nins-14-590029 Oc obe 8, 2020 Time: 18:47 # 1
ORIGINAL RESEARCH
published: 14 Oc obe 2020
doi: 10.3389/ nins.2020.590029
Edi ed by:
Ma in Wi ,
Cen e o T ansdisciplina y
Neu oscience Ros ock, Ge many
Re iewed by:
And eas W ee,
Uni e si y o Ros ock, Ge many
Ve onica Alexand a An ipo a,
Medical Uni e si y o G az, Aus ia
Alexande Hawli schka,
Uni e si y Hospi al Ros ock, Ge many
*Co espondence:
Ca alina Requejo
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Neu odegene a ion,
a sec ion o he jou nal
F on ie s in Neu oscience
Recei ed: 31 July 2020
Accep ed: 24 Sep embe 2020
Published: 14 Oc obe 2020
Ci a ion:
Requejo C, López-de-Ipiña K,
Ruiz-O ega JÁ, Fe nández E,
Cal o PM, Mo e a-He e as T,
Miguelez C, Ca dona-G i oll L,
Cepeda H, Ugedo L and La uen e JV
(2020) Changes in Day/Nigh Ac i i y
in he 6-OHDA-Induced Expe imen al
Model o Pa kinson’s Disease:
Explo ing P od omal Bioma ke s.
F on . Neu osci. 14:590029.
doi: 10.3389/ nins.2020.590029
Changes in Day/Nigh Ac i i y in he
6-OHDA-Induced Expe imen al
Model o Pa kinson’s Disease:
Explo ing P od omal Bioma ke s
Ca alina Requejo1,2*, Ka mele López-de-Ipiña3,4, José Ángel Ruiz-O ega5,6,
Elsa Fe nández3, Pila M. Cal o3, Te esa Mo e a-He e as5,6, C is ina Miguelez5,6,
Lau a Ca dona-G i oll1, Hodei Cepeda1, Luisa Ugedo5,6 and José Vicen e La uen e1
1LaNCE, Depa men o Neu oscience, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain, 2Depa men
o Neu ology, Icahn School o Medicine a Moun Sinai, The F iedman B ain Ins i u e, New Yo k, NY, Uni ed S a es, 3EleKin
Resea ch G oup, Depa men o Sys ems Enginee ing and Au oma ion, Uni e si y o he Basque Coun y (UPV/EHU),
Donos ia, Spain, 4Depa men o Psychia y, Uni e si y o Camb idge, Camb idge, Uni ed Kingdom, 5Depa men
o Pha macology, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain, 6Au onomic and Mo emen Diso de s Uni ,
Neu odegene a i e diseases, Bioc uces Heal h Resea ch Ins i u e, Ba akaldo, Spain
The sea ch o expe imen al models mimicking an ea ly s age o Pa kinson’s disease
(PD) be o e mo o mani es a ions is undamen al in o de o explo e ea ly signs
and ge a be e p ognosis. In e es ingly, ou p e ious s udies ha e indica ed ha
6-hyd oxydopamine (6-OHDA) is a sui able model o induce an ea ly degene a ion o
he nig os ia al sys em wi hou any g oss mo o impai men . Conside ing ou p e ious
indings, we aim o implemen a no el sys em o moni o a s a e in as ia al injec ion o
6-OHDA o de ec and analyze physiological changes unde lying p od omal PD. Twen y
male Sp ague-Dawley a s we e unila e ally injec ed wi h 6-OHDA (n= 10) o saline
solu ion (n= 10) in o he igh s ia um and placed in en iched en i onmen cages whe e
he ac i i y was moni o ed. A e 2 weeks, he amphe amine es was pe o med be o e
he sac i ice. Immunohis ochemis y was de eloped o he mo phological e alua ion
and wes e n blo analysis o assess molecula changes. Home-cage moni o ing
e ealed beha io al changes in esponse o 6-OHDA adminis a ion including signi ican
hype ac i i y and hypoac i i y du ing he ligh and da k phase, espec i ely, u ning ou
in a change o he ci cadian iming. A p eclinical s age o PD was unc ionally con i med
wi h he amphe amine es . Mo eo e , he loss o y osine hyd oxylase exp ession was
signi ican ly co ela ed wi h he mo o esul s, and 6-OHDA induced ea ly p oapop o ic
e en s. Ou indings p o ide e idence o a no el p od omal 6-OHDA model ollowing a
cus omized moni o ing sys em ha could gi e insigh s o de ec non-mo o de ici s and
molecula a ge s o es neu op o ec i e/neu o es o a i e agen s.
Keywo ds: 6-hyd oxydopamine, ci cadian hy hms, home-cage moni o ing sys em, p od omal bioma ke s,
p od omal Pa kinson’s disease symp oms, beha io , non-mo o de ici s, a
Abb e ia ions: ADN, Axodend i ic ne wo k densi y; AKT, P o ein kinase B; AUC, A ea Unde he Cu e; CL, Con ala e al;
EE, En iched en i onmen ; ERK, Ex acellula signal- egula ed kinase; GFAP, Glial ib illa y acidic p o ein; IL, Ipsila e al;
NMS, Non-mo o symp oms; 6-OHDA, 6-hyd oxydopamine; PD, Pa kinson’s disease; REM, Rapid eye mo emen ; NREM,
Non- apid eye mo emen ; RBD, Rapid eye mo emen sleep beha io diso de ; ROC, Recei e Ope a ing Cha ac e is ics;
ROS, Reac i e oxygen species; SN, subs an ia nig a; SN , Subs an ia nig a e icula e; TH, Ty osine hyd oxylase; TH-i ,
Ty osine hyd oxylase-immuno eac i i y; Tpm, Tu ns pe minu e; VTA, Ven al egmen al a ea.
