In vivo assessment of cortical astrocyte network dysfunction during autoimmune demyelination

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Jou nal o Neu ochemis y, 2025; 169:e16305
h ps://doi.o g/10.1111/jnc.16305
Jou nal o Neu ochemis y
SPECIAL ISSUE As ocy es in Cogni ion
ORIGINAL ARTICLE OPEN ACCESS
In Vi o Assessmen o Co ical As ocy e Ne wo k
Dys unc ion Du ing Au oimmune Demyelina ion:
Co ela ion Wi h Disease Se e i y
A.Mo eno- Ga cía1,2,3 | R.Se a 4,5 | F.Julio- Kalajzic4,5 | A.Be nal- Chico1,2,3 | A.M.Ba aiba 1,2,3 | C.Ma u e1,6 |
G.Ma sicano4,5 | S.Ma o1,2,3
1Depa men o Neu osciences, Uni e si y o he Basque Coun y UPV/EHU, Leioa, Spain | 2Achuca o Basque Cen e o Neu oscience, Leioa,
Spain | 3Neu oinmunology G oup, Biobizkaia Heal h Resea ch Ins i u e, Ba akaldo, Spain | 4Cen o de In es igación Biomédica en Red Sob e
En e medades Neu odegene a i as (CIBERNED), Mad id, Spain | 5INSERM, U1215 Neu oCen e Magendie, Bo deaux, F ance | 6Uni e si y o Bo deaux,
Bo deaux, F ance
Co espondence: G. Ma sicano (gio anni.ma sicano@inse m. ) | S. Ma o (susana.ma [email protected])
Recei ed: 11 Oc obe 2024 | Re ised: 2 Janua y 2025 | Accep ed: 3 Janua y 2025
Funding: This wo k was unded by he Ins i u o de Salud Ca los III (PI21/00629, o S.M.) and co ounded by he Eu opean Union, Basque Go e nmen
(PIBA_2023_1_0046; 2023111031; IT1473- 22, o S.M.; CannaMe HD o S.M. and G.M.; IT1203- 19, o C.M.), ARSEP Founda ion (ARSEP- 1310 o S.M. and
G.M.), INSERM ( o G.M.), he Eu opean Resea ch Council (MiCaB a, ERC- 2017- AdG- 786467; CaMeLS, ANR- 23- CE16- 0022- 01; o G.M. o G.M.), Fonda ion
pou la Reche che Medicale (FRM, DRM20101220445 o G.M.), Region Aqui aine (CanB ain, AAP2022A- 2021- 16763610 and - 17219710 o G.M.); F ench
S a e/Agence Na ionale de la Reche che (HippObese, ANR- 23- ce14- 0004- 03; ERA- Ne Neu on CanShank, ANR- 21- NEU2- 0001- 04, o G.M.), La Caixa
Resea ch Heal h 2023 (PsychoCannabis, HR23- 00793, o G.M.), Minis e io de Ciencia e Inno acion(PID2019- 109724RB- 100 o C.M.), and he Pos doc o al
P og am o he Basque Go e nmen ( o A.M.B.).
Keywo ds: as ocy e| calcium| mul iple scle osis| soma osenso y co ex
ABSTRACT
Co ical damage and dys unc ion is a pa hological hallma k o mul iple scle osis (MS) ha co ela es wi h he se e i y o physical
and cogni i e disabili y. As ocy es pa icipa e in MS pa hobiology h ough a a ie y o mechanisms, and abno mal as ocy ic
calcium signaling has been poin ed as a pa hogenic mechanism o co ical dys unc ion in MS. Howe e , in i o e idence sup-
po ing de egula ion o as ocy e calcium- dependen mechanisms in co ical MS is s ill limi ed. He e, we applied ibe pho om-
e y o he longi udinal analysis o spon aneous and senso y- e oked as ocy e ne wo k ac i i y in he soma osenso y co ex o
mice in an expe imen al au oimmune encephalomyeli is (EAE). We ound ha eely mo ing EAE mice exhibi spon aneously
occu ing as ocy e calcium signals o inc eased du a ion and educed ampli ude. Concomi an ly, co ical as ocy es in EAE
mice esponded o senso y s imula ion wi h calcium e en s o dec eased ampli ude. The eme gence o abe an as ocy e calcium
signals in he soma osenso y co ex pa alleled he onse o neu ological symp oma ology, and changes in he ampli ude o bo h
spon aneous and e oked esponses we e selec i ely co ela ed o he se e i y o neu ological de ici s. These esul s highligh he
imbalance o as ocy e ne wo k ac i i y in he b ain co ex du ing au oimmune in lamma ion and u he suppo he ele ance
o as ocy e- based pa hobiology as an unde lying mechanism o co ical dys unc ion in MS.
