TYPE O iginal Resea ch
PUBLISHED 18 Oc obe 2022
DOI 10.3389/ nana.2022.1004702
OPEN ACCESS
EDITED BY
Daniel A. Pe e son,
Rosalind F anklin Uni e si y
o Medicine and Science, Uni ed S a es
REVIEWED BY
Pankaj Gau ,
Geo ge own Uni e si y, Uni ed S a es
Sand a Mon agud Rome o,
Uni e si y o Valencia, Spain
*CORRESPONDENCE
Joan Sallés
[email p o ec ed]
RECEIVED 27 July 2022
ACCEPTED 22 Sep embe 2022
PUBLISHED 18 Oc obe 2022
CITATION
Echeaza a L, Ba ondo S, Ga cía del
Caño G, Bonilla-Del Río I,
Egaña-Hugue J, Puen e N,
A e xabala X, Mon aña M, López de
Jesús M, González-Bu gue a I,
Saumell-Esnaola M, Goicolea MA,
G andes P and Sallés J (2022)
Up- egula ion o CB1cannabinoid
ecep o s loca ed a glu ama e gic
e minals in he medial p e on al
co ex o he obese Zucke a .
F on . Neu oana . 16:1004702.
doi: 10.3389/ nana.2022.1004702
COPYRIGHT
© 2022 Echeaza a, Ba ondo, Ga cía
del Caño, Bonilla-Del Río,
Egaña-Hugue , Puen e, A e xabala,
Mon aña, López de Jesús,
González-Bu gue a, Saumell-Esnaola,
Goicolea, G andes and Sallés. This is
an open-access a icle dis ibu ed
unde he e ms o he C ea i e
Commons A ibu ion License (CC BY).
The use, dis ibu ion o ep oduc ion in
o he o ums is pe mi ed, p o ided
he o iginal au ho (s) and he copy igh
owne (s) a e c edi ed and ha he
o iginal publica ion in his jou nal is
ci ed, in acco dance wi h accep ed
academic p ac ice. No use, dis ibu ion
o ep oduc ion is pe mi ed which
does no comply wi h hese e ms.
Up- egula ion o CB1
cannabinoid ecep o s loca ed
a glu ama e gic e minals in he
medial p e on al co ex o he
obese Zucke a
Ley e Echeaza a1,2, Se gio Ba ondo3,4,5,
Gon zal Ga cía del Caño5,6, I zia Bonilla-Del Río7,8 ,
Jon Egaña-Hugue 7,8 , Nago e Puen e7,8 , Xabie A e xabala6,
Ma io Mon aña3, Maide López de Jesús3,5,
Imanol González-Bu gue a5,6, Miquel Saumell-Esnaola3,5,
Ma ía A ánzazu Goicolea9, Ped o G andes7,8,10 and
Joan Sallés3,4,5*
1Depa men o Physiology, Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU),
Vi o ia-Gas eiz, Spain, 2Bioa aba, Disposi i os Mó iles pa a el Con ol de En e medades C ónicas,
Vi o ia-Gas eiz, Spain, 3Depa men o Pha macology, Facul y o Pha macy, Uni e si y o he Basque
Coun y (UPV/EHU), Vi o ia-Gas eiz, Spain, 4Cen o de In es igación Biomédica en Red de Salud
Men al, Mad id, Spain, 5Bioa aba, Neu o a macología Celula y Molecula , Vi o ia-Gas eiz, Spain,
6Depa men o Neu osciences, Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU),
Vi o ia-Gas eiz, Spain, 7Depa men o Neu osciences, Facul y o Medicine and Nu sing, Uni e si y
o he Basque Coun y (UPV/EHU), Leioa, Spain, 8Achuca o Basque Cen e o Neu oscience,
Science Pa k o he Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain, 9Depa men o
Analy ical Chemis y, Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU),
Vi o ia-Gas eiz, Spain, 10Di ision o Medical Sciences, Uni e si y o Vic o ia, Vic o ia, BC, Canada
The p esen s udy desc ibes a de ailed neu oana omical dis ibu ion map
o he cannabinoid ype 1 (CB1) ecep o , along wi h he biochemical
cha ac e iza ion o he exp ession and unc ional coupling o hei cogna e
Gi/op o eins in he medial p e on al co ex (mPCx) o he obese Zucke a s.
The CB1 ecep o densi y was highe in he p elimbic (PL) and in alimbic
(IL) sub egions o he mPCx o obese Zucke a s ela i e o hei lean
li e ma es which was associa ed wi h a highe pe cen age o CB1 ecep o
immunoposi i e exci a o y p esynap ic e minals in PL and IL. Also, a highe
exp ession o CB1 ecep o s and WIN55,212-2-s imula ed [35S]GTPγS binding
was obse ed in he mPCx bu no in he neoco ex (NCx) and hippocampus
o obese a s. Low- equency s imula ion in laye s II/III o he mPCx induced
CB1 ecep o -dependen long- e m synap ic plas ici y in IL o a ea obese
Zucke bu no lean a s. O e all, he ele a ed 2-AG le els, up- egula ion o
CB1 ecep o s, and inc eased agonis -s imula ed [35S]GTPγS binding s ongly
F on ie s in Neu oana omy 01 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
sugges ha hype ac i i y o he endocannabinoid signaling akes place a he
glu ama e gic e minals o he mPCx in he obese Zucke a . These findings
could endo se he impo ance o he CB1 ecep o s loca ed in he mPCx in
he de elopmen o obesi y in Zucke a s.
KEYWORDS
obese Zucke a , CB1 ecep o , Gi/op o eins, 2-AG le els, medial p e on al co ex,
glu ama e gic e minals, exci a o y synapses, long- e m po en ia ion
In oduc ion
The gene ically obese Zucke ( a/ a) a is one o he
a ailable animal models used o s udy he gene ic and
neu ochemical ac o s ha con ibu e o ood consump ion
and hype phagia (Guillaume-Gen il e al.,1990;di Ma zo
e al.,2001;Simle e al.,2006;Rasmussen e al.,2010).
The beha io al pheno ype o he obese Zucke a , which
de elops inna ely due o a gene ically de e mined lep in
ecep o de ec ha esul s in lep in esis ance and obesi y,
is associa ed wi h inc eased le els o appe i e-s imula ing
signals including endocannabinoids (di Ma zo e al.,2001).
Mo eo e , du ing adul hood o he obese Zucke a , an
inc eased exp ession o cannabinoid ype 1 (CB1) ecep o s
occu s inna ely in se e al co ical a eas (Thanos e al.,
2008b;Za a e e al.,2008a) ha a e ema kably ela ed o
eeding beha io . In e es ingly, obese Zucke a s exhibi a
highe sensi i i y o cannabinoid d ugs han lean a s (Vicke s
e al.,2003;Rasmussen and Huskinson,2008;Se ano e al.,
2008;Smi h and Rasmussen,2010;Boomhowe e al.,2013;
Buckley and Rasmussen,2014). Fu he mo e, a bo h low and
high esponse equi emen se ings o ood consump ion,
imonaban , and o he CB1 ecep o an agonis s educe ood
in ake (Vicke s e al.,2003) and a enua e he ein o cing
p ope ies o pala able ood (Rasmussen and Huskinson,2008;
Rasmussen e al.,2010), supp essing ood- ein o ced beha io
(Rasmussen e al.,2012;Buckley and Rasmussen,2014) and
inc easing sensi i i y o esponse equi emen s o suc ose
Abb e ia ions: 2-AG, 2-a achidonoylglyce ol; [35S]GTPγS, guanosine
5-o-(3-[35S] hio- iphospha e; AC, an e io cingula e co ex; CaMKII,
calcium/calmodulin-dependen p o ein kinase II; CB1, ecep o
(cannabinoid ype 1 ecep o ); DAG, diacylglyce ol; DAGL, diacylglyce ol
lipase; DG, Den a e gy us; eCB-eLTD, endocannabinoid-media ed
exci a o y long- e m dep ession; EGTA, e hylene glycol bis (2-
aminoe hyl e he ) e aace ic acid; EPSP, field exci a o y pos synap ic
po en ial; IL, in alimbic co ex; LC/MS-MS, liquid ch oma og aphy
and mass spec ome y; LFS, low- equency s imula ion; LH, la e al
hypo halamic a ea; LTD, long- e m dep ession; LTP, long- e m
po en ia ion; MAGL, monoacylglyce ol lipase; Mop, p ima y mo o
co ex; mPCx, medial p e on al co ex; NAcc, nucleus accumbens;
NCx, neoco ex; PI, phosphoinosi ides; PLC-β1, phospholipase C-β1;
PMSF, phenylme hylsul onyl fluo ide; PTX, pic o oxin; PL, p elimbic
co ex; VTA, en al egmen al a ea.
