an eed in he ligh o he p esen esul s.
CONCLUSIONS
In his wo k, he e ec o an o al o mula ion o co inine
plus KO o p e en he cogni i e and dep essi e-like beha io
induced by ch onic es ain s ess. The esul s show ha a
he dose es ed, he co inine plus KO p e en ed dep essi e-
like beha io , memo y impai men and he as ocy es dec ease
induced by RS, and sugges s ha his o mula ion may
be use ul in humans and non-p ima es mammals su e ing
om es ain s ess due o aging o o he pa hological and
auma ic condi ions. Clinical s udies a e needed o con i m his
hypo hesis.
AUTHOR CONTRIBUTIONS
All au ho s lis ed ha e made a subs an ial, di ec and in ellec ual
con ibu ion o he wo k, and app o ed i o publica ion.
FUNDING
This wo k was suppo ed by he Fondo de Ciencia y Tecnologia
(FONDECYT) de Chile, (g an 1150194) and he Uni e sidad
San Sebas ian, Chile.
ACKNOWLEDGMENTS
This ma e ial is he esul o wo k suppo ed wi h esou ces
and he use o acili ies om he Uni e sidad San Sebas ian
(Chile). The con en s do no necessa ily ep esen he iews
o he Depa men o Ve e ans A ai s o he Uni ed S a es
Go e nmen .
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