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Requejo e al. 6-OHDA Induced P od omal PD Symp oms
INTRODUCTION
Pa kinson’s disease (PD) is a complex neu ologic diso de
in which no only mo o impai men s occu , bu also o he
non-mo o symp oms (NMS) play a ele an ole, including
hyposmia, sleep diso de s, dep ession, cons ipa ion and cogni i e
de ici (Schapi a and Tolosa, 2010;Man i e al., 2018;Wein aub
e al., 2018). Mos o hem o en appea ea lie han he mo o
symp oma ology, du ing he so-called p od omal s age and
wo sen ollowing he disease’s p og ession (Schapi a e al., 2017).
Indeed, NMS may mani es se e al yea s p io o he onse o
mo o symp oms, e en up o 20 yea s be o e he diagnosis, and
he p e alence can a y be ween he pa ien s (Schapi a e al.,
2017;Rees e al., 2019). In his con ex , al hough he e a e no
speci ic NMS o PD, he p esence and combina ion o a ious
NMS as well as he co ela ion wi h an ea ly dopamine deple ion
may be use ul o ea ly diagnosis (Be g e al., 2013;Schapi a
e al., 2017). Rema kably, sligh mo o symp oms (<40–60%
o dopamine gic neu onal cell loss) ha e been also associa ed
o he p od omal s age when hey do no mee he c i e ia
o he clinical diagnosis o PD (Mahlknech e al., 2015).
Thus, i is essen ial o es ablish a co ela ion be ween he ea ly
mani es a ions o PD (p od omal s age) wi h he clinical and
pa hological s age o an ea ly diagnosis o PD (Mahlknech e al.,
2015;Liepel -Sca one e al., 2017;She ba e al., 2018).
Thus, s udies abou ea ly e en s o he disease a e eme ging
in o de o ind some bioma ke o an ea ly-p emo o diagnosis
(Mille and O’Callaghan, 2015). Howe e , he neu oana omical
and e iological backg ound o NMS in PD emain o be elucida ed
(Schapi a e al., 2017). One o he i s NMS ha is su e ed by a
high pe cen age o he pa ien s is he sleep diso de , such as sleep
agmen a ion, excessi e day ime sleepiness o “REM beha io
diso de ” (RBD), which is a pa hology based on inc easing
muscle one du ing he REM ( apid eye mo emen ) sleep (De
Lazza i e al., 2018;De Cas o Medei os e al., 2019). Among he
mechanisms unde lying sleep dis u bance, he dis up ion in he
ci cadian sys em is a ibu ed as igge ing ac o (Willison e al.,
2013). Indeed, g owing e idences suppo an associa ion be ween
ci cadian dis up ion and PD, sugges ing ha he dopamine
deple ion may lead o ci cadian hy hm i egula i ies including
he al e a ion o he ci cadian con ol o he es /ac i i y hy hms
(Tanaka e al., 2012;De Lazza i e al., 2018).
In he sea ch o p eclinical models o PD, he e is a main
challenge o ep oducing hose unc ional and pa hological
changes ha could p o ide he sca old o ind ou abou he
a ge o he design o neu op o ec i e he apies be o e he
p og ession o he disease (Du y and Jenne , 2011;Ko and
Beza d, 2017;La uen e e al., 2017;Ia ko e al., 2020). In
his con ex , he well-known model o he 6-hyd oxydopamine
(6-OHDA) which is widely used o s udy mo o de ici s, can also
be use ul o iden i ying non-mo o and ea ly mo o impai men s
specially when combined wi h ad anced echnological de ices
(G andi e al., 2018). In his model, he si e o adminis a ion
and ime elapsed a e he injec ion a e c i ical o de e mining
he ex en and ime cou se o he lesion (Ki ik e al., 2000;
Deumens e al., 2002). In ou hands, his model is also use ul
o s udying he p eclinical phase o PD, as in as ia al injec ion
o 6-OHDA in adul male a s induced an ea ly degene a ion o
he nig os ia al sys em wi hou any g oss mo o impai men as
well as up egula ion o caspase-3 and down egula ion o su i al
signaling pa hways (Requejo e al., 2018a,b).
The e o e, in he p esen s udy we aimed o de ec p od omal
changes in he beha io ollowing an expe imen al model o PD
based on he in as ia al injec ion o 6-OHDA wi h a sho ime
o e olu ion (2 weeks) in adul male a s housed in moni o ed
en iched en i onmen (EE) cages.
MATERIALS AND METHODS
Expe imen al Design and Housing
Condi ions
Twen y adul (3-mon h-old) male Sp ague-Dawley a s,
weigh ing 280–300 g a he ime o su ge y, we e ob ained
om Ha lan Labo a o ies, S, A, (Ba celona, Spain). Ra s we e
andomly assigned o he ollowing g oups: Sham g oup
(saline-lesioned a s, n= 10) as con ol and 6-OHDA g oup
(6-OHDA-lesioned a s, n= 10). A e s e eo axic injec ion
o 6-OHDA o saline solu ion, he animals we e housed o 2
weeks in moni o ed en iched en i onmen cages (10 animals pe
cage) consis ing o la ge cage (790 mm ×460 mm ×640 mm)
wi h wo loo s, which we e connec ed by a plas ic amp and
an ex e nal unning wheel (Figu e 1A and Supplemen a y
Figu e S1). A 12 h ligh /12 h da k cycle was es ablished wi h
access o ood and wa e ad libi um. The EE cages we e de eloped
by ou esea ch g oup and hey we e designed o hos up o 12
a s as well as housing ood eede s, wa e eeding bo les, wo
semi-closed ooms, a wide-open a ea, and a amp o access a
unway si ua ed on he uppe loo . In addi ion o his, hese
EE cages also p o ide ou ga es ha may be opened o connec
o o he cages, unways, o exe cise wheels. Cu en EE cages
we e supplied wi h an au oma ic eco ding sys em. This no el
sys em consis s, basically, o an in a ed came a si ua ed abo e
e e y cage and connec ed o a aspbe y de ice p og ammed
o collec pic u es accu a ely e e y 30 s (Figu e 1A). In ac , a
Raspbe y Pi (Raspbe y Pi 2 Model B, Raspbe y Pi p oduc s,
Uni ed Kingdom) is a s andalone embedded compu e ha
measu es he size o a c edi ca d. I has enough powe o un a
linux ope a ing sys em and connec i o se e al ex e nal de ices
and senso s. In his case, i was connec ed o Pi NoIR Came a
V2 (Raspbe y Pi p oduc s, Uni ed Kingdom) ha p o ided a
clea image o he cage e en a nigh ’s da k due o he in a ed
ligh ing ha was se up h oughou he oom (Supplemen a y
Figu e S1). This in-house cus omized au oma ic sys em
con inuously eco ded a pic u e e e y 30 s and sa ed i in an
SD Ca d. A he end o he expe imen , mo e han 60 gigaby es
in pic u es we e collec ed and p ocessed in o de o measu e
changes in he ac i i y (Figu e 1B).