A. Mo eno- Ga cía and R. Se a sha ed i s au ho ship.
G. Ma sicano and S. Ma o sha ed senio au ho ship.
[Co ec ion added on 14 May 2025, a e i s online publica ion: The copy igh line was changed.]
Abb e ia ions: Ca . no, Ca alog numbe ; CNS, Cen al ne ous sys em; Dpi, Days pos immuniza ion; EAE, Expe imen al au oimmune encephalomyeli is; GFAP, Glial ib illa y acidic p o ein;
GFP, G een luo escen p o ein; MAD, Median absolu e de ia ion; MOG, Myelin oligodend ocy e glycop o ein; MS, Mul iple scle osis; PBS, Phospha e- bu e ed saline; RRID, Resea ch Resou ce
Iden i ie ; SEM, S anda d e o o he mean.
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.
© 2025 The Au ho (s). Jou nal o Neu ochemis y published by John Wiley & Sons L d on behal o In e na ional Socie y o Neu ochemis y.
2 o 10 Jou nal o Neu ochemis y, 2025
1 | In oduc ion
Mul iple scle osis (MS) is a widely p e alen neu ological dis-
o de o he cen al ne ous sys em (CNS) ha in ol es neu-
oin lamma o y and neu odegene a i e mechanisms a ec ing
bo h he b ain and he spinal co d. The main neu opa hological
hallma ks o MS include demyelina ion, in lamma ion, as o-
cy ic gliosis, and neu odegene a ion, wi h i e e sible neu oax-
onal damage unde lying disease symp oma ology and clinical
p og ession (Mahad, T app, and Lassmann2015). Pa ien s wi h
MS mani es a b oad ange o symp oms ha include abno mal-
i ies in senso y pe o mance, including numbness, bu ning and
inc eased pain sensi i i y, a igue, spas ici y, u ina y incon i-
nence, and isual de ec s, among o he s, whose neu oana omical
and cellula basis emains poo ly unde s ood. A ailable neu o-
pa hological and neu oimaging e idence poin s o he ce eb al
co ex as a pa icula ly ulne able egion o MS and possible
subs a um o disabili y wo sening: co ical pa hology is p esen
a he ea lies s ages o he disease, co ela es wi h he se e i y o
physical disabili y, and is associa ed wi h he cogni i e impai -
men s ha a ec 35%–90% o he pa ien s acco ding o a iables
such as age, gende , and s age o disease p og ession (Calab ese
e al.2012; Lazza o o e al.2024; Ma goni e al.2023). De ec s
in soma osenso y co ical ac i i y, in pa icula , ha e been iden-
i ied bo h in MS pa ien s and animal models o he disease and
is associa ed wi h di e se clinical signa u es such as impai ed
limb unc ion, a igue, and pain (Madsen e al.2022; La ypo
e al.2024; Tecchio e al.2014; Po e e al.2016).
As ocy es a e a unc ionally complex glial cell popula ion ha
suppo s neu onal unc ion h ough a a ie y o mechanisms,
including he elease o molecules ha ine- une synap ic s uc-
u e and unc ion and he homeos a ic con ol o he ex acel-
lula milieu du ing as - synap ic ansmission. S udies o e he
pas wo decades ha e endowed as ocy es wi h he abili y o
modula e neu on exci abili y h ough he elease o neu oac i e
molecules based on in acellula calcium a ia ions induced
by incoming neu o ