Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease

RESEARCH ARTICLE
Al e ed glial exp ession o he cannabinoid 1 ecep o in he
subiculum o a mouse model o Alzheime 's disease
I zia Te adillos
1,2
| I zia Bonilla-Del Río
1,2
| Nago e Puen e
1,2
|
Mai ane Se ano
1,2
| Amaia Mimenza
1,2
| Lei e Lekunbe i
1,2
|
Ilazki Anau -Lusa
1,2
| Lei e Regue o
1,2
| Inmaculada Ge ikagoi ia
1,2
|
Samuel Ruiz de Ma ín Es eban
3
| Cecilia J. Hilla d
4
| Ma ía T. G ande
3
|
Julián Rome o
3
| Izaskun Elezga ai
1,2
| Ped o G andes
1,2
1
Depa men o Neu osciences, Facul y o
Medicine and Nu sing, Uni e si y o he
Basque Coun y UPV/EHU, Leioa, Spain
2
Achuca o Basque Cen e o Neu oscience,
Leioa, Spain
3
Facul y o Expe imen al Sciences, Uni e sidad
F ancisco de Vi o ia, Pozuelo de Ala c
on, Spain
4
Depa men o Pha macology and Toxicology,
Neu oscience Resea ch Cen e , Medical
College o Wisconsin, Milwaukee,
Wisconsin, USA
Co espondence
Ped o G andes, Depa men o Neu osciences,
Facul y o Medicine and Nu sing, Uni e si y o
he Basque Coun y UPV/EHU, Leioa, Spain.
Email: [email p o ec ed]us
Funding in o ma ion
Eusko Jau la i za, G an /Awa d Numbe s:
IT1230-19, IT1620-22; Minis e io de Ciencia e
Inno aci
on, G an /Awa d Numbe s:
PID2019-107548RB-I00,
PID2019-108992RB-I00; Resea ch and
Educa ion Componen o he Ad ancing a
Heal hie Wisconsin Endowmen a he
Medical College o Wisconsin
Abs ac
The al e a ion o he endocannabinoid one usually associa es wi h changes in he
exp ession and/o unc ion o he cannabinoid CB
1
ecep o . In Alzheime 's disease
(AD), amyloid be a (Aβ)-con aining agg ega es induce a ch onic in lamma o y
esponse leading o eac i i y o bo h mic oglia and as ocy es. Howe e , how his
glial esponse impac s on he glial CB
1
ecep o exp ession in he subiculum o a
mouse model o AD, a b ain egion pa icula ly a ec ed by la ge accumula ion o
plaques and concomi an subcellula changes in mic oglia and as ocy es, is unknown.
The CB
1
ecep o localiza ion in bo h glial cells was in es iga ed in he subiculum o
male 5xFAD/CB
2EGFP/ /
(AD model) and CB
2EGFP/ /
mice by immuno-elec on
mic oscopy. The indings e ealed ha glial CB
1
ecep o s su e ema kable changes
in he AD mouse. Thus, CB
1
ecep o exp ession inc eases in eac i e mic oglia in
5xFAD/CB
2EGFP/ /
, bu emains cons an in as ocy es wi h CB
1
ecep o labeling
ising p opo ionally o he pe ime e o he eac i e as ocy es. No leas , he CB
1
ecep o localiza ion in mic oglial p ocesses in he subiculum o con ols and closely
su ounding amyloid plaques and dys ophic neu i es o he AD model, suppo s p e-
ious sugges ions o he p esence o he CB
1
ecep o in mic oglia. These indings on
he co ela ion be ween glial eac i i y and he CB
1
ecep o exp ession in mic oglial
cells and as ocy es, con ibu e o he unde s anding o he ole o he endocannabi-
noid sys em in he pa hophysiology o Alzheime 's disease.
KEYWORDS
as oglia, endocannabinoid sys em, immuno-elec on mic oscopy, mic oglia, neu odegene a ion
I zia Te adillos and I zia Bonilla-Del Río sha e i s au ho ship.
Recei ed: 2 Augus 2022 Re ised: 23 Oc obe 2022 Accep ed: 18 No embe 2022
DOI: 10.1002/glia.24312
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any
medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made.
© 2022 The Au ho s. GLIA published by Wiley Pe iodicals LLC.
866 Glia. 2023;71:866–879.
wileyonlinelib a y.com/jou nal/glia
1|INTRODUCTION
As ocy es and mic oglia ac i i y associa ed wi h ch onic in lamma ion
induced by Aβ-con aining agg ega es esul s in abno mal mo phology
and p oli e a ion o bo h glial cells in AD (Beni o e al., 2003;McAlpine
e al., 2021; Smi e al., 2021). The po en ial o cannabinoids o a ge
se e al p ocesses in ol ed in AD pa hogenesis is ega ded as a he a-
peu ic s a egy (Casa ejos e al., 2013; Chen e al., 2012;Eljaschewi sch
e al., 2006; Tala ico e al., 2019), in pa because he main cannabinoid
CB
1
ecep o is localized in b ain egions and cells, including glia,
a ec ed by he disease. We ha e p e iously es ima ed ha abou
56% o he CB
1
ecep o labeling localizes o GABAe gic e minals,
12% o glu ama e gic e minals, 6% o as ocy es, 15% o mi o-
chond ia and he es o o he cells and compa men s o be de e -
mined (Bonilla-Del Rίoe al.,2019;Bonilla-DelRíoe al.,2021).
The exp ession o CB
1
ecep o s in as ocy es seems o be eg-
ula ed by mul iple ac o s and can a y unde di e en b ain condi-
ions, o example, ansien ecep o po en ial anilloid 1 knock ou
mice show a signi ican dec ease in CB
1
densi y in as ocy es
(Egaña-Hugue e al., 2021), acu e Δ-9- e ahyd ocannabinol expo-
su e causes CB
1
inc ease in hese glial cells (Bonilla-Del Río
e al., 2021), o adolescen binge d inking signi ican ly dec eases
CB
1
in as ocy es in he adul b ain (Bonilla-Del Rίoe al.,2019).
Al hough he impac ha as oglial dys unc ion may ha e in AD
is s ill poo ly unde s ood (Smi e al., 2021; Ve kh a sky &
Nede gaa d, 2018), pieces o e idence sugges ha changes in he
endocannabinoid sys em occu in as ocy es nea AD lesions. Thus,
as ocy es closely associa ed wi h Aβagg ega es show mo e in e -
media e ilamen p o eins and hype ophy o cell bodies (Esca in
e al., 2019;Smi e al.,2021). These as ocy es clea as well as
deg ade Aβagg ega es and elease p o-in lamma o y molecules
(Fa ina e al., 2007) which can be diminished by endocannabinoids
ac ing on CB
1
ecep o s localized in hese glial cells (Me na-
Lau en & Ma sicano, 2015). Also, high a y acid amide hyd olase
(FAAH) le els, he main deg ading enzyme o he endocannabinoid
anandamide, ha e been ound in as ocy es a ound neu i ic plaques
(Aba e e al., 2021;Beni oe al.,2003). Howe e , he eal impac o
he p og ession o Alzheime 's disease on he exp ession o CB
1
ecep o s in as ocy es su ounding he lesions is unknown.
Mic oglia is ano he impo an playe in he pa hogenesis o
Alzheime 's disease. Cannabinoids p e en Aβ-induced neu ode-
gene a ion by educing mic oglial ac i i y. Bo h CB
1
and CB
2
ecep-
o sexp essedinmic ogliaa ein ol edin hisac ionas hey
inhibi neu oin lamma ion by p e en ing eac i e oxygen species
(ROS) o ma ion and cy okines elease by hese cells (Casa ejos
e al., 2013; Ma ín-Mo eno e al., 2011; Ramí ez e al., 2005;
Tala ico e al., 2019). Mic oglia also elici s a signi ican inc ease in
endocannabinoid p oduc ion ha , in u n, ac i a es mo e CB
1
and
CB
2
ecep o s and hei signaling cascades, ampli ying he an i-
in lamma o y and p o ec i e mic oglial pheno ype (Du y
e al., 2021; Mecha e al., 2016). In ac , he numbe and Aβphago-
cy ic capaci y o mic oglial cells dec ease in mice lacking CB
2
ecep-
o s (de Ma ín e al., 2022).
The e a e pieces o e idence indica ing ha mic oglia cons i u-
i ely exp esses CB
1
ecep o s ha media e some o he cannabi-
noid e ec s in es ing mic oglial cells (Kaplan, 2013; Na a o
e al., 2018; Ribei o e al., 2013;S ella,2009;Thione al.,2018),
and egula e neu oin lamma ion in a sex-dependen manne
(De Meij e al., 2021). CB
1
ecep o s ha e been no iced in cul u ed
mic oglia (Ca lisle e al., 2002; Facchine i e al., 2003;Klege is
e al., 2003; Molina-Holgado e al., 2002; Sinha e al., 1998;
S e ano e al., 1996; Waksman e al., 1999;Wal e e al.,2003),
and speci ic an i-CB
1
an ibodies de ec ed some sca e ed CB
1
sig-
nal in mic oglia in he hypo halamic a cua e nucleus o emales
(De Meij e al., 2021).
CB
1
ecep o exp ession inc eases in many in lamma o y and
neu odegene a i e diseases like AD (Bisogno & Di Ma zo, 2010;
Ribei o e al., 2013); howe e , e y li le is known abou he localiza-
ion and exp ession o cannabinoid ecep o s in glial cells in AD. We
hypo hesize in his s udy ha CB
1
ecep o exp ession in glia is
al e ed in he subiculum o a mouse model o AD, a b ain egion pa -
icula ly a ec ed by la ge accumula ion o plaques, as a esul o
concomi an subcellula changes in mic oglia and as ocy es. Ou
indings show ha CB
1
ecep o s in mic oglial cells su e ema kable
modi ica ions in 5xFAD/CB
2EGFP/ /
mice, a mu ine model o AD
ecen ly epo ed o ha e an inc ease in CB
2
ecep o s in mic oglia
ela ed o dys ophic neu i es (Ruiz de Ma ín Es eban e al., 2022),
simila ly o he CB
2
ise obse ed in plaque-associa ed mic oglia
(Beni o e al., 2003,2007).
2|MATERIAL AND METHODS
2.1 |E hics s a emen
The p o ocols o animal ca e and use we e app o ed by he Com-
mi ee o E hics o Animal Wel a e o he Uni e si y o he Basque
Coun y (M20/2020/109) and we e in acco dance o he Eu opean
Communi ies Council Di ec i e o Sep embe 22, 2010 (2010/63/
EU) and Spanish egula ions (Real Dec e o 53/2013, BOE
08-02-2013). E o s we e made o minimize he numbe and su -
e ing o animals.
2.2 |Expe imen al animals
Expe imen s we e done in 6.5–7.5-mon h-old male CB
2EGFP/ /
mice,
con ols and co-exp essing i e AD mu a ions (5xFAD; Oakley
e al., 2006) p e iously used in ou labo a o y o he localiza ion o
CB
2
ecep o s (Ruiz de Ma ín Es eban e al., 2022). The CB
2EGFP/ /
mice we e gene a ed a he Genoway acili ies (Lyon, F ance) by
designing a a ge ing s a egy consis ing o he inse ion o an
enhanced g een luo escen p o ein (EGFP) epo e gene, p eceded
by an in e nal ibosomal en y si e sequence (IRES) in o he 30
un ansla ed egion (UTR) o he mouse cn 2 gene. This esul ed in
exp ession o he epo e gene (EGFP) unde he con ol o he
TERRADILLOS ET AL.867
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mouse endogenous cn 2 p omo e , and ansc ip ion o he same
bicis onic mRNA as he CB
2
ecep o p o ein. In addi ion, hese mice
co-exp essed 5xFAD mu a ions a he same ime. The 5xFAD mice
wi h a C57BL/6J backg ound we e pu chased om Jackson Labo a-
o y (Ba Ha bo , Main, USA). To ob ain he co-exp ession, 5xFAD
mice we e ma ed wi h CB
2EGFP/ /
mice o a leas i e gene a ions o
gene a e 5xFAD/CB
2EGFP/ /
mice (L
opez e al., 2018). This 5xFAD
model does no seem o exp ess he mu a ion ha causes he neu o i-
b illa y degene a ion (Oblak e al., 2021), bu i may occu indi ec ly
h ough neu onal degene a ion and Aβ1–42 deposi s.
2.3 |B ain issue p ocessing
Mice we e anes he ized wi h ke amine/xylazine (100 mg/10 mg/kg
body weigh , in ape i oneal injec ion) and subsequen ly pe used
ansca dially a oom empe a u e (RT) h ough he le en icle. Fi s
wi h phospha e bu e ed saline (PBS) 0.1 M (pH 7.4) o 20 s, and hen
wi h he ixa i e solu ion composed o 4% o maldehyde, 0.2% pic ic
acid and 0.1% glu a aldehyde in PBS 0.1 M (pH 7.4) o 10–15 min,
wi h a ixa i e solu ion olume o 80 ml pe mouse. The b ains we e
hen emo ed om he skull and pos - ixed in he ixa i e solu ion o
app oxima ely 1 week a 4C. Subsequen ly, hey we e s o ed in 1:10
dilu ed ixa i e solu ion a 4C wi h 0.025% sodium azide. B ain ib o-
sec ions we e cu co onally a 50 μm and s o ed wi h 1 ml o phos-
pha e bu e (PB) 0.1 M (pH 7.4) wi h 0.025% sodium azide a 4C.
2.4 |Immunohis ochemis y o ligh mic oscopy
B ain sec ions con aining he subiculum we e collec ed in PB 0.1 M
(pH 7.4) a RT, p e-incuba ed wi h a blocking solu ion o 10% bo ine
se um albumin (BSA), 0.1% sodium azide and 0.5% i on X-100 in 1x
is-bu e ed saline (TBS 1x) (pH 7.4) o 30 min a RT, and incuba ed
wi h he ollowing p ima y polyclonal an ibodies: guinea pig an i-CB
1
ecep o (1:100; CB
1
-GP-A 530; AB_2571593, F on ie Ins i u e Co.,
l d), abbi an i-ionized calcium-binding adap e molecule 1 (Iba1,
1:500; 019-19741; AB_839504, FUJIFILM Wako Pu e Chemical Co -
po a ion), abbi an i-glu ama e aspa a e anspo e 1 (An i-A522
[GLAST] EAAT1, 0.3 μg/ml; Ab#314; AB_231456, kindly gi ed by
P o . Niels Ch is ian Danbol , Uni e si y o Oslo). They we e p epa ed
in blocking solu ion and gen ly shaken o 2 days a 4C o 1 day
a RT. Then, sec ions we e washed wi h 1% BSA and 0.5% i on
X-100 in TBS 1x o 30 min, and incuba ed wi h a bio inyla ed an i-
guinea pig (1:200, Bio in-SP-A iniPu e Goa An i-Guinea Pig IgG;
AB_2337394, Jackson Immuno Resea ch), o bio inyla ed an i- abbi
seconda y an ibody (1:200, Bio in-SP-A iniPu e Donkey An i-Rabbi
IgG; AB_2340593, Jackson Immuno Resea ch) dilu ed in he washing
solu ion o 1 h on a shake a RT. They we e washed wi h 1% BSA
and 0.5% i on X-100 in TBS 1x (30 min). Tissue was incuba ed wi h
he a idin-bio in pe oxidase complex (ABC; 1:50, Eli e, Vec o Labo a-
o ies, Bu lingame, CA, USA) p epa ed in he washing solu ion, o 1 h
a RT. Samples we e washed wi h 1% BSA and 0.5% i on X-100 in
TBS 1x (3 x 1 min) and las ly wi h PB 0.1 M (pH 7.4) and 0.5% i on
X-100 (2 x 10 min). Labeling was e ealed wi h 0.05% diaminobenzi-
dine (DAB) in PB 0.1 M (pH 7.4) con aining 0.5% i on X-100 and
0.01% hyd ogen pe oxide o 3.5 min a RT. This was ollowed by
washes in PB 0.1 M (pH 7.4) wi h 0.5% i on X-100 (3 x 1 min,
2 x 10 min). Tissue sec ions we e moun ed on gela inized slides, d ied
and dehyd a ed in g aded e hanol o 5 min each. A e insing wi h
xylene (3 x 5 min), he slides we e co e slipped wi h DPX. The subi-
culum was examined and pho og aphed wi h a Zeiss AxioCam ligh
mic oscope coupled o a Zeiss AxioCam HRc came a.
2.5 |Immunohis ochemis y o elec on
mic oscopy
The p o ocol is al eady published (Puen e e al., 2019). Fou o i e sec-
ions pe b ain con aining he subiculum we e selec ed. They we e p e-
incuba ed in a blocking solu ion (1 ml/well) o 10% BSA, 0.02% saponin
and 0.1% sodium azide in TBS 1x (pH 7.4), o 30 min on he shake a
RT. Tissue was hen incuba ed wi h a p ima y guinea pig an i-CB
1
ecep o an ibody (1:100) in combina ion wi h a abbi an i-GLAST an i-
body (0.3 μg/ml) o a abbi an i-Iba1 an ibody (1:500). The solu ion
con ained 10% BSA in TBS 1x, 0.1% sodium azide and 0.004% saponin.
Incuba ion was pe o med on a shake o 2 days a 4C ollowedby
washes in 1% BSA/TBS 1x. Then, sec ions we e incuba ed wi h 1.4 nm
gold-conjuga ed goa an i-guinea pig IgG an ibody (Fab agmen ,
1:100, #2055, Nanop obes, Inc., Yaphank, NY, USA). They we e also
incuba ed wi h bio inyla ed an i- abbi IgG an ibody (1:200) dilu ed in
1% BSA/TBS 1x wi h 0.004% saponin on a shake o 4 h a RT. Tissue
was washed in 1% BSA/TBS 1x on a shake a RT and incuba ed wi h
ABC (1:50) p epa ed in washing solu ion o 1.5 h a RT. Sec ions we e
washed in 1% BSA/TBS 1x, kep o e nigh a 4C and pos - ixed wi h
1% glu a aldehyde in TBS 1x (1 ml/well) o 12 min a RT. Then, hey
we e washed in double dis illed wa e and gold pa icles we e sil e -
in ensi ied wi h he HQ Sil e ki (#2012, Nanop obes, Inc., Yaphank,
NY, USA) in he da k o 12 min a RT. A e in ensi ica ion, he sec-
ions we e washed i s in double dis illed wa e and hen wi h PB
0.1 M (pH 7.4) o 30 min. The bio inyla ed an ibody was e ealed
wi h 0.05% DAB p epa ed in PB 0.1 M (pH 7.4) con aining 0.5% i on
X-100 and 0.01% hyd ogen pe oxide o 3.5 min a RT, ollowed by
se e al washes in PB 0.1 M (pH 7.4). They we e osmica ed (1% osmium
e oxide in PB 0.1 M, pH 7.4) in he da k o 20 min, washed in PB
0.1 M (pH 7.4), dehyd a ed in g aded e hanol, clea ed in p opylene oxide,
p e-embedded in 1:1 p opylene oxide/Epon 812 esin on a shake o e -
nigh a RT and embedded in pu e Epon 812 esin. Immunogold labeling
was isualized wi h a ligh mic oscope in sec ions con aining he subicu-
lum,and issuepo ionswi hgoodandconsis en CB
1
ecep o labeling
we e iden i ied and immed down o ul a hin sec ioning. The p oce-
du e has al eady been desc ibed in de ail (Bonilla-Del Rίoe al.,2019;
Bonilla-Del Río e al., 2021; Gu ié ez-Rod íguez e al., 2018; Puen e
e al., 2019). Th ee o ou semi- hin sec ions (0.7 μm- hick) we e
ob ained wi h a his o-diamond kni e (Dia ome USA) and s ained wi h
1% oluidine blue. To u he s anda dize he condi ions, only he
868 TERRADILLOS ET AL.
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i s i e ul a hin sec ions we e cu (50 nm- hick) wi h an ul a-
diamond kni e (Dia ome USA), collec ed on o nickel mesh g ids
and coun e s ained wi h 2.5% lead ci a e o 20 min a RT. Elec on
mic og aphs we e andomly aken wi h a Hamama su FLASH digi al
came a inse ed in a ansmission elec on mic oscope (JEOL JEM 1400
Plus). Sampling was always ca e ully and accu a ely done using he ol-
lowing ana omical coo dina es o delimi he subiculum: in e au al,
0.40/0.00 mm; b egma, 3.40/3.80 mm (F anklin & Paxinos, 2008).
2.6 |An ibodies speci ici y
Expe imen s we e always pe o med unde he same condi ions. In
addi ion, nega i e con ols omi ing he p ima y an ibodies we e done.
Fu he mo e, he CB
1
ecep o an ibody was es ed in CB
1
/b ain
issue (Ma sicano e al., 2002) by double p e-embedding immunogold
(CB
1
) and immunope oxidase (Iba1 o GLAST) me hod o elec on
mic oscopy (Figu e 1). The an i-A522 (EAAT1 [GLAST]) an ibody
(Ab#314) a ge ing he C- e minal esidues 522–541 o a EAAT1
(Hu e al., 2020) was used o iden i y as ocy ic compa men s. GLAST
was es ic ed o as ocy es and localized in acellula ly wi h no
de ec able labeling in ne e e minals, as p e iously desc ibed (Leh e
e al., 1995; Schmi e al., 1997). Fu he mo e, GLAST dis ibu ion
is e y simila in oden s and humans (Li e al., 2012), so he use o
an i-GLAST an ibody is a good app oach o label as ocy es in he AD
model. The speci ici y o he Iba1 an ibody has been con i med in p e-
ious s udies (Delcamb e e al., 2016; Szabo & Gulya, 2013). GLAST
can be exp essed in bo h mic oglia and as ocy es unde ce ain
condi ions (Bescho ne e al., 2007). Expe imen s we e conduc ed o
igu e his ou . S ikingly, GLAST and Iba1 we e no seen o co-
localize in a subicula a ea o 3506 μm
2
analyzed in CB
2EGFP/ /
and o
4743 μm
2
in 5xFAD/CB
2EGFP/ /
(da a no shown). Taken oge he , we
could easonably conclude ha GLAST and Iba1 a e selec i e ma ke s
o as ocy e and mic oglia, espec i ely, in he 6.5–7.5 mon h-old-
CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
mice s udied.
2.7 |Quan i a i e and s a is ical assessmen
To ensu e homogeneous labeling be ween all samples, only he i s
1.5 μm om he sec ion su ace o each specimen we e conside ed
o he analysis. A ea, pe ime e , numbe o p ocesses and CB
1
ecep-
o exp ession in as ocy es, we e s udied in 5596 μm
2
o i e
CB
2EGFP/ /
, and in 7681 μm
2
o se en 5xFAD/CB
2EGFP/ /
mice. In
addi ion, he a ea, pe ime e and numbe o mic oglial p ocesses we e
measu ed in 7900 μm
2
o se en CB
2EGFP/ /
, and in 10,793 μm
2
o
10 5xFAD/CB
2EGFP/ /
mice. Fo he s udy o CB
1
ecep o s in mic o-
glia, 6345 μm
2
in i e CB
2EGFP/ /
and 7268 μm
2
in se en 5xFAD/
CB
2EGFP/ /
mice, we e analyzed.
CB
1
ecep o labeling in as ocy es and mic oglia was assessed in
GLAST- and Iba1-immunoposi i e p ocesses, espec i ely. The p opo -
ion o cell compa men s wi h CB
1
ecep o signal was hen abula ed.
Posi i e labeling was conside ed when a leas one immunopa icle was
wi hin 30 nm o he memb ane s udied. CB
1
ecep o densi y (pa i-
cles/μm memb ane) was also de e mined by coun ing gold pa icles in
he posi i e compa men s. Memb ane leng h (pe ime e ) was mea-
su ed wi h he Image-J so wa e (NIH; SCR_003070).
All alues we e gi en as mean ± S.E.M. using a s a is ical so wa e
package (G aphPad P ism 8, SCR_002798, G aphPad So wa e, Inc.,
San Diego, USA). The no mali y es (Kolmogo o –Smi no no mali y
es ) was always applied be o e s a is ics was done. Da a we e ana-
lyzed by non-pa ame ic o pa ame ic es s: Mann–Whi ney U es o
S uden 's Unpai ed - es (*p< .05).
Mino con as and b igh ness adjus men s we e made o he ig-
u es using Adobe Pho oshop (Adobe Pho oshop, SCR_014199, CS3,
Adobe Sys ems, San Jose, CA, USA) and Gimp (GNU Image Manipula-
ion P og am, SCR_003182).
3|RESULTS
3.1 |Glial mo phology in he subiculum o
CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
mice
Changes in s aining densi y we e de ec ed in mic oglial and as ocy ic
cells (Figu e 2a–d). Mic oglia iden i ied by Iba1 in CB
2EGFP/ /
FIGURE 1 Subiculum o CB
1
knock ou mice (CB
1
/). Double
immunogold and immunope oxidase me hod o elec on mic oscopy.
An ibodies we e es ed in CB
1
/mice (a–d). Simul aneous labeling
o CB
1
(gold) and Iba1 (a and b; DAB immunodeposi s in b own) o
GLAST (c and d; DAB in pink). No CB
1
ecep o signal is obse ed.
Scale ba s: 50 nm
TERRADILLOS ET AL.869
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(Figu e 2a,a1) occupied a much la ge a ea in 5xFAD/CB
2EGFP/ /
(Figu e 2b,b1). This inc ease seemed o ela e o bo h he numbe o
mic oglial cells and he hickness o hei p ocesses (Figu e 2a1,b1). In
he elec on mic oscope, only sca e ed mic oglial p ocesses we e
obse ed in con ols (Figu e 3a,b), while nume ous Iba1-posi i e p o-
cesses su ounding plaques (Figu e 3c, ) and dys ophic neu i es
(Figu e 3c1,c3- ) we e seen in 5xFAD/CB
2EGFP/ /
mice. The a ea o
he mic oglial p ocesses was signi ican ly inc eased in 5xFAD/
CB
2EGFP/ /
(0.3229 ± 0.05282 μm
2
; ***p< .0001) ela i e o CB
2EGFP/ /
(0.1000 ± 0.01754 μm
2
;Figu e5a), as i was he o al mic oglial a ea pe
sample no malized o 100 μm
2
(5xFAD/CB
2EGFP/ /
: 2.074 ±
0.5156 μm
2
; ***p< .0001; CB
2EGFP/ /
: 0.3485 ± 0.06955 μm
2
;
Figu e 5b). In addi ion, a signi ican inc ease in he pe ime e o he
mic oglial p olonga ions was de ec ed in 5xFAD/CB
2EGFP/ /
(2.200 ±
0.1248 μm; ***p< .0001) e sus CB
2EGFP/ /
(1.260 ± 0.1036 μm;
Figu e 5c) and e lec ed in he pe ime e o he o al mic oglial p ocesses
pe sample no malized o 100 μm
2
(15.73 ± 1.929 μmin5xFAD/
CB
2EGFP/ /
; 4.334 ± 0.5345 μminCB
2EGFP/ /
; ***p< .0001; Figu e 5d).
Finally, signi ican changes we e also no iced in he numbe o mic oglial
p ocesses (7.260 ± 0.6304/100 μm
2
in 5xFAD/CB
2EGFP/ /
; 3.413 ±
0.4092/100 μm
2
in CB
2EGFP/ /
;***p< .0001; Figu e 5e).
As o as ocy es, GLAST s aining seen in CB
2EGFP/ /
(Figu e 2c,c1)
was mo e in ense in 5xFAD/CB
2EGFP/ /
(Figu e 2d,d1). By using GLAST-
DAB, he a ea, pe ime e and numbe o as ocy ic elemen s we e ana-
lyzed in he elec on mic oscope (Figu e 4). As ocy ic p ocesses su -
ounded dys ophic neu i es and plaques in 5xFAD/CB
2EGFP/ /
(Figu e 4b–d) and showed a signi ican inc ease in hei a ea (5xFAD/
CB
2EGFP/ /
: 0.2598 ± 0.01853 μm
2
;CB
2EGFP/ /
: 0.1565 ± 0.006515 μm
2
;
***p< .0001; Figu e 5a). Howe e , no di e ences we e obse ed in he
o al a ea pe sample occupied by as ocy ic p ocesses no malized o
100 μm
2
(5xFAD/CB2
EGFP/ /
: 8.993 ± 0.8664 μm
2
;CB2
EGFP/ /
: 7.415
± 0.6552 μm
2
;p: .1711; Figu e 5b). The e was also a g ea inc ease in he
pe ime e o he as ocy ic p ocesses (5xFAD/CB
2EGFP/ /
: 2.833
± 0.08486 μm; CB
2EGFP/ /
: 2.116 ± 0.04741 μm; ***p< .0001; Figu e 5c).
Ne e heless, no di e ences in he o al pe ime e o as ocy ic p ocesses
pe samplewe ede ec ed(5xFAD/CB
2EGFP/ /
: 99.89 ± 8.087 μm;
CB
2EGFP/ /
: 100.1 ± 6.811 μm; p: .9820; Figu e 5d). Consis en wi h
hese esul s, a signi ican dec ease in he numbe o as ocy ic p ocesses
in 5xFAD/CB
2EGFP/ /
(35.52 ± 2.661 pe 100 μm
2
) ela i e oCB
2EGFP/ /
(47.33 ± 2.709 pe 100 μm
2
) was no iced (**p: .0036; Figu e 5e).
O e all, ou analyses in he subiculum o he Alzheime 's model
e eal ha he la ge a ea o he mic oglia co ela es wi h an inc ease
in size and numbe o hei p ocesses, while as ocy ic p ojec ions a e
ewe bu bigge (Figu e 5a–e).
3.2 |CB
1
ecep o s in CB
2EGFP/ /
and 5XFAD/
CB
2EGFP/ /
subiculum
The CB
1
ecep o labeling obse ed in CB
2EGFP/ /
(Figu e 2e,e1) was
mo e pa chy in 5xFAD/CB
2EGFP/ /
showing many delimi ed ound
a eas wi h much lowe o insigni ican s aining, p obably co espond-
ing o neu i ic plaques su ounded by a neu opil wi h mo e CB
1
ecep-
o immuno eac i i y (Figu e 2 , 1).
FIGURE 2 Subiculum o CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
mice showing Iba1, GLAST and CB
1
immunos aining. A idin-bio in pe oxidase
me hod o ligh mic oscopy. Iba1 in CB
2EGFP/ /
(a, a1) is d as ically inc eased in 5xFAD/CB
2EGFP/ /
mic oglia (b, b1). GLAST in CB
2EGFP/ /
(c, c1)
is weake han in 5xFAD/CB
2EGFP/ /
(d, d1). Dense CB
1
ecep o s aining in CB
2EGFP/ /
(e, e1) changes o a weake and mo e pa chy appea ance
in 5xFAD/CB
2EGFP/ /
( , 1). Scale ba s: 200 μm(a– ) and 50 μm (a1– 1)
870 TERRADILLOS ET AL.
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3.3 |Mic oglial CB
1
ecep o localiza ion in
CB
2EGFP/ /
and 5XFAD/CB
2EGFP/ /
subiculum
CB
1
ecep o pa icles we e localized o memb anes o Iba1-posi i e
mic oglial p ocesses in bo h CB
2EGFP/ /
(Figu e 3b2) and 5xFAD/
CB
2EGFP/ /
mice (Figu e 3c– ). The analysis e ealed a signi ican
inc ease in CB
1
-posi i e mic oglial p ocesses in 5xFAD/CB
2EGFP/ /
(0.9942 ± 0.1259 CB
1+
p ocesses/100 μm
2
) compa ed o CB
2EGFP/ /
(0.3254 ± 0.07758 CB
1+
p ocesses/100 μm
2
; ***p< .0001; Figu e 6a,
le ). In addi ion, a s ike inc ease in he p opo ion o CB
1
-posi i e
FIGURE 3 Double p e-embedding immunogold (CB
1
) and immunope oxidase (Iba1) me hod o elec on mic oscopy in he subiculum o
CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
mice. In CB
2EGFP/ /
, a ew slende Iba1 posi i e mic oglial elemen s a e obse ed (a, b) (DAB immunodeposi s in
b own). Howe e , hick p ocesses o eac i e mic oglia appea in 5xFAD/CB
2EGFP/ /
(c– ) su ounding plaques (in pu ple; c, ) and dys ophic
neu i es (in u quoise; c– ). No ice memb ane CB
1
pa icles (o ange a ows) in mic oglial p ocesses o CB
2EGFP/ /
(b2) and 5xFAD/CB
2EGFP/ /
(c– ),
wi h pa icula abundance in 5xFAD/CB
2EGFP/ /
.CB
1
ecep o labeling is also in memb anes o exci a o y e minals (g een a ows and p o iles in a1,
b2 and d1), inhibi o y e minals (yellow a ows and p o iles in b1, d2) and mi ochond ia (blue a ows and p o iles in a1 and d2), in bo h mu an s.
Scale ba s: 2 μm
TERRADILLOS ET AL.871
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elemen s was de ec ed in 5xFAD/CB
2EGFP/ /
mice (6.27 ± 1.15%;
CB
2EGFP/ /
: 3.79 ± 2.10%; **p: .0033; Figu e 6b, le ). Howe e , CB
1
ecep o densi y in he posi i e mic oglial p ocesses was signi ican ly
educed in 5xFAD/CB
2EGFP/ /
(69.44 ± 7577 pa icles/100 μmo
memb ane; CB
2EGFP/ /
: 135.5 ± 24.78/100 μm o memb ane; **p:
.0023; Figu e 6c, le ). Fu he mo e, signi ican di e ences in he o al
numbe o mic oglial CB
1
pa icles pe 100 μm
2
we e obse ed
be ween 5xFAD/CB
2EGFP/ /
(1.31 ± 0.18 pa icles) and CB
2EGFP/ /
FIGURE 4 Double p e-embedding immunogold (CB
1
ecep o ) and immunope oxidase (GLAST) me hod o elec on mic oscopy in he
subiculum o CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
mice. GLAST-posi i e as ocy ic p ocesses (DAB immunodeposi s in pink) seen in CB
2EGFP/ /
(a) a e hicke in 5xFAD/CB
2EGFP/ /
(b–d). Obse e as ocy ic elemen s su ounding dys ophic neu i es ( u quoise in b–d) and close o a plaque
(pu ple in d) in he 5xFAD/CB
2EGFP/ /
.CB
1
pa icles ( ed a ows) localize o plasma memb anes o GLAST-posi i e as ocy ic p ocesses in
CB
2EGFP/ /
(a) and 5xFAD/CB
2EGFP/ /
(b–d). Typical CB
1
ecep o labeling is also obse ed in memb anes o exci a o y e minals (g een a ows
and p o iles in a2, b1), inhibi o y e minals (yellow a ows and p o iles in a2, b2) and mi ochond ia (blue a ows and p o iles in a2), in bo h
CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
. Scale ba s: 2 μm
872 TERRADILLOS ET AL.
10981136, 2023, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24312 by Readcube (Lab i a Inc.), Wiley Online Lib a y on [06/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
(0.48 ± 0.13 pa icles; ***p: .0009; Figu e 6d, le ). Finally, he e we e
no di e ences in he numbe o CB
1
pa icles pe posi i e mic oglial
p ocess be ween bo h mu an s (5xFAD/CB
2EGFP/ /
: 1.333 ± 0.08347
pa icles/p ocess; CB
2EGFP/ /
: 1.450 ± 0.2112 pa icles/p ocess;
p: .7736; Figu e 6e, le ).
3.4 |As oglial CB
1
ecep o localiza ion in
CB
2EGFP/ /
and 5XFAD/CB
2EGFP/ /
subiculum
The CB
1
ecep o was localized o memb anes o GLAST-posi i e
as ocy ic p ocesses in bo h mu an s (Figu e 4a–d), as p e iously
epo ed (Bonilla-Del Rίo e al., 2019; Bonilla-Del Río e al., 2021;
Bosie e al., 2013; Gu ié ez-Rod íguez e al., 2018; Han e al., 2012).