Two weeks a e he in as ia al injec ion he amphe amine
es was pe o med o e alua ing mo o de ici s and wi h
he pu pose o con i ming ha no a p esen ed mo e han
5 u ns pe minu e ( pm) in o de o ge a p eclinical model
(Supplemen a y Table S1). This c i e ium was es ablished
due o he ac ha i has p e iously been shown ha
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Requejo e al. 6-OHDA Induced P od omal PD Symp oms
FIGURE 1 | Expe imen al design and moni o ing analysis. (A) Schema ic ep esen a ion illus a ing he imeline o he expe imen s. Ra s ecei ed 6-OHDA (6-OHDA
g oup) o saline solu ion (Sham g oup) injec ions in o he igh s ia um, and immedia ely a e he lesion, hey we e placed in moni o ed EE cages o 2 weeks. These
EE cages we e supplied wi h an au oma ic eco ding sys em ha collec pic u es o he EE cages e e y 30 s unde a egula 12 h:12 h ligh /da k cycle in o de o
de ec ac i i y changes du ing he ligh and da k phases. In a ed ligh ing was also se up o ake clea pic u es du ing he da k phase. Two weeks la e , he
amphe amine es was pe o med o assess mo o symp oms, and hen, a s we e ansca dially pe used o decapi a ed o he his ological and biochemical
analysis, espec i ely. (B) Da a acquisi ion, p e-p ocessing and analysis wo k low. The p ocess was di ided in wo phases. (1) Image p e-p ocessing (in g een): ame
di e ence, g ay le el con e sion, noise smoo hing and bina iza ion). (2) Image analysis (in o ange): clus e ing analysis and cen oid calcula ion, ac i i y analysis.
6-OHDA, 6-hyd oxydopamine; EE, en iched en i onmen .
a s o a ing mo e han 5 pm p esen ed a lesion anging
om 50 o 90% which may p edic mo o impai men
(Bjö klund and Dunne , 2019). A e he amphe amine
es , a s we e sac i iced by ansca dial pe usion o he
his ological e alua ion o by decapi a ion o he biochemical
analysis (Figu e 1A).
All he expe imen al p o ocols we e e iewed and app o ed
by he E hical Commi ee and Animal Wel a e o he Uni e si y
o he Basque Coun y (UPV/EHU, CEBA M20/2015/024,
app o al da e 12/28/2015), and in acco dance wi h he
Eu opean Communi y Council Di ec i e on “The P o ec ion
o Animals Used o Scien i ic Pu poses” (2010/63/EU) and
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Requejo e al. 6-OHDA Induced P od omal PD Symp oms
wi h Spanish Law (RD 53/2013) o he ca e and use o
labo a o y animals.
6-OHDA Lesions
Ra s we e lesioned as we p e iously desc ibed (Requejo
e al., 2018a,b). B ie ly, hi y minu es be o e su ge y, a s
we e p e- ea ed wi h desip amine (25 mg/kg, i.p., Sigma,
S . Louis, Uni ed S a es) and pa gyline (50 mg/kg, i.p.,
Sigma, S . Louis, Uni ed S a es) in o de o p ese e he
no ad ene gic sys em and he deg ada ion o he oxin,
espec i ely. Then, a s we e deeply anaes he ized wi h
iso lu ane inhala ion (1.5–2%; Es e e Química, Ba celona,
Spain) and placed in he s e eo axic ame (Da id Kop R
Ins umen s). 6-OHDA (3 µg/µl, in 0.02% asco bic acid)
o saline solu ion (0.9% NaCl) was in used using a 10 µl-
Hamil on sy inge i ed wi h a 26-gauge needle a a a e o
0.5 µl/min by a single sy inge in usion pump (KDS Scien i ic,
MA, Uni ed S a es).
To gene a e a mild lesion o he nig os ia al pa hway, h ee
injec ions o 2.5 µl o he 6-OHDA solu ion (a o al olume o
7.5 µl) we e adminis e ed a h ee coo dina es in o he igh
s ia um, ela i e o he b egma and du a, wi h he oo hba
se a −2.4: an e opos e io (AP) +1.3 mm, mediola e al
(ML) +2.8 mm, do so en al (DV) −4.5 mm; AP −0.2 mm,
ML +3.0 mm, DV −5.0 mm and AP −0.6 mm, ML +4.0 mm,
DV −5.5 mm, acco ding o Paxinos and Wa son a las (Paxinos
and Wa son, 2013). The needle was le in place o an addi ional
5 min o allow he oxin o di use in o he s uc u e, and hen
i was slowly e ac ed. 6-OHDA was p epa ed daily o each
su ge y session and changed e e y 2–3 h. The 6-OHDA solu ion
was kep on ice and p o ec ed om ligh du ing he su ge y o
a oid oxida ion, which would be indica ed by a colo change
(clea o b own-pink colo ). Con ol a s ecei ed he same
olume o saline solu ion in he same manne . In addi ion, a sho
ime o e olu ion (2 weeks) was elapsed be o e sac i ice.
Moni o ing Analysis
The in-house cus omized au oma ic sys em based on he image
p ocessing wi h he in a ed Came a Module 2 (Pi NoIR;
Raspbe y Pi P oduc s, Uni ed Kingdom) was used o he
analysis o he a g oup beha io . A schema ic diag am
o he wo king p ocedu e o image p ocessing is desc ibed
in Figu e 1B.
This p ocess was di ided in wo phases: image p e-p ocessing
and image analysis.
1. In he i s phase ideo sequences we e acqui ed by a high-
quali y came a and p e-p ocessed by an own oolbox in
MATLAB o c ea e se ies o bina y image ames o he
a g oup ac i i y. The Toolbox includes: (1) unc ion o
image p ocessing, (2) Ac i i y analysis (cen oid analysis),
and (3) Pe iod analysis among o he s. Thus, he ac i i y
a eas (mo ion de ec ion) we e calcula ed based on a ame
di e ence me hod (Manchanda and Sha ma, 2016;Senga
and Mukhopadhyay, 2016). F ame absolu e di e ence was
calcula ed be ween wo consecu i e ames wi h a sampling
pe iod o 30 s. Images we e con e ed om Red G een Blue
(RGB) which is a s anda d o ma o colo image, in o g ay
scale, a e ha in bina y images and hen by a h eshold
il e ex ac ed he main ac i i y a ea o he sys em ( a s,
and objec s mo ed by he a s). Finally, ac i i y a eas we e
de ined applying mo phological il e s o educe he noise
and o smoo hing he images. In pa icula , mo phological
il e s we e applied using MATLAB unc ion bwmo ph:
Bwmo ph (I ,’open’);% Remo e peaks
Bwmo ph (I ,’close’);% Remo e hole
2. Du ing he second phase he ajec o ies o he animal
g oup is gene a ed by he sys em cen oid ( he a e age
posi ion o all he ac i i y a eas in he bina y image)
e olu ion. The cen oid is es ima ed by k-means algo i hm
o e he bina ized ame se ies (Egui aun e al., 2018;
López-de-Ipiña e al., 2019). Thus, wi hin each ame, he
coo dina es o he cen e s o e e y objec we e calcula ed,
and K-means we e applied o ind he cen e o he
en i e g oup, ha is he “cen oid.” A cen oid wi h wo
coo dina es:
C=(xc,yc)
3. The g oup ajec o y consis s o he e olu ion o ha
cen oid om he i s ame o he las one.
Finally, he a g oups’ beha io was desc ibed and modeled
by he ollowing pa ame e s:
1. Numbe o ac i i y a eas in he bina y images.
2. Shannon en opy o he cen oid ajec o y. Shannon
en opy is a main concep in in o ma ion heo y and is
a measu e o a e age unce ain y (in o ma ion con en ).
En opy in biosignals gi es in o ma ion abou he sys em
e olu ion and beha io and can be applied o analyze
pa hological beha io s (Egui aun e al., 2018;López-de-
Ipiña e al., 2019).
Veloci y, speed and accele a ion de ined as:
cx =1xc/1
cy =1yc/1 (1)
being speed he absolu e alue o eloci y. An accele a ion:
acx =1 cx/1
acy =1 cy/1 (2)
Amphe amine-Induced Ro a ion Tes
Two weeks a e in as ia al injec ion he amphe amine es
was de eloped ollowing he p e iously desc ibed me hodology
o his beha io al es (Miguelez e al., 2011). B ie ly,
D-amphe amine (5 mg/kg in 0.9% NaCl; Sigma-Ald ich,
S . Louis, Uni ed S a es) was in ape i oneally adminis e ed
and he animals we e placed in an indi idual ci cula cage
( o ame e ). In he p esen s udy, we used a highe dose o
D-amphe amine in o de o ge o a ional beha io due o he
lesion was mino (Bjö klund and Dunne , 2019). A e 15 min
o la ency, he o al numbe o ull ipsila e al (IL) o a ions
was eco ded du ing 90 min (Mul icoun e LE3806; Ha ad
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Requejo e al. 6-OHDA Induced P od omal PD Symp oms
Appa a us, Hollis on, MA, Uni ed S a es) in o de o include he
en i e esponse pe iod and a oid di e ences in a iabili y due
o he di e ences in he pha macokine ics o in he dopamine
elease kine ics be ween a s (Miguelez e al., 2011;Bjö klund
and Dunne , 2019). Da a we e exp essed as he numbe o
u ns pe minu e.
Mo phological Analysis
Mo phological e alua ion was pe o med as we p e iously
desc ibed (Requejo e al., 2015, 2018a,b).
Tissue P ocessing o His ological E alua ion
A e beha io al es , i e a s om each g oup we e
in ape i oneally anes he ized wi h chlo al hyd a e a 20%
(Re : 141,975, Pan eac Quimica SA, Ba celona, Spain) and
ansca dially pe used wi h 0.9% sodium chlo ide ollowed by
4% pa a o maldehyde (PFA) in 0.1 M phospha e-bu e ed saline
(PBS) pH 7.4. B ains we e emo ed, pos - ixed o e nigh in he
same ixa i e solu ion and nex ans e ed in o a c yop o ec i e
solu ion con aining 30% suc ose and 0.1M PBS pH 7.4.50 µm
se ial co onal sec ions con aining s ia um and subs an ia nig a
(SN) we e ob ained wi h a eezing mic o ome and collec ed
ollowing he Paxinos and Wa son a las (Paxinos and Wa son,
2013) in 0.6% sodium azide in 0.1 M PBS pH 7.4 o s o age and
u he analysis.