(Rasmussen and Huskinson,2008;Rasmussen e al.,2012).
Finally, consis en wi h s udies showing ha CB1-specific d ugs
affec impulsi i y- ela ed p ocesses in a s (Pa ij e al.,2007)
and humans (McDonald e al.,2003), he obese Zucke a s
beha e mo e impulsi ely han hei lean li e ma es in ood
consump ion (Boomhowe e al.,2013). In iew o p e ious
epo s ha he p ope unc ioning o he oden medial
p e on al co ex (mPCx), a b ain egion associa ed wi h
decision making (Royall e al.,2002), depends on he balance
be ween exci a o y and inhibi o y synap ic ansmission (E/I
balance) (Yizha e al.,2011), i is concei able ha changes
in he ne impac o CB1 ecep o -media ed effec s on E/I
balance may con ibu e o he beha io al defici s obse ed in
he obese Zucke a . In his sense, i is s ill unknown o
wha ex en he up- egula ion o b ain co ical CB1 ecep o s
desc ibed in p e ious epo s (Thanos e al.,2008b;Za a e
e al.,2008a) is associa ed wi h neu ons o he exci a o y
o inhibi o y ype in he mPCx. Fu he mo e, al hough ou
p e ious s udies in his model showed inc eased CB1 ecep o
exp ession in he on al co ex and ela ed limbic a eas o he
obese Zucke a (Za a e e al.,2008a,b), CB1immuno eac i i y
was dis ibu ed no only in i s canonical loca ion a axon
e minals bu also in con o e sial soma odend i ic loca ions
(Za a e e al.,2008a). We discuss he e his disc epancy on
he basis o a ecen s udy om ou labo a o y ha un a els
echnical issues ele an o he specific immunohis ochemical
de ec ion o he CB1 ecep o in he oden b ain (Echeaza a
e al.,2021) and p o ides a amewo k o in e p e pas
and u u e esul s de i ed om he use o diffe en an i-CB1
ecep o an ibodies.
Wi h his e idence in mind, we easoned ha he obese
Zucke a , as a beha io al pheno ype o impulsi e app oach
o ood, p o ides an excellen oppo uni y o u he assess
changes in he key elemen s o he endocannabinoid sys em
in he mPCx, a key b ain egion o he cogni i e con ol
o subs ance use beha io (Volkow e al.,2003). He e, we
compa ed he exp ession le els o CB1 ecep o s and o he
membe s o he Gαi/o amily o G p o ein subuni s, as well as
analyzed CB1 ecep o -Gαi/o unc ional coupling in he mPCx
o obese and lean Zucke a s and ex ended he analyses o
neoco ex (NCx) and hippocampus o e i y whe he possible
F on ie s in Neu oana omy 02 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
changes in he pa ame e s analyzed a e egion-specific o ,
on he con a y, a gene al phenomenon. Simul aneously, we
de e mined he le els o 2-a achidonoylglyce ol (2-AG), as
he mos abundan endogenous ligand in ol ed in e og ade
cannabinoid signaling a synapses (S ella e al.,1997;Gaoe al.,
2010;Tanimu a e al.,2010;Yoshino e al.,2011), o gain insigh s
in i s b ain dis ibu ion pa e n in ela ion wi h he pa e n o
CB1 ecep o exp ession, as p e iously epo ed in male Wis a
s ain a s (Bisogno e al.,1999). In ac , i has been epo ed
an inc eased accumula ion o 2-AG in he hypo halamus o
obese Zucke a s wi h espec o hei lean con ols (di Ma zo
e al.,2001). Ou esul s add expe imen al suppo o he
hypo hesis ha he obese Zucke a may ep esen a p eclinical
model o ulne abili y o obesi y, concei ably linked wi h
an al e ed endocannabinoid sys em-dependen E/I balance a
he mPCx.
Ma e ials and me hods
Animals
Fo y 12-week-old male lean and obese Zucke a s we e
used in his s udy. Six een animals (8 pe geno ype) we e
used o immunohis ochemis y and elec onic mic oscopy,
12 (6 pe geno ype) we e used o bo h Wes e n blo
analysis and unc ional binding s udies, 6 (3 pe geno ype)
we e used o elec ophysiological s udies, and he emaining
6 (3 pe geno ype) o liquid ch oma og aphy and mass
spec ome y (LC/MS-MS) analysis (Figu e 1). Animals we e
pu chased om Cha les Ri e Labo a o ies España S.A.
(Ba celona, Spain), housed in a con olled en i onmen
(22 ±2◦C: 12 h ligh -da k cycle) wi h ood and wa e
p o ided ad libi um, and allowed o acclima e o a leas
2 weeks be o e culling be ween 10:00 and 12:00 AM. Animal
handling was ca ied ou in acco dance wi h he EU Di ec i e
2010/63/EU and expe imen al p ocedu es we e app o ed by
he E hics/Animal Commi ee o he Uni e si y o he Basque
Coun y (CEBA/199/2011/GARCIA DEL CAÑO). All effo s
we e made o minimize animal suffe ing and o educe he
numbe o animals used.
D ugs and chemicals
Guanosine 5-o-(3-[35S] hio- iphospha e) ([35S]GTPγS,
1000–1400 Ci/mmol) was pu chased om Pe kin-
Elme (Ba celona, Spain). P o ease inhibi o cock ail
(phenylme hylsul onyl fluo ide -PMSF- and iodoace amide),
GDP, GTP, GTPγS, and WIN 55,212-2 we e pu chased om
Sigma Chemical (S . Louis, MO, USA). Pic o oxin (PTX) and
AM251 we e pu chased om Toc is Bioscience (B is ol, UK).
Fo he endocannabinoid de e mina ions, 2-AG and 1-AG, and
hei deu e a ed analog, 2-AG-d5and 1-AG-d5, we e ob ained
om Cayman Chemicals. Wa e (H2O), ace oni ile, o mic
acid, e hylace a e, and hexane (all o Fluka LC-MS g ade) we e
ob ained om Sigma Ald ich.
Immunohis ochemis y o ligh
mic oscopy and p eembedding
immunogold o elec on mic oscopy
Ra s we e anes he ized wi h an o e dose o cho al hyd a e
(1 g/kg, i.p.; Pan eac Química S.A., Cas ella del Vallés,
Ba celona, Spain) and pe used ansca dially a a cons an
flow o 30 ml/min (Heidolph Ins umen s GmbH & Co. KG,
Pumpd i e PD 5106, Schwabach, Ge many) o 1 min wi h
0.1 M phospha e-buffe ed saline (PBS, pH 7.4), ollowed by
5 min pe usion wi h fixa i e solu ion made up o ei he
4% pa a o maldehyde in 0.1 M phospha e buffe (PB pH
7.4), o ligh mic oscopy, o 4% pa a o maldehyde, 0.2%
pic ic acid and 0.1% glu a aldehyde in PB, o elec on
mic oscopy. The ea e , b ains we e emo ed and pos -fixed
o e nigh in he same fixa i e o 4 h. B ains in ended
o ob ain sec ions o ligh mic oscopy we e imme sed
in a c yop o ec i e solu ion (30% suc ose in 0.1 M PB,
pH 7.4) o 48 h.