No signi ican di e ences we e de ec ed in he numbe o CB
1
-
posi i e as ocy ic p olonga ions be ween bo h mice (CB
2EGFP/ /
:
8.661 ± 0.8977 CB
1+
p ocesses/100 μm
2
;5xFAD/CB
2EGFP/ /
:7.967±
1.224 CB
1+
p ocesses/100 μm
2
;p: .3094; Figu e 6a, igh ). Likewise,
he pe cen age o CB
1
-posi i e as ocy ic b anches was s a is ically
simila be ween 5xFAD/CB
2EGFP/ /
(21.24 ± 2.37%) and CB
2EGFP/ /
(17.75 ± 1.21%; p: .2303; Figu e 6b, igh ). The e was nei he di e -
ences in CB
1
ecep o densi y in he as ocy ic posi i e p ocesses
(5xFAD/CB
2EGFP/ /
: 29.15 ± 2.220 pa icles/100 μmo memb ane;
CB
2EGFP/ /
: 37.57 ± 2.970 pa icles/100 μm o memb ane; p: .2209;
Figu e 6c, igh ), no in he numbe o as ocy ic CB
1
pa icles pe
100 μm
2
(5xFAD/CB
2EGFP/ /
: 12.78 ± 2.174 pa icles; CB
2EGFP/ /
:
11.63 ± 1.265; p: .6716; Figu e 6d, igh ). Howe e , he numbe o
CB
1
pa icles pe posi i e as ocy ic p ocesses was signi ican ly
highe in 5xFAD/CB
2EGFP/ /
(1.603 ± 0.05081 pa icles/p ocess)
han in CB
2EGFP/ /
(1.343 ± 0.03909 pa icles/p ocess; ***p: .0005;
Figu e 6e, igh ).
Al oge he , he numbe o mic oglial p ocesses exp essing CB
1
ecep o s inc eases and he la ge as ocy ic p o iles ha e mo e CB
1
ecep o s in he subiculum o he Alzheime 's mouse model (Figu e 6a–e).
FIGURE 5 Mo phological pa ame e s
o mic oglia and as ocy es in he
subiculum o CB
2EGFP/ /
and 5xFAD/
CB
2EGFP/ /
mice. (a) Mic oglial and
as ocy ic p ocesses a ea. (b) Glial a ea
(mic oglia and as ocy es) no malized o
100 μm
2
. (c) Mic oglial and as ocy ic
p ocesses pe ime e . (d) Glial pe ime e
(mic oglia and as ocy es) no malized o
100 μm
2
. (e) Numbe o glial p ocesses
(mic oglia and as ocy es) in 100 μm
2
.
Da a we e analyzed by non-pa ame ic o
pa ame ic es s (Mann–Whi ney U- es
o S uden 's - es ). Mann–Whi ney U-
es o S uden 's - es . p< .05*; p< .01**;
p< .001***; p< .0001****. All da a a e
ep esen ed as mean ± SEM
TERRADILLOS ET AL.873
10981136, 2023, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24312 by Readcube (Lab i a Inc.), Wiley Online Lib a y on [06/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
4|DISCUSSION
We de ec ed in he elec on mic oscope he p esence o plaques
and a mul i ude o dys ophic neu i es in he 5xFAD/CB
2EGFP/ /
mice ha con i ms he use ulness o his animal model o s udying
he pa hophysiology o AD (Ruiz de Ma ín Es eban e al., 2022).
We also obse ed an o e mic oglial and as ocy ic eac i i y wi h
an inc ease in he a ea and pe ime e o hei p ocesses, demon-
s a ing he exis ence o signi ican al e a ions in he subicula
cy oa chi ec u e. Then we s udied he exp ession o he majo can-
nabinoid CB
1
ecep o in glial cells in he subiculum o 5xFAD/
CB
2EGFP/ /
and CB
2EGFP/ /
mice. The main indings we e ha CB
1
ecep o exp ession conspicuously changes in mic oglial cells bu
ecep o densi y emains s eady in as ocy es despi e he eac i i y
o he as ocy ic p ocesses in he AD mouse. No leas , he localiza-
ion o CB
1
ecep o s in mic oglial p ocesses in he subiculum o
con ols and closely su ounding amyloid plaques and dys ophic
neu i es in he subiculum o he AD model, suppo s he p esence
o CB
1
in mic oglia. The disc ee amoun o CB
1
ecep o s in as o-
cy es has been e ealed accu a ely by immuno-elec on mic oscopy
(Bonilla-Del Río e al., 2021; Gu ié ez-Rod íguez e al., 2018;
Puen e e al., 2019), a echnique ha has also been p o en in his
s udy o be op imal o he localiza ion o CB
1
ecep o s in
mic oglia.
FIGURE 6 S a is ical assessmen o he CB
1
ecep o localiza ion in subicula as ocy es and mic oglia o CB
2EGFP/ /
and 5xFAD/CB
2EGFP/ /
mice. (a) Numbe o mic oglial (le ) and as ocy ic ( igh ) CB
1
posi i e p ocesses pe 100 μm
2
. (b) Pe cen age o CB
1
posi i e mic oglial (le ) and
as ocy ic ( igh ) p ocesses. (c) CB
1
densi y in posi i e mic oglial (le ) and as ocy ic ( igh ) elemen s pe 100 μm. (d) Mic oglial (le ) and as ocy ic
( igh ) CB
1
pa icles pe 100 μm
2
. (e) CB
1
ecep o labeling pe mic oglial (le ) and as ocy ic ( igh ) p ocess. Da a we e analyzed by non-
pa ame ic o pa ame ic es s (Mann–Whi ney U- es o S uden 's - es ). Mann–Whi ney U- es o S uden 's - es . p< .05*; p< .01**;
p< .001***; p< .0001****. All da a a e ep esen ed as mean ± SEM
874 TERRADILLOS ET AL.
10981136, 2023, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24312 by Readcube (Lab i a Inc.), Wiley Online Lib a y on [06/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License