TH Immunos aining
Ty osine hyd oxylase (TH) immunohis ochemical s aining was
de eloped on ee- loa ing co onal slices. B ie ly, sec ions we e
ea ed wi h 3% H202and 10% me hanol in po assium phospha e
bu e ed saline (KPBS), p eincuba ed wi h 5% no mal goa se um
(NGS) and 1% T i on X-100 in KPBS (KPBS-T) o 1 h a
oom empe a u e (RT), and la e incuba ed o e nigh a 4◦C
wi h abbi polyclonal an i-TH (Re : AB-152, Millipo e; 1:1,000)
in KPBS/T con aining 5% NGS, ollowed by incuba ion wi h
a seconda y bio inyla ed goa an i- abbi IgG an ibody (Re :
BA1000, Vec o Labo a o ies, Bu lingame, CA; 1:200) in 2.5%
NGS KPBS/T o 2 h. A e wa d, all sec ions we e p ocessed
wi h he a idin-bio in-pe oxidase complex o 1 h using a
comme cial ki (Re . PK-6102, Eli e ABC ki , Vec o Labo a o ies,
Bu lingame, CA) and he eac ion was shown by using 3,3-
diaminobenzidine (DAB).
Images we e isualized, cap u ed and analyzed a 4x and 40x
magni ica ion by an Olympus BX-50 pho omic oscope.
Double Immuno luo escence S aining o Caspase-3
and NeuN
Co onal sec ions we e incuba ed wi h abbi an i-caspase
3 (H-277) (Re : sc-7148, San a C uz Bio echnology Inc.,
Spain; 1:50) and monoclonal mouse an i-NeuN (Re : MAB377,
Chemicon In e na ional, Inc., Spain; 1:100) dilu ed in 5%
bo ine se um albumin (BSA) and 0.1% T i on X-100 in
PBS o e nigh a 4◦C. Sec ions we e subsequen ly incuba ed
wi h seconda y an ibodies conjuga ed o Alexa Fluo -488
(Re : A11029; In i ogen; 1:400) and Alexa Fluo -568 (Re :
A11036 In i ogen; 1:400) o 1 h a oom empe a u e in
da kness. Immunos ained sec ions we e u he eincuba ed wi h
Hoechs o nuclea coun e s aining o 10 min, hen slices
we e washed, moun ed on glass slides and co e slipped using
Vec ashield moun ing medium o luo escence (Re : x-0517;
Vec o labo a o ies).
Images we e inally analyzed a 20x magni ica ion using
Olympus Fluo iew FV500 con ocal mic oscope.
S e eological Analysis
The unbiased s e eological analysis was pe o med as p e iously
desc ibed (Requejo e al., 2015, 2017, 2018a,b) by using a
compu e ized image analysis sys em (Me ca o Image Analysis
sys em, Explo a No a, La Rochelle, F ance) connec ed o an
Olympus BX-50 pho omic oscope. Fo his pu pose, a o al o
7–8 sec ions in a 1: 8 se ies we e analyzed o co e he en i e
s ia um and SN o each animal.
In b ie , in o de o e alua e he TH-immuno eac i i y (i )
in he IL s ia um, he olume o he p ese ed s ia um was
calcula ed using he Ca alie i me hod (Howa d and Reed, 2004)
a ailable on Me ca o image analysis sys em by delimi ing he
nega i e a eas and he en i e s ia um a 4x magni ica ion, and
mul iplying hese measu emen s by he hickness o he slices
and he in e sec ional dis ance (Requejo e al., 2015, 2018a,b).
Values we e exp essed as he pe cen age o TH-i olume o he
IL s ia um espec o he o al olume o he IL one.
Changes in he densi y o dopamine gic neu ons and
axodend i ic ne wo k (ADN) we e e alua ed in bo h hemisphe es
h ough quan i ying he TH-i neu onal densi y in he en i e SN
and he TH-i ADN densi y in he SN e icula a (SN ) using
a s e eological ool (an op ical ac iona o ) p o ided by he
Me ca o image analysis sys em (Wes e al., 1991). Once he
egion o in e es was ou lined a 4x magni ica ion, p obes o
50 ×50 µm sepa a ed by 100 µm we e launched in o he SN. TH-
i neu ons and ADN inside he p obe, o c ossing he igh side o
he X–Y axis, we e coun ed a 40x magni ica ion in he en i e SN
and SN , espec i ely. Da a we e exp essed as he pe cen age o
TH-i neu ons o ADN p esen s on he IL side (lesioned side) s.
he con ala e al (CL) hemisphe e (non-lesioned hemisphe e).
The s udy o he opological dis ibu ion was pe o med
ollowing he p e iously desc ibed app oach (Requejo e al.,
2015, 2017). Fo each animal, sec ions con aining he s ia um
and he SN we e e alua ed a h ee ep esen a i e os o-caudal
le els, espec i ely, acco ding o Paxinos and Wa son’s A las
(Paxinos and Wa son, 2013): os al (b egma +0.70 mm), middle
(b egma, −0.26 mm), and caudal (b egma, −0.80 mm) s ia al
sec ions we e conside ed o de e mine he TH-i olume; and
os al (b egma, −5.20 mm), middle (b egma, −5.60), and caudal
(b egma, −6.04) nig al sec ions we e examined o es ima e he
TH-i neu onal and ADN densi y.