Sec ions o immunohis ochemical s aining o ligh
mic oscopy we e c yosec ioned a 40 μm using a mic o ome
(Lei z-We zla 1310, We zla , Ge many) equipped wi h a
empe a u e senso (5MP BFS-Physi emp Con olle , Cli on,
NJ, USA) and p ocessed o immunohis ochemical s aining
as p e iously desc ibed (Mon aña e al.,2012;Echeaza a
e al.,2021) using an affini y-pu ified goa polyclonal
an ibody aised agains he C- e minal 31 amino acids
(443–473) o he mouse CB1 ecep o (CB1-Go-A 450;
F on ie Science Co. L d, Hokkaido, Japan) (Table 1). Fo
elec on mic oscopy, 50 μm- hick ib a ome sec ions o he
mPCx we e collec ed in 0.1 M phospha e buffe (pH 7.4) a
oom empe a u e and p ocessed o p e-embedding sil e -
in ensified immunogold using he CB1-Go-A 450 an ibody
(Table 1) as p e iously desc ibed (Bonilla-Del Río e al.,2021).
De ails o he immunohis ochemical and p e-embedding
sil e -in ensified immunogold me hods a e p o ided in
Supplemen a y Me hods.
Ligh mic oscopy imaging and
semiquan i a i e analysis o he
CB1-immunos aining
B ain sec ions p ocessed o immunohis ochemis y we e
examined wi h an Olympus BX50F ligh mic oscope (Olympus,
Tokyo, Japan) connec ed o a high- esolu ion digi al came a
(Olympus and So Imaging Sys ems, Tokyo, Japan). All
F on ie s in Neu oana omy 03 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
FIGURE 1
Diag am summa izing he wo kflow, sample size and p ocessing, and expe imen al app oaches o answe he ou main ques ions ( amed
boxes a op) posed in his s udy. 2-AG, 2-a achidonoylglyce ol; eCB, endocannabinoid; EPSP, field exci a o y pos synap ic po en ial esponse;
IGS-EM, immunogold-sil e s aining o elec on mic oscopy; IHC, immunohis ochemis y; LC/MS-MS, liquid ch oma og aphy and mass
spec ome y; mPCx, medial p e on al co ex; WB, Wes e n blo .
TABLE 1 P ima y an ibodies used.
Ta ge Dilu ion (ng/mL) Hos and
clonali y
Pu i y and
iso ype
Immunizing an igen Sou ce, ca alog
IHC IGS-EM WB
CB1
ecep o
1000 2000 200 Goa
polyclonal
Immunogen
affini y-pu ified IgG
31 amino acids a
he C- e minus o mouse CB1
F on ie Ins i u e Co., L d.,
CB1-Go-A 450
Gαo
subuni
— — 40 Rabbi
polyclonal
P o ein A/G
affini y-pu ified IgG
Pep ide wi hin a highly
di e gen domain o a Gαo
San a C uz Bio ech. Inc.,
sc-387
Gαi1
subuni
— — 40 Rabbi
polyclonal
P o ein A/G
affini y-pu ified IgG
Pep ide wi hin a highly
di e gen domain o a Gαi1
San a C uz Bio ech. Inc.,
sc-391
Gαi2
subuni
— — 200 Rabbi
polyclonal
P o ein A/G
affini y-pu ified IgG
Pep ide wi hin a highly
di e gen domain o a Gαi2
San a C uz Bio ech. Inc.,
sc-7276
Gαi3
subuni
— — 5 Rabbi
polyclonal
P o ein A/G
affini y-pu ified IgG
Pep ide mapping
a he C- e minus
o a Gαi3
San a C uz Bio ech. Inc.,
sc-262
PLC-β1— — 32 Mouse
monoclonal
P o ein A/G
affini y-pu ified IgG1
Amino acids 4–159 o a
PLC-β1
BD T ansduc ion Labs.,
610924
IHC, immunohis ochemis y; IGS-EM, immunogold-sil e s aining o elec on mic oscopy; WB, Wes e n blo . An ibody manu ac u e s: San a C uz Bio echnology, San a C uz, CA,
USA; BD T ansduc ion Labo a o ies, San Diego, CA, USA; F on ie Ins i u e Co., L d., Hokkaido, Japan
mic oscope images we e digi ized wi h a g ayscale esolu ion
o 16 bi s/pixel using CellA so wa e o image acquisi ion
(Olympus and So Imaging Sys ems, Tokyo, Japan) and
sa ed in TIFF o ma . Mic oscope images used o illus a ion
we e expo ed as 8-bi g ayscale TIFF files and compiled
and labeled using Adobe Pho oshop CS3 so wa e (San Jose,
CA, USA).
Images used o semiquan i a i e analysis o he op ical
densi y (OD) o CB1-immunos aining in lean and obese
Zucke a s we e acqui ed unde iden ical condi ions (exposu e
ime and in ensi y o illumina ion) wi h a 10×objec i e and
a g ayscale esolu ion o 16 bi s/pixel. Measu emen s we e
pe o med in se e al a eas o he co ex and hippocampal
o ma ion o lean and obese Zucke a s on mic oscope
images acqui ed. Wi hin he co ex, p elimbic (PL) and
in alimbic (IL) sub egions o mPCx, p ima y mo o co ex
(MOp), and an e io cingula e co ex (AC) we e chosen
o analysis. Wi hin he hippocampal o ma ion, analyses
we e pe o med in den a e gy us (DG) and fields CA1,
CA2, and CA3 o he Ammon’s ho n. Quan ifica ion was
ca ied ou using he measu e unc ion o ImageJ image
analysis so wa e (ImageJ, NIH, Be hesda, MD, USA) (de ails
a e gi en in Supplemen a y Me hods and Supplemen a y
Figu es 1A,B).
F on ie s in Neu oana omy 04 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
Elec on mic oscopy imaging and
quan ifica ion o CB1-immunogold
pa icles
Ul a hin sec ions o 50 nm we e collec ed on mesh
nickel g ids, s ained wi h 2.5% lead ci a e o 20 min, and
examined in a Philips EM208S elec on mic oscope. Tissue
p epa a ions we e pho og aphed using a digi al came a coupled
o he elec on mic oscope. Images used o illus a ion we e
expo ed as 8-bi g ayscale TIFF files and compiled and
labeled using Adobe Pho oshop CS3 so wa e (San Jose,
CA, USA).
Sampling was pe o med accu a ely and in he same way
o all he animals s udied. Be o e he ul a- hin sec ioning,
immunogold-labeled sec ions we e isualized unde he
ligh mic oscope in o de o selec po ions o he IL and
PL a eas displaying good and ep oducible CB1 ecep o
immunolabelling. Elec on mic og aphs we e aken a
22,000 ×using a Digi al Mo ada Came a om Olympus.
Analyses we e pe o med on he fi s fi e 50 nm-ul a hin
sec ions ob ained wi hin he fi s 1.5 μm om he su ace
o he issue block. To de e mine he p opo ion o CB1
ecep o posi i e e minals, posi i e labeling was conside ed
when a leas one CB1 ecep o immunopa icle was wi hin
∼30 nm om he memb ane o he specific compa men
unde s udy. The exci a o y and inhibi o y synapses we e
iden ified on he basis o hei ul as uc u al ea u es.
Exci a o y synapses a e asymme ical wi h pos synap ic
densi ies and p esynap ic axon e minals con aining abundan ,
clea , and sphe ical synap ic esicles. Inhibi o y synapses
a e symme ical wi h slende pos synap ic memb anes and
axon e minals con aining pleomo phic synap ic esicles. To
de e mine he pe cen ages o exci a o y and inhibi o y e minals
con aining CB1 ecep o pa icles and he CB1 ecep o densi y
(pa icles/μm memb ane) in bo h ypes o e minals elec on
mic oscope images we e analyzed on ImageJ. S a is ics we e
done on G aphPad P ism 8.
Slice p epa a ion and ex acellula field
eco dings
Lean and obese Zucke a s we e anes he ized wi h
isoflu ane (2–4%) and b ains we e apidly emo ed and placed
in a suc ose-based solu ion a 4◦C ha con ained (in mM):
87 NaCl, 75 suc ose, 25 glucose, 7 MgCl2, 2.5 KCl, 0.5 CaCl2,
and 1.25 NaH2PO4. Co onal ib a ome sec ions (300 μm
hick, Leica Mic osis emas S.L.U.) we e collec ed, eco e ed a
32–35◦C be o e being placed in he eco ding chambe , and
supe used (2 ml/min) wi h a ificial ce eb ospinal fluid (aCSF)
con aining (in mM): 130 NaCl, 11 glucose, 1.2 MgCl2, 2.5 KCl,
2.4 CaCl2, 1.2 NaH2PO4, and 23 NaHCO3, equilib a ed wi h
95% O2/5% CO2. All expe imen s we e ca ied ou a 32–35◦C.
Pic o oxin (PTX; 100 μM, Toc is Bioscience, B is ol, UK) was
added o he aCSF o block GABAA ecep o s.
Fo ex acellula field eco dings, a glass- eco ding pipe e
was filled wi h aCSF. The s imula ion and eco ding elec odes
(bo osilica e glass capilla ies, Ha a d appa a us UK capilla ies
30–0062 GC100T-10) we e placed in IL laye s II/III o
he mPCx. To e oke field exci a o y pos synap ic po en ial
esponses ( EPSPs), epe i i e con ol s imuli we e deli e ed
a 0.1 Hz (S imulus Isola e ISU 165, Cibe ec, Spain;
con olled by aMas e -8, A.M.P.I.). An Axopa ch-200B (Axon
Ins umen s/Molecula De ices, Union Ci y, CA, USA) was
used o eco d he da a fil e ed a 1–2 kHz, digi ized a 5 kHz on
a DigiDa a 1440A in e ace, collec ed on a PC using Clampex
10.0 and analyzed using Clampfi 10.0 (all ob ained om
Axon Ins umen s/Molecula De ices, Union Ci y, CA, USA).
A he s a o each expe imen , an inpu -ou pu cu e was
cons uc ed. S imula ion in ensi y was selec ed o baseline
measu emen s ha yielded be ween 40 and 60% o he maximal
ampli ude esponse. To induce eCB-eLTD o glu ama e gic
inpu s, a low- equency s imula ion (LFS, 10 min a 10 Hz)
p o ocol was applied ollowing he eco ding o a s eady baseline
as desc ibed p e iously (Puen e e al.,2011;Peñasco e al.,
2020). The magni ude o he EPSP a ea o eCB-eLTD was
calcula ed as he pe cen age change be ween he baseline a ea
(a e aged exci a o y esponses o 10 min be o e LFS) and
he las 10 min o s able esponses, eco ded 30 min a e
he end o he LFS. A leas h ee a s we e used o each
expe imen al condi ion.
Tissue sampling o wes e n blo ,
[35S]GTPγS binding, and 2-AG
measu emen s
Ra s we e anes he ized wi h an o e dose o cho al hyd a e
(1 g/kg, i.p.; Pan eac Química S.A.) and sac ificed animals by
decapi a ion. Immedia ely a e culling, b ains we e emo ed
and co ical samples we e dissec ed on an ice-chilled glass
pla e unde a s e eomic oscope (Nikon, SMZ800, Nikon
Ins umen s Eu ope, B.V Ams el een, Ne he lands), using
he midline, he co pus callosum and he hinal fissu e as
ana omical landma ks (Supplemen a y Figu e 2). Immedia ely
a e dissec ion, co ical and hippocampal samples we e ozen
in isopen ane p e-cooled o –80◦C. Samples o mPCx and
NCx om 12 animals (six lean and six obese Zucke a s)
we e used o Wes e n blo analysis and [35S]GTPγS binding
assays. Wes e n blo was also pe o med in hippocampal
samples om six lean and six obese Zucke a s. Samples
o p e on al, an e io and pos e io NCx om six animals
( h ee lean and h ee obese Zucke a s) we e used o 2-
AG measu emen s by LC/MS-MS analysis. Samples o mPCx,
NCx, and hippocampus in ended o Wes e n blo analysis and
[35S]GTPγS binding assays we e pooled om g oups o six
F on ie s in Neu oana omy 05 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
animals o he same geno ype, hus gi ing wo pools pe co ical
egion and geno ype. Samples in ended o LC/MS-MS we e
ea ed indi idually.
Tissue ac iona ion o wes e n blo
and [35S]GTPγS binding
F ozen issue samples we e hawed in ice-cold
homogeniza ion buffe : 20 mM T is-HCl, 0.32 M suc ose,
1 mM e hylene glycol bis (2-aminoe hyl e he ) e aace ic acid
(EGTA), and p o ease inhibi o s (1 mM phenylme hylsul onyl
fluo ide -PMSF- and 0.5 mM iodoace amide), pH 7.5. Tissue was
homogenized in 20 olumes o he same buffe and cen i uged
a 1,100 ×g o 10 min a 4◦C. The pelle con aining nuclei and
cell deb is was disca ded and he supe na an was cen i uged
a 14,000 ×g o 30 min a 4◦C. The esul ing pelle was
e-suspended in he same olume, dis ibu ed in 1 ml aliquo
in o 1.5 ml Eppendo ubes, and cen i uged a 40,000 ×g
o 10 min. The supe na an was disca ded and he aliquo s o
he final washed pelle s o c ude memb anes (P2) we e s o ed
a –80◦C un il use. The p o ein con en in P2 pelle s and he
p o ein concen a ion in Cy ac ion we e de e mined by he
B ad o d me hod wi h he Bio-Rad dye eagen (He cules, CA,
USA) using bo ine γ-globulin as s anda d.
Wes e n blo ing and linea eg ession
analysis o p o ein exp ession
Wes e n blo analysis was pe o med as p e iously desc ibed
(Mon aña e al.,2012;Saumell-Esnaola e al.,2021) wi h mino
modifica ions. CB1cannabinoid ecep o , Gi/oα-subuni s, and
phospholipase C-β1(PLC-β1) we e de ec ed by immunoblo ing
wi h he ollowing an ibodies: goa polyclonal an i-CB1 ecep o
(CB1-Go-A 450; F on ie Science Co. L d, Hokkaido, Japan),
abbi polyclonal an i-Gαo(sc-387; San a C uz Bio echnology,
San a C uz, CA, USA), abbi an i-Gαi1(sc-391; San a C uz
Bio echnology), abbi polyclonal an i-Gαi2(sc-7276; San a
C uz Bio echnology), abbi polyclonal an i-Gαi3(sc-262;
San a C uz Bio echnology) and mouse monoclonal an i-PLC-
β1(BD T ansduc ion Labo a o ies, San Diego, CA, USA)
(Table 1). De ails o he Wes e n blo p ocedu e a e gi en in
Supplemen a y Me hods.
Immuno eac i e signals p oduced by inc easing amoun s
o o al P2 p o ein we e analyzed by linea eg ession. Fi s ,
he linea ange o de ec ion o he CB1 ecep o , Gαo,
and Gαi1−3subuni s, and PLC-β1p o eins was gene a ed
by immunoblo ing inc easing amoun s ( o al p o ein) o P2
ac ion, ollowed by densi ome ic analysis using he gel
analysis ool o ImageJ so wa e. Fo each Wes e n blo assay,
wo samples om each geno ype (each sample co esponding
o a pool om h ee a s) we e hawed and dena u ed.
Inc easing amoun s o o al p o ein om sample pools o
lean and obese Zucke a s we e esol ed and p ocessed in
pa allel o immunoblo . Thus, densi ome ic analysis o specific
immuno eac i e bands p o ided one aw in eg a ed OD densi y
alue pe p o ein load poin and sample pool.
WIN55,212-2-s imula ed [35S]GTPγS
specific binding o b ain memb anes
The [35S]GTPγS binding assays we e pe o med ollowing
he p ocedu e desc ibed elsewhe e o mice and human b ain
memb anes (Saumell-Esnaola e al.,2021). B iefly, b ain co ical
and hippocampal memb anes we e hawed, and incuba ed
a 30◦C o 2 h in [35S]GTPγS-incuba ion buffe (0.5 nM
[35S]GTPγS, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl,
0,2 mM DTT, 50 μM GDP, and 50 mM T is-HCl, pH
7.4). The cannabinoid CB1 ecep o agonis WIN 55,212-2
(10−9–10−5M, eigh concen a ions) was added o de e mine
ecep o -s imula ed [35S]GTPγS binding. Non-specific binding
was defined in he p esence o 10 μM unlabeled GTPγS. Basal
binding was assumed o be he specific [35S]GTPγS binding in
he absence o agonis . The eac ions we e e mina ed by apid
acuum and fil a ion h ough Wha man GF/C glass fibe fil e s
and he emaining bound adioac i i y was measu ed by liquid
scin illa ion spec ome y as desc ibed abo e.
Measu emen o co ical b ain
endogenous 2-AG le els by liquid
ch oma og aphy and mass
spec ome y
Samples (95–120 mg we weigh ) we e weighed in o
bo osilica e ubes con aining 2 ml ice-cold 0.1 M o mic acid and
we e homogenized wi h he aid o a 5 mm-s eel ball using he
Digi al Sonifie (Model S250 B anson, USA) o 1 cycle o 10 s a
10% ampli ude. Aliquo s (50 μl) o he homogena e we e placed
in o silanized mic ocen i uge ubes con aining ice-cold 0.1 M
o mic acid and we e spiked wi h 20 μl ace oni ile con aining
he in e nal s anda ds (deu e a ed 2-AG-d5and deu e a ed 1-
AG-d5, final concen a ion 100 nM) and wi h 10 μlo he
app op ia e concen a ion o 2-AG in i s na u al o m, o gi e
a final olume o 500 μl. E hylace a e/hexane (1,000 μl; 9:1, / )
was added o ex ac he co ical homogena e, again wi h he aid
o he Digi al Sonifie o 1 cycle o 10 s a 10% ampli ude. Then,
he ubes we e cen i uged o 10 min a 10,000 ×gand 4◦C, and
he uppe (o ganic) phase was emo ed, e apo a ed o d yness
unde a gen le s eam o ni ogen a 37◦C, and e-dissol ed in
500 μl ace oni ile.
Analyses we e pe o med as p e iously desc ibed (Schul e
e al.,2012) on an LC-MS/MS sys em based on Agilen
echnologies (Wilming on) consis ing o a 6410 T iple Quad
F on ie s in Neu oana omy 06 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
mass spec ome e equipped wi h an elec osp ay ioniza ion
sou ce ope a ing in posi i e ion mode, and a 1200-se ies bina y
pump sys em. 2-AG was sepa a ed wi h a Phenomenex Luna
2.5 μm C18(2)-HST column, 100 ×2 mm, combined wi h a
Secu i y Gua d p e-column (C18, 4 ×2 mm; Phenomenex) wi h
sol en s A (0.1% o mic acid in 20:80 ace oni ile/wa e , / ) and
B (0.1% o mic acid in ace oni ile), using he ollowing g adien :
55–90% B (0–2 min), hen held a 90% B (2–7.5 min) and e-
equilib a ed a 55% B (7.5–10 min). The column empe a u e
was 25◦C, he flow a e was 0.3 mL/min, he injec ion olume
was 10 μL and he needle was insed o 60 s using a
flushpo wi h Wa e /Ace oni ile (80:20) as he eluen . The
elec osp ay ioniza ion in e ace was ope a ed using ni ogen
as a nebulize and desol a ion gas, and using he ollowing
se ings: empe a u e 350◦C, nebulize p essu e 40 psi, and
capilla y ol age + 4,800 V. The ollowing p ecu so - o-p oduc
ion ansi ions we e used o mul iple- eac ion moni o ing
(MRM): 2-AG and 1-AG m/z 379.4→287; 2-AG-d5and 1-
AG-d5m/z 384→287. Dwell imes we e 20 ms; pause be ween
MRM ansi ions was 5 ms. Da a acquisi ion and analysis we e
pe o med using MassHun e So wa e.
The de e mina ion o he endogenous 2-AG le els by
LC-MS/MS was ca ied ou as desc ibed p e iously (Schul e
e al.,2012) using a s a egy o iso ope dilu ion combined
wi h s anda d addi ion echniques de eloped in ou labo a o y
(Ga cía Del Caño e al.,2015) in o de o ge an accu a e
quan ifica ion o he endogenous le els o 2-AG. Thus, we
spiked in o each aliquo o he b ain co ical homogena e he
same amoun o he in e nal s anda d solu ion (2-AG-d5), while
2-AG in i s na u al o m was spiked wi h inc eased amoun s
(s anda d addi ions: 50, 100, 150, 200, 300, and 400 nM) in o
he se ies o subsamples ubes excep he fi s one. In hese
condi ions, i is possible o find a linea ela ionship wi h an
excellen co ela ion coefficien be ween he concen a ion o
2-AG spiked o he homogena e and he a io o he ob ained
a eas o 2-AG and 2-AG-d5. Finally, he in e cep on he x-axis
p o ides he endogenous le el o 2-AG in he aliquo o he b ain
co ical homogena e. Since 2-AG unde goes apid isome iza ion
o 1-AG unde common expe imen al condi ions (Zoe ne e al.,
2011), all he samples we e also spiked wi h he in e nal s anda d
1-AG-d5(100 nM), which allowed us o analyze o wha ex en
he 1-AG isome con ibu es o he analy ical measu emen . In
ou hands, he le els o 1-AG usually ep esen 5% o 2-AG and
we e no included in he o al 2-AG amoun s.
S a is ical analysis
The s a is ical analysis was pe o med using G aphPad
P ism 8 so wa e (G aphPad So wa e Inc., San Diego, CA,
USA). All alues a e gi en as mean ±s anda d e o o
he mean (SEM) unless o he wise indica ed. The significance
le el was se a p<0.05 o all compa isons. Diffe ences
be ween lean and obese Zucke a s in ela i e OD o
CB1-immunos aining we e analyzed by wo-way analysis o
a iance (ANOVA), wi h geno ype (lean and obese) and co ical
a ea (MOp, mPCx, AC, PL, and IL), o geno ype (lean
and obese) and hippocampal egion (CA1, CA2, CA3, DG)
as he main ac o s, ollowed by Bon e oni pos hoc es .
Da a ob ained by immunogold-sil e s aining and elec on
mic oscopy (pe cen ages o exci a o y and inhibi o y e minals
con aining CB1 ecep o pa icles and he CB1 ecep o
densi y) we e analyzed using pa ame ic o non-pa ame ic
wo- ailed S uden ’s - es o one-way ANOVA wi h Bon e oni
pos hoc analysis. The po en ial a iabili y be ween a s o he
same g oup was s a is ically analyzed, finding no diffe ences
be ween hem, so all da a om each condi ion we e pooled.
Elec ophysiological da a we e fi s analyzed by Shapi o–Wilk
and Kolmogo o –Smi no o no mali y. In gene al, s a is ical
significance be ween condi ions (baseline e sus a e d ug o
s imula ion p o ocol o bo h) was es ed using pa ame ic
( wo- ailed S uden ’s - es ) o non-pa ame ic (Mann–Whi ney
es ). To analyze OD densi y alues o immuno eac i e bands
p oduced by inc easing amoun s o o al p o ein by Wes e n
blo , he in eg a ed OD alues ob ained om he bands a he
maximum amoun o p o ein om samples o lean Zucke
a s we e a e aged and aken as a e e ence o calcula e he
pe cen age OD o each band’s in eg a ed OD. Bes -fi lines we e
gene a ed using G aphPad P ism and he s a is ical significance
o he diffe ence be ween slopes was analyzed by an F- es .
Indi idual WIN 55,212-2 concen a ion- esponse cu es we e
fi ed by non-linea eg ession o he ou -pa ame e Hill
equa ion, using G aph Pad P ism 8. The s a is ical significance
o diffe ences be ween he means o he pa ame e es ima es was
e alua ed by S uden ’s - es . Because he affini y cons an s a e
ob ained expe imen ally o ha e a log no mal dis ibu ion, EC50
alues a e loga i hmically ans o med o s a is ical analysis
(Ch is opoulos,1998). The significance o diffe ences be ween
mean alues o 2-AG le els de e mined by LC/MS-MS was
analyzed by unpai ed wo- ailed S uden ’s - es .
Resul s
CB1 ecep o exp ession is selec i ely
up- egula ed in in alimbic and
p elimbic a eas o he medial
p e on al co ex o obese Zucke a s
The CB1 ecep o immunos aining pa e n in he neoco ex
(Figu e 2) and a ious subco ical egions (Supplemen a y
Resul s and Supplemen a y Figu e 3) o he lean Zucke
a ma ched he desc ibed p e iously in he a (Ege o á
and Elphick,2000;Bodo e al.,2005;Deshmukh e al.,2007;
Echeaza a e al.,2021). Thus, in NCx, a laye -specific
F on ie s in Neu oana omy 07 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
FIGURE 2
CB1-immunohis ochemical s aining pa e n in he neoco ex o he lean Zucke a . (A) Low magnifica ion mic og aph o he p ima y mo o
neoco ical a ea showing he laye dis ibu ion o CB1-immunos aining. (B–D) Highe magnifica ion images o a eas amed in A o illus a e he
immunos aining pa e n in laye s II (B),Va(C), and VI (D). A owheads indica e CB1-immunoposi i e in e neu ons. Scale ba s =100 μmin(A);
25 μmin(D) (applies o B–D).
immunolabelling pa e n was clea ly obse ed, being ma kedly
dense in laye s II-III, uppe pa o laye V (Va) and laye VI
han in he es (Figu e 2A). CB1-immunoposi i e s uc u es
consis ed p incipally o axon-like p ofiles con aining a icosi ies
(Figu es 2B–D), along wi h cell soma a, which we e spa sely
dis ibu ed in laye s II-III and, o a lesse ex en , in laye VI
(Figu es 2B,D). Simila o he NCx, he dis ibu ion o CB1-
immunos aining wi hin he medial p e on al co ex showed a
ma ked laye ed pa e n in he lean Zucke a , al hough i was
s onge compa ed o neoco ical a eas. Immunos aining was
dense in he uppe pa o laye s II–III and V, and in laye
VI han in he es and inc eased g adually owa d IL suba ea
(Figu es 3A,C,E,G). CB1-immunos aining in mPCx o obese
Zucke a s was quali a i ely he same as ha obse ed in lean
a s, bu immuno eac i i y was conside ably mo e in ense in all
suba eas and laye s (Figu es 3B,D,F,H). Densi ome ic analysis
o immunos aining in MOp and mPCx sub egions ollowed
by wo-way ANOVA e ealed a significan diffe ence be ween
lean and obese geno ypes [F(1,40)=34.68, p<0.0001], wi h
no effec o co ical a ea ac o on he obse ed diffe ence
[F(4,40)=0.87, P=0.4890]. Pos hoc Bon e oni’s es e ealed
a significan ly highe CB1-immuno eac i i y in mPCx, PL, and
F on ie s in Neu oana omy 08 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
IL o obese Zucke a s (p<0.05, 0.01, and 0.005, espec i ely,
Figu e 4A). A simila analysis was pe o med in hippocampi
o lean and obese Zucke a s. The dis ibu ion o CB1-
immunos aining was i ually iden ical in all egions o he
hippocampal o ma ion o bo h geno ypes wi h no appa en
diffe ences in s aining in ensi y (Figu e 5), which was confi med
by densi ome ic analysis o CB1-immuno eac i i y in issue
sec ions (Figu e 4B).
Nex , we pe o med immunoblo analysis o CB1 ecep o
exp ession in samples o mPCx, NCx, and hippocampus
om lean and obese Zucke a s. Linea eg ession analysis
gene a ed by densi ome ic analysis o CB1-immuno eac i i y
in immunoblo s o inc easing amoun s o c ude memb anes
om lean and obese Zucke a s e ealed ha exp ession o he
CB1 ecep o was inc eased by 57% in mPCx o obese a s in
compa ison wi h lean a s [slope diffe ences, F(1,36)=10.210,
p=0.0029] (Figu es 6A,C), whe eas no changes we e obse ed
in NCx [slope diffe ences, F(1,34)=0.071, p=0.7904]
(Figu es 6B,D) and hippocampus (no shown).
Inc eased CB1 ecep o -labeled
exci a o y e minals in he medial
p e on al co ex o obese Zucke a s
CB1-immunoposi i e pa icles we e localized in p esynap ic
e minals making symme ic and asymme ic synapses in
he IL and PL sub egions o he mPCx o lean and obese
Zucke a s. In IL, he pe cen age o exci a o y (asymme ic)
e minals con aining CB1 ecep o pa icles (Figu es 7A,B)
was significan ly inc eased in obese (43.73 ±1.57%) ela i e
o lean a s (29.95 ±1.35%) (Mann–Whi ney U=31951,
nlean =308, nobese =298, p<0.0001) (Figu e 7C). In
con as , he pe cen age o inhibi o y e minals wi h CB1
ecep o labeling (Figu es 7D,E) was i ually iden ical be ween
lean (62.19 ±4.26%) and obese (61.31 ±3.59%) (Mann–
Whi ney U=9321, nlean =111, nobese =171, P=0.7785)
(Figu e 7C). As expec ed, hese alues we e conside ably highe
han he p opo ion o labeled exci a o y e minals (Figu e 7C).
Fu he mo e, he densi y o CB1pa icles was much highe a
inhibi o y (lean: 4.24 ±0.28; obese: 3.61 ±0.176 pa icles/μm)
han a exci a o y e minals (lean: 0.45 ±0.01; obese: 0.46 ±0.01
pa icles/μm). Howe e , no significan diffe ences in CB1-
ecep o densi y we e obse ed be ween lean and obese Zucke
a s in exci a o y (Mann–Whi ney U=90938, nlean =364,
nobese =523, p=0.2578) o inhibi o y e minals (Mann–
Whi ney U=7751, nlean =107, nobese =162, p=0.1427)
(Figu e 7F).
In PL, he pe cen age o exci a o y e minals con aining
CB1immunopa icles (Figu es 7G,H) was also significan ly
inc eased in obese Zucke (34.71 ±1.77%) ela i e o lean
a s (25.39 ±1.60%) (Mann–Whi ney U=9665, nlean =136,
nobese =168, P=0.0186) (Figu e 7I). The pe cen age o
inhibi o y e minals wi h CB1 ecep o pa icles was simila in
bo h geno ypes (lean: 68.75 ±3.24%; obese: 74.78 ±3.20%)
(Mann–Whi ney U=5502, nlean =112, nobese =101,
P=0.6776), and again conside ably highe han he p opo ion
o CB1-posi i e exci a o y e minals in lean and obese Zucke
a s (Figu es 7I–K). Finally, he densi y o CB1 ecep o labeling
was much highe a inhibi o y (lean: 4.24 ±0.28; obese:
4.02 ±0.19 pa icles/μm) han a exci a o y e minals (lean:
0.45 ±0.011; obese: 0.52 ±0.013 pa icles/μm), (Figu e 7L)
howe e , i was only significan ly inc eased in exci a o y
e minals o he obese Zucke a s (Mann–Whi ney U=8973,
nlean =140, nobese =186, p<0.0001) (Figu e 7L).
Long e m synap ic plas ici y a
exci a o y synapses in laye s II/III o
in alimbic sub egion o lean and
obese medial p e on al co ex
To in es iga e whe he CB1 ecep o changes in exci a o y
e minals impac synap ic plas ici y, a low- equency
s imula ion (LFS, 10 Hz, 10 min) known o elici long
e m dep ession o he exci a o y synap ic ansmission (eLTD)
(La ou cade e al.,2007;Puen e e al.,2011) was applied o
mPCx laye s II/III o lean and obese Zucke a s. In lean
a s, a significan dec ease o mean EPSP ampli ude was
obse ed (74.02 ±6.88% EPSP ela i e o baseline; =3.091,
p<0.0149, d =8) (Figu es 8A,D). Unexpec edly, he same
p o ocol igge ed exci a o y long- e m po en ia ion (eLTP)
in obese Zucke a s, as shown by a significan inc ease in he
mean EPSP (138.8 ±18.27% EPSP ela i e o baseline; Lean
e sus Obese: =2.858, P=0.0170, d =10) (Figu es 8A,D).
No ewo hy, he CB1 ecep o in e se agonis AM251 (4 μM)
did no affec LFS-induced eLTD in lean a s (67.37 ±5.07%
EPSP ela i e o baseline; Lean e sus Lean + AM251:
=0.6729, p=0.5261, d =6) (Figu es 8B,D), whe eas
supp essed eLTP in obese a s (76.14 ±13.44% EPSP ela i e o
baseline; Obese e sus Obese + AM251: =2.761, p=0.0172,
d =12) (Figu es 8C,D). Taken oge he , hese esul s show
changes in CB1 ecep o -dependen long- e m plas ici y a IL
exci a o y synapses o obese Zucke a s.
The unc ional esponse o CB1
ecep o s o WIN 55,212-2 is inc eased
in memb anes o he medial p e on al
co ex o obese Zucke a s
To de e mine whe he he diffe ences in CB1 ecep o
exp ession we e co ela ed wi h diffe ences in ecep o coupling
o hei cogna e Gαi/op o eins, [35S]GTPγS binding assays
we e pe o med in b ain co ical memb anes ob ained om
F on ie s in Neu oana omy 09 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
FIGURE 11
2-AG le els in b ain co ical egions. Rep esen a i e illus a ion o he me hod o iso opic dilu ion combined wi h s anda d addi ion echniques
o de e mine endogenous 2-AG le els in b ain co ex. Th ee sepa a e expe imen s, each wi h 21 subsamples (aliquo s o he b ain co ical
homogena e), we e pe o med in iplica e. P io o addi ion o e hylace a e/hexane o ex ac ion, 2-AG-d5and 1-AG-d5(bo h, 100 nM), and
2-AG s anda d a a ying amoun s (0, 50, 100, 150, 200, 300, and 400 nM) we e added o he subsamples. Resul s o he no malized signal
s eng h o 2-AG/2-AG-d5 om lean (◦) and obese Zucke p e on al co ex (•) a e p esen ed wi h indi idual bes fi lines. The lines a e
essen ially pa allel bu ha e di e en in e cep s. Each da a poin is he mean ±SD o a iplica e in one expe imen al assay. Th ee independen
assays we e pe o med o ob ain da a shown in Table 3.
diffe ences in po ency and/o in insic ac i i y o he CB1
ecep o owa d diffe en Gαi/osub ypes (Glass and No hup,
1999;P a he e al.,2000;Mukhopadhyay and Howle ,
2005). Fo ins ance, s imula ion o CB1 ecep o s by WIN
55,212-2 esul s in he ac i a ion o a dis inc pa e n o a
leas fi e diffe en Gαi/osubuni s in se e al b ain egions
and, mos impo an ly, he concen a ion o WIN 55,212-2
equi ed o hal maximally ac i a e indi idual G p o eins in
he ce ebellum a ied o e a 30- old ange o diffe en Gαi/o
subuni s (P a he e al.,2000). The e o e, we a e emp ed o
specula e ha he igh wa d shi in he concen a ion- esponse
cu e o WIN55,212-2-s imula ed [35S]GTPγS binding in
mPCx memb anes o obese Zucke a s is ela ed o he
ec ui men o a popula ion o Gαi/op o eins in which
he con ibu ion o Gαi2has been educed and has been
compensa ed by o he Gαi/op o eins wi h lowe affini y o he
WIN55,212-2-bound CB1 ecep o . A de ailed pha macological
analysis compa ing he po encies and efficacies o a ious
cannabinoid agonis s, and especially he endogenous agonis 2-
AG, in ac i a ing indi idual Gαi/op o eins should help add ess
his ques ion and is cu en ly an expe imen al objec i e in
ou labo a o y.
Ou esul s also show ha 2-AG le els in co ical a eas o
obese Zucke a s a e inc eased by 2- o 4- old as compa ed
o hei lean li e ma es. P e ious esul s also showed and
inc eased accumula ion (2- old) o 2-AG in he hypo halamus
o obese Zucke a s wi h espec o hei lean con ols (di
Ma zo e al.,2001). Mo eo e , acco ding o ou p esen and
p e ious da a (di Ma zo e al.,2001), bo h he pa e n o he
egional diffe ences in he le els o 2-AG (i.e., hypo halamus
e sus on al co ex) and he absolu e hypo halamic and co ical
2-AG le els (nmoles/g b ain issue) in lean Zucke a s a e
simila o hose epo ed in Sp ague–Dawley and Wis a a s
(Bisogno e al.,1999;di Ma zo e al.,2001;Richa dson e al.,
2007). The biosyn hesis o 2-AG in ol es only one amily o
lipids, he sn-1-acyl-2-a achidonoylglyce ols (DAGs), which a e
mos ly p oduced by he hyd olysis o phosphoinosi ides (PI)
ia PI-specific PLC ac i i y. DAGs a e used as biosyn he ic
p ecu so s o 2-AG h ough he ac ion o ei he o he wo sn-
2-selec i e DAG lipases DAGLαand DAGLβ(di Ma zo and de
Pe ocellis,2012). Wi h espec o he inac i a ion o 2-AG, he
p ominen ole o monoacylglyce ol lipase (MAGL) has been
unequi ocally es ablished (Dinh e al.,2002,2004). Al hough
he mechanism o inc eased 2-AG le els in he p e on al co ex
o he obese Zucke a s epo ed he e is s ill unclea inc eased
le els o bo h DAGL and MAGL ac i i ies we e epo ed in
he spinal co d o obese Zucke a s (Fa ooqui e al.,1987).
Addi ionally, ecen ly i has been defined a no el mechanism o
DAGLα egula ion by calcium/calmodulin-dependen p o ein
kinase II (CaMKII) (Shonesy e al.,2013). The au ho s show
ha CaMKIIαbinds o and phospho yla es DAGLα, and
inhibi s 2-AG syn hesis in i o. Mo eo e , beha io al s udies
indica e ha CaMKIIαis a nega i e egula o o 2-AG signaling
in i o (Shonesy e al.,2013). In e es ingly, down- egula ion
o phospho yla ed and o al CaMKII has been epo ed in he
hippocampal CA1 egion o obese Zucke a s (Alzoubi e al.,
2005) and in animal models o ch onic psychosocial s ess
(Ge ges e al.,2004).
The da a discussed so a suppo he hypo hesis ha
he obese Zucke a s may ep esen a p eclinical model
F on ie s in Neu oana omy 16 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
o gene ic ulne abili y o obesi y, due o hype ac i i y o
endocannabinoid signaling in he mPCx ha would shi he
E/I balance, hus inc easing he inhibi o y one in his b ain
a ea. P e ious s udies using condi ional mu an mice lacking
CB1 ecep o s ei he in co ical glu ama e gic neu ons (Glu-
CB1-KO) o GABAe gic neu ons (GABA-CB1-KO) (Mono y
e al.,2006,2007) e ealed some clues o explain how CB1
ecep o s loca ed in exci a o y and inhibi o y e minals o
mPCx could con ibu e o he beha io al pheno ype o he
obese Zucke a . When he condi ional mu an mice Glu-
CB1-KO and GABA-CB1-KO we e subjec ed o expe imen al
p o ocols ha allowed o assessing ood consump ion, he
opposi e pheno ypes appea ed clea ly (La enê e e al.,2009;
Bellocchio e al.,2010). Glu-CB1-KO mice we e hypophagic, and
he pheno ype was pha macologically ela ed o he glu ama e
ac ions on NMDA ecep o s. Con e sely, GABA-CB1-KO mice
we e mo e hype phagic han hei wild- ype li e ma es, and
he pheno ype was a consequence o an inc eased GABA-
A ecep o ac i a ion (Bellocchio e al.,2010). Mo eo e ,
when he expe imen al p o ocol was designed o assess he
beha io al inhibi ion componen o impulsi i y (Pa ij e al.,
2007), he esul s sugges ed ha he dele ion o CB1 ecep o s
in glu ama e gic neu ons leads o an inc eased beha io al
inhibi ion in he app oach o no el pala able ood, whe eas
GABA-CB1-KO mice did no show any sa e y beha io owa d
no el ood, and immedia ely consumed he maximal amoun
(La enê e e al.,2009). As discussed by he au ho s (La enê e
e al.,2009;Bellocchio e al.,2010), a educ ion o glu ama e gic
ansmission h ough ac i a ion o he endocannabinoid sys em
would dec ease beha io al inhibi ion, and he eby inc ease
impulsi i y in he app oach o ood. In suppo o hese
findings, mos ecen da a demons a ed ha he lack o
CB1 ecep o s in do sal elencephalic glu ama e gic neu ons
ab oga ed he o e consump ion o pala able ood and he
de elopmen o obesi y (Ruiz de Azua e al.,2021)by
p omo ing a esilien pheno ype o ood addic ion (Domingo-
Rod iguez e al.,2020). Consis en ly, in s anda d oden
animals, he sys emic blockade o glu ama e gic ansmission
leads o an inc ease in impulsi e beha io (Mi jana e al.,2004;
Flo esco e al.,2008).