Biochemical Analysis
Wes e n Blo ing
A e he beha io al es , 5 a s o each g oup we e anes he ized
wi h chlo al hyd a e a 20% (Re : 141,975, Pan eac Quimica
SA, Ba celona, Spain) and decapi a ed wi h a oden
guillo ine o ob ain esh b ain issue. IL and CL s ia um
and SN we e collec ed by mic odissec ion and quickly ozen
(Chiu e al., 2007). Fo his pu pose, once b ains we e emo ed,
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co onal slices con aining he s ia um and SN we e cu ou
and subjec ed o biopsy wi h a small biopsy needle. Fi s ly, he
ce ebellum was sepa a ed, and he b ain was cu bi-hal in o
he igh and le hemisphe e, nex cu was o emo ing he
ol ac o y bulb a he le el o he op ic chiasm, and he ou h cu
was a he le el o he pi ui a y s alk, sepa a ing he wo co onal
slices, one o hem con aining he s ia um and he o he one
he SN. The on al co ex om he slice con aining s ia um
was emo ed in he i h cu , ob aining he s ia um. The SN
was collec ed in he six h, sepa a ing he co ex om he slice.
Samples we e s o ed a −80◦C un il he analysis.
B ain slices we e manually homogenized (1:20 w/ ) in
lysis bu e [10 mM phospha e-bu e ed (PB) (pH 7.4), 5 mM
e hylenediamine e aace ic acid (EDTA), 5 mM e hyleneglycol-
bis(2-aminoe hyle he )- e aace ic acid (EGTA), 1 mM
di hio ei ol (DTT)] con aining a p o ease inhibi o cock ail
(Re : P-8340, Sigma-Ald ich, Spain). Samples we e cen i uged
(13,000 pm a 4◦C o 15 min) and soluble p o eins we e
eco e ed in he supe na an s and quan i ied using he Bio-Rad
P o ein Assay (Re : 500-0006, Bio-Rad Labo a o ies SA, Spain)
based on B ad o d’s me hod (B ad o d, 1976).
As p e iously desc ibed (Requejo e al., 2018a,b), o each
sample 20 µg o p o eins was loaded in o polyac ylamide
CRITERION TGX 12% gels (Bio-Rad Labo a o ies Inc., Spain)
o elec opho esis and hen ans e ed o a PVDF memb ane in
a T ans-Blo Tu bo T ans e Sys em (Bio-Rad, Uni ed S a es) o
7 min. Memb anes we e incuba ed wi h he ollowing p ima y
an ibodies: abbi an i-Phospho-p o ein kinase B (AKT) (Se
473) (1:1,000), abbi an i-AKT (1:1,000), abbi an i-Phospho-
p44/42 MAPK (ex acellula signal- egula ed p o ein kinases 1
and 2 (ERK 1/2)] (Th 202/204) (1:1,000), abbi an i-P44/42
MAPK (ERK 1/2) (1:1,000) (all o hem om Cell Signaling
Technology Inc., Uni ed S a es), abbi an i-caspase 3 (H-277)
(1:1,000) (San a C uz Bio echnology Inc., Spain), abbi an i-
β-Ac in (1:2,000) (Sigma-Ald ich, Spain) and abbi an i-Be a-
Tubulin (1:1,000) (No us Biologicals, Uni ed S a es) a 4◦C
o e nigh . A e wa d hey we e incuba ed wi h an i- abbi IgG
pe oxidase conjuga ed seconda y an ibodies (1:2,000) (Sigma-
Ald ich, Spain) o 2 h a RT and immunoblo s we e de eloped
wi h an enhanced chemiluminescence ki (GE Heal hca e Li e
Science, Uni ed Kingdom). The luminescence o he eac ion
p oduc was de ec ed in a pe sonal scanne , LI-COR C-DiGi (LI-
COR, Bonsai Ad anced Technologies SL, Spain), and isualized
bands we e analyzed wi h Image S udio Li e 4.0 so wa e (LI-
COR, Bonsai Ad anced Technologies SL, Spain). β-Ac in and
β- ubulin we e used as loading con ols.
S a is ical Analysis
All esul s we e exp essed as he mean ±SEM (s anda d
e o mean). S a is ical analysis was de eloped wi h G aphPad
P ism ( 5; G aphPad So wa e, Inc., Uni ed S a es) and SPSS
S a is ics ( 20; IBM Co po a ion, A monk, NY, Uni ed S a es).
P io o he analysis, he Shapi o–Wilk es was used o assess
he no mal dis ibu ion o he samples, and Le ene’s es was
used o de e mine he homogenei y o a iance. Mann-Whi ney
U- es was pe o med o assess di e ences be ween g oups and
wi hin g oups in he moni o ing analysis. The ROC (Recei e
Ope a ing Cha ac e is ics) cu e was pe o med and he AUC
(A ea Unde he Cu e) was calcula ed in o de o measu e
he accu acy o he model which a e a me ics o checking
classi ica ion models (Hanley and McNeil, 1982;Fawce , 2006),
in his case: Sham g oup and 6-OHDA g oup. The beha io al
da a, s e eology and densi ome y esul s we e analyzed by means
o he wo- ailed unpai ed S uden ’s - es o compa e di e ences
be ween g oups. A one-way analysis o a iance (ANOVA)
ollowed by Tukey’s mul iple-compa isons es was used o es
he di e ences be ween os o-caudal g adien s wi hin each
expe imen al g oup. The co ela ion was examined by Pea son
p oduc -momen co ela ion coe icien . Values P<0.05 we e
conside ed s a is ically signi ican .
RESULTS
Beha io al E alua ion
The moni o ing o EE cages allows ob aining da a abou
ac i i y and cen oid ea u es (speed, accele a ion and en opy)
du ing he ligh /da k cycle. Rema kably, 6-OHDA-lesioned a s
dec eased signi ican ly he numbe o ac i i y a eas du ing he
da k phase, which is he ac i e pe iod o he oden ci cadian
cycle, compa ing o Sham g oup (∗∗∗p<0.0001, Mann-Whi ney
U- es ) (Figu e 2B). Mo eo e , he 6-OHDA g oup also showed
a signi ican hype ac i i y du ing he ligh phase ( es ing pe iod)
in compa ison wi h con ol a s which may be ela ed o a
sleep beha io dis up ion (∗∗∗p<0.0001, Mann-Whi ney U- es ;
Figu es 2A,B). O e all, hese esul s sugges ed ha animals om
he 6-OHDA g oup swi ched hei ansi ional hy hms espec
o he con ol g oup. Fu he mo e, he e ec o 6-OHDA on
he dis up ion o ci cadian hy hms was also suppo ed by a
signi ican di e ence in he ac i i y be ween bo h ligh and da k
phases which was also seen in Sham g oup (###p<0.0001 ac i i y
a ea mean in ligh cycle s. ac i i y a ea mean in da k phase
wi hin he same g oup, Mann-Whi ney U- es ; Figu e 2B).
In line wi h hese esul s, al hough Sham g oup showed
mo e ema kable changes be ween da k and ligh cycles in
e ms o speed and accele a ion han 6-OHDA g oup, en opy
was signi ican ly highe in he 6-OHDA g oup (6-OHDA
g oup s. Sham g oup, ∗∗∗p<0.0001, Mann-Whi ney U- es ;
Figu es 2C–E). The ac ha en opy was highe suppo ed and
con i med he incipien sleep pa hological condi ion (Figu e 2E).
Bo h g oups also showed signi ican di e ences be ween bo h
da k and ligh phases in all he examined pa ame e s ela ed
o he cen oid e olu ion indica ing di e en beha io s in bo h
phases acco ding o he ligh o da k pe iod (###p<0.0001
wi hin he same g oup, Mann-Whi ney U- es ; Figu es 2C–E).
Besides, he 6-OHDA e ec on he accele a ion and speed
in bo h phases poin ed o a di icul y in he mo emen
no iced in bo h phases.
Thus, his is a model ha allows in eg a ing in o ma ion abou
bo h da k and ligh phases.
Acco dingly, he e iciency o his model was con i med by he
esul s ob ained by he ROC cu e and i s AUC which measu e
he accu acy o he model by Logis ic Reg ession. In he ROC
cu e he ue posi i e a e (Sensi i i y) is ep esen ed in unc ion
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FIGURE 2 | Ci cadian hy hm dis up ion in 6-OHDA-induced a s. (A) Diag am illus a ing he mo ion ac i i y signal in 6-OHDA (uppe panel) and Sham (bo om
panel) g oups du ing he whole manusc ip . A e wa d, he da a a e p ep ocessed and selec ed in o de o ob ain o he s a is ical analysis, app op ia ed balanced
da ase s o he ligh and da k pe iods o he wo g oups. Blue: mo ion ac i i y signal, G een: smoo hed signal o con ol g oup, Red: smoo hed signal o 6-OHDA
g oup, Yellow: ligh le el. G aphs depic signi ican ac i i y changes (B), he cen oid ea u es including he mean o cen oid speed (C), he mean o cen oid
accele a ion (D) and he en opy mean (E) be ween 6-OHDA and Sham g oups du ing he ligh /da k cycle and wi hin he same g oup. Da a a e exp essed as
mean ±SEM. Gene al analysis was pe o med by conside ing 12 h o da k/ligh pe iods. S a is ical di e ences appea (p<0.05) o Mann-Whi ney U- es be ween
bo h g oups and wi hin he same g oup o bo h ligh and da k phases (6-OHDA g oup s. Sham g oup, ***p<0.001; and ligh cycle s. da k cycle wi hin he same
g oup, ###p<0.001).
o he alse posi i e a e (1-Speci ici y) o di e en ope a ing
poin s, ea u es o he sys em, whe e an AUC o 100% would
ep esen he highes speci ici y and sensibili y o he model o
sepa a e bo h g oups. Ou da a ga e a ROC cu e (each poin
on he cu e ep esen s a sensi i i y/speci ici y) ha eached a
signi ican AUC o 0.854 gi ing 85% o sensibili y and speci ici y
o his model (0.854 ±0.0047, p=0;Figu e 3).
On he o he hand, minimal mo o de ici s we e obse ed
in he amphe amine es (Supplemen a y Table S1). Al hough
animals inc eased signi ican ly he numbe o IL u ns pe minu e
( pm) espec o con ol g oup, (1.93 ±0.39 pm s. 0.32 ±0.076
pm; (17)= 4.292, p= 0.0005, Unpai ed S uden ’s - es ), no
a s u ned mo e han 3.98 u ns pe minu e (0.79–3.98 pm;
Supplemen a y Figu e S2).
The e o e, a p od omal s age would be suppo ed by he mild
p esence o mo o symp oms.
Mo phological E alua ion
Once con i med he unc ional changes displayed by 6-OHDA
g oup 2 weeks a e 6-OHDA adminis a ion, we also assessed
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Requejo e al. 6-OHDA Induced P od omal PD Symp oms
FIGURE 3 | ROC cu e o linea eg ession. Modeling o 6-OHDA and Sham g oups. ROC cu e (each poin on he cu e ep esen s a sensi i i y/speci ici y) ha
eached a signi ican AUC o 0.854 gi ing 85% o sensibili y and speci ici y o his model (0.854 ±0.0047, p= 0). ROC, Recei e Ope a ing Cha ac e is ics;
6-OHDA, 6-hyd oxydopamine; AUC, A ea Unde he ROC Cu e.
whe he 6-OHDA exe ed a his ological e ec ha we had
p e iously obse ed 3 weeks a e 6-OHDA adminis a ion
(Requejo e al., 2018a,b).
6-OHDA-Induced Mild Nig os ia al Dopamine gic
Degene a ion A e a Sho E olu ion Time
Analysis o TH-immunos ained sec ions om he s ia um and
SN con i med ha , a his su i al ime, 6-OHDA induced
a mode a e TH-i ibe loss ei he in he s ia um o in
he SN (Figu e 4). In addi ion, dopamine gic neu ons
in he SN we e no no ably educed (Figu es 4E,G).