One in e es ing ques ion a ising om he esul s p esen ed
he e is ela ed o he po en ial impac ha he hype ac i i y o
CB1 ecep o signaling a he glu ama e gic e minals o he PL
and IL sub egions o mPCx could ha e on he fi ing ac i i y
o subco ical monoamine gic neu ons (i.e., en al egmen al
a ea-VTA). I is in his ame o easoning, i mus be kep in
mind ha bo h he cell body o he mesolimbic dopamine gic
neu ons and hei e minal p ojec ion a eas (i.e., nucleus
accumbens, NAcc) a e inne a ed by glu ama e gic affe en s
om he p e on al co ex (Sesack e al.,1989;Takagishi and
Chiba,1991;Be endse e al.,1992). Al hough he mechanisms
and pa hways by which mPCx modula es VTA dopamine
neu ons p ojec ing o he NAcc need o be cla ified, p e ious
esul s ha e demons a ed ha di ec glu ama e injec ed in o he
mPCx selec i ely inc eased bu s fi ing o single dopamine cells
in he VTA a ea and enhanced he elease o dopamine om
ne e e minals in he NAcc (Mu ase e al.,1993). Mo eo e ,
he specific pha macological blockade o NMDA ecep o s in he
mPCx is able o p oduce he neu ochemical and mo o changes
associa ed wi h he dys unc ion o he co icos ia al ci cui ,
including an inc ease in dopamine and ace ylcholine elease
in he NAcc and also an inc ease in mo o ac i i y (del A co
e al.,2008). Impo an ly, he chemogene ic inhibi ion o he
glu ama e gic neu onal ac i i y o a specific p elimbic-nucleus
accumbens pa hway, which is indeed also unde he con ol
o CB1 ecep o s, induces compulsi e ood seeking (Domingo-
Rod iguez e al.,2020). Al hough we ocus on he ole o
he p e on al co ex on inhibi o y con ol and impulsi i y
(Volkow e al.,2008a,b;Boeka and Lokken,2011), we a e awa e
ha he unc ions o he p e on al co ex ha con ibu e o
obesi y could be hose ha a e sensi i e o modula ion by he
neu o ansmi e dopamine ia s ia al p e on al pa hways.
Clinical neu oimaging s udies in obese subjec s ha e p o ided
e idence ha he associa ion o s ia al D2dopamine ecep o s
wi h inhibi o y con ol and wi h impulsi i y is media ed in
pa by hei modula ion o p e on al egions (Eps ein e al.,
2007;Volkow e al.,2008b). Simila ly, p eclinical s udies ha e
shown ha animals wi h low s ia al D2dopamine ecep o
le els, including he obese Zucke a , a e mo e impulsi e
han hei li e ma es wi h highe D2dopamine ecep o le els
(Dalley e al.,2007;Thanos e al.,2008a;Boomhowe e al.,
2013). Finally, addi ional s udies a e needed o cla i y whe he
he obse ed up- egula ion o CB1 ecep o s a glu ama e gic
e minals o he PL and IL sub egions o he medial p e on al
co ex epo ed he e se es an adap i e pu pose, as i has
been demons a ed in a mu ine model o obesi y wi h lep in
deficiency (C is ino e al.,2013). In ha s udy, au ho s epo ed
ha o exigenic neu ons o he la e al hypo halamic a ea (LH)
o he ob/ob mice unde go a shi om p edominan con ol by
CB1-exp essing exci a o y o CB1-exp essing inhibi o y inpu s,
a phenomenon ha was pa ly e e sed by lep in adminis a ion
ia he mTOR b anch o he lep in ecep o signaling cascade
(Co a e al.,2006).
In conclusion, he esul s o he p esen s udy show ha
he Zucke lines (lean and obese) ep esen wo homogeneous
popula ions ha can be used o es he hypo hesis on
he in ol emen o hype ac i i y o he medial p e on al
endocannabinoid sys em a glu ama e gic e minals in he
indi idual ulne abili y o obesi y.
Da a a ailabili y s a emen
The o iginal con ibu ions p esen ed in his s udy a e
included in he a icle/Supplemen a y ma e ial. Fu he
inqui ies can be di ec ed o he co esponding au ho .
F on ie s in Neu oana omy 17 on ie sin.o g
Echeaza a e al. 10.3389/ nana.2022.1004702
E hics s a emen
The animal s udy was e iewed and app o ed by Commi ee
o E hics o Animal Wel a e o he Uni e si y o he Basque
Coun y (Re . CEBA/199/2011/GARCIA DEL CAÑO).
Au ho con ibu ions
LE, SB, GG, IB-D, JE-H, NP, XA, MM, ML, IG-B, MS-E, MG,
and JS pe o med o con ibu ed o he expe imen s. SB, GG,
IB-D, JE-H, NP, and MG pe o med he analyses. GG, PG, MG,
and JS designed he s udy and d a ed he fi s manusc ip . All
au ho s e iewed and app o ed he final manusc ip .
Funding
This wo k was suppo ed by g an s o he Basque
Go e nmen (g an s numbe s IT1230-19 and IT1620-22 o PG),
he Spanish Go e nmen (g an numbe s CTQ2017-85686-R o
MG and JS) and he Ins i u o de Salud Ca los III, Cen o de
In es igación Biomédica en Red de Salud Men al, CIBERSAM.
The unde s had no ole in s udy design, da a collec ion and
analysis, decision o publish, o p epa a ion o he manusc ip .
Acknowledgmen s
We wish o hank o Se icio Gene al de Análisis, SGIKER
o skill ul echnical assis ance wi h LC/MS-MS expe imen s.
JS is a membe o he Socie a Ca alana de Biologia, a
subsidia y socie y o he Ins i u d’Es udis Ca alans (Ba celona,
Ca alonia). We also hank all membe s o PG labo a o y o
hei help ul commen s, sugges ions, and discussions du ing he
pe o mance o his s udy.
Conflic o in e es
The au ho s decla e ha he esea ch was conduc ed
in he absence o any comme cial o financial ela ionship
ha could be cons ued as a po en ial conflic o
in e es .
Publishe ’s no e
All claims exp essed in his a icle a e solely hose o he
au ho s and do no necessa ily ep esen hose o hei affilia ed
o ganiza ions, o hose o he publishe , he edi o s and he
e iewe s. Any p oduc ha may be e alua ed in his a icle, o
claim ha may be made by i s manu ac u e , is no gua an eed
o endo sed by he publishe .
Supplemen a y ma e ial
The Supplemen a y Ma e ial o his a icle can be ound
online a : h ps://www. on ie sin.o g/a icles/10.3389/ nana.
2022.1004702/ ull#supplemen a y-ma e ial
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