Ros o-caudal s udy o TH-i in he SN indica ed ha
he numbe o he su i al TH-i neu ons as well as he
TH-i axodend i ic ne wo k (ADN) in 6-OHDA g oup
inc eased os o-caudally. In e es ingly, one-way ANOVA
ollowed by Tu key pos hoc es indica ed signi ican
di e ences (*p<0.05) be ween os al and caudal le els
wi hin he 6-OHDA g oup ega ding he numbe o TH-
i neu ons (35.61 ±8.89% o TH-i neu onal cells a he
os al sec ions s. 70.12 ±7.03% o TH-i neu onal cells
a he caudal sec ions; p= 0.031). Howe e , ega ding he
numbe o TH-i AND no signi ican di e ences we e
ound be ween os al and caudal sec ions [52.24 ±6.32%
o TH-i ADN a he os al sec ions s. 76.86 ±5.29% o
TH-i o ADN a he caudal sec ion; One-way ANOVA,
F(2,9)= 3.800, p= 0.063; Figu es 4G,H]. In addi ion o his,
no opological dis ibu ion in he TH-i s ia al ibe s was
app ecia ed (Figu e 4D).
Co ela ions Be ween Mo o and Mo phological
Changes
The numbe o IL amphe amine-induced o a ions was s ongly
co ela ed wi h he pe cen age o TH-i neu onal cell loss
( =−0.94) and i was s a is ically signi ican (p≤0.05)
(Figu e 5B). On he o he hand, he TH-i e minal loss ei he
in s ia um o in SN also showed a co ela ion wi h he inc ease
in he numbe o IL o a ions pe minu e close o be s a is ically
signi ican ( =−0.84, p= 0.073 o TH-i IL s ia al olume and
=−0.93, p= 0.065 o TH-i AND; Figu es 5A,C).
Biochemical Analysis
In o de o assess changes in apop osis, he exp ession o caspase-
3 in neu onal cells was mo phologically e alua ed by double
immunos aining agains NeuN (nuclea ma ke o neu onal cells)
and caspase-3 (apop osis ma ke ). In addi ion, caspase-3 le els
we e also biochemically analyzed by wes e n blo in he s ia um
and SN (Figu e 6).
Double immuno luo escence e ealed highe caspase-3
immuno eac i i y and lowe NeuN immuno eac i i y in
6-OHDA-lesioned a s compa ed o Sham g oup in he IL
s ia um as well as in he IL SN. In ac , 6-OHDA induced a
ema kable apop osis in neu onal cells due o a conside able
amoun o NeuN posi i e cells co-localized wi h caspase-3 in
6-OHDA g oup (Figu es 6A,C).
Fu he mo e, immunoblo esul s we e consis en wi h he
ou comes showed by immunos aining. The exp ession o
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Requejo e al. 6-OHDA Induced P od omal PD Symp oms
FIGURE 4 | TH-i loss in he 6-OHDA-induced p eclinical model. (A,B) Ros o-caudal dis ibu ion o co onal sec ions co esponding o he CL and IL s ia um and
SN immunos ained wi h y osine hyd oxylase (TH) in Sham and 6-OHDA g oups. (C,D) G aphs show he e ec s o 6-OHDA on he loss o he TH-i olume in he IL
s ia um compa ing o Sham g oup (***p<0.001, Unpai ed S uden ’s - es .) and no signi ican changes we e ound along he os o-caudal axis. (E,F) TH-i
neu onal densi y and TH-i ADN densi y dec ease signi ican ly ollowing 6-OHDA adminis a ion compa ing o Sham g oup in he SN (***p<0.001, Unpai ed
S uden ’s - es ). (G,H) Topological analysis shows a selec i e ulne abili y in he SN o 6-OHDA dec easing he TH-i neu onal and he TH-i ADN densi y
os o-caudally (*p<0.05 os al sec ion s. caudal sec ion wi hin 6-OHDA g oup, One-way ANOVA). Da a a e p esen ed ei he as he pe cen age o TH-i
ipsila e al s ia al olume espec o he o al ipsila e al one (C,D) o as he pe cen age o TH-i neu ons (E,G) o TH-i ADN (F,H) emaining in he IL side espec o
he CL side. Scale ba : 2 mm (A),1mm(B). CL, con ala e al; IL, ipsila e al; TH-i , y osine hyd oxylase immuno eac i i y; 6-OHDA, 6-hyd oxydopamine; ADN,
axodend i ic ne wo k; SN, subs an ia nig a.
FIGURE 5 | Mo pho- unc ional co ela ion be ween amphe amine-induced o a ions and dopamine gic neu ons in SN in addi ion o dopamine gic e minals in bo h
s ia um and SN a e 6-OHDA adminis a ion. (A) Amphe amine-induced IL o a ions co ela e nega i ely wi h he % o TH-i s ia al olume IL, (B) also wi h he % o
TH-i neu onal densi y and (C) wi h he % o TH-i ADN densi y. Signi icance. p<0.05, IL o a ions s. TH-i neu ons. SN, subs an ia nig a; 6-OHDA,
6-hyd oxydopamine; TH-i , y osine hyd oxylase immuno eac i i y; IL, ipsila e al.
caspase-3 was up egula ed in 6-OHDA-lesioned a s in he
s ia um and in SN ollowing a simila pa e n (127.9 ±5.1%
and 127.9 ±26.08%, espec i ely), while caspase-3 ac i a ion was
ba ely e idenced in Sham g oup (103.8 ±7.6% in he s ia um
and 90.7 ±7.57% in he SN). Howe e , s a is ically signi ican
di e ences be ween bo h g oups we e only ound in he s ia um
[ (7)= 2.426, p= 0.045, Unpai ed S uden ’s - es ; Figu es 6B,D].
Howe e , no signi ican changes we e seen in he p o-
su i al signaling p o ein le els (AKT and ERK) be ween
6-OHDA and Sham g oups 2 weeks pos injec ion, despi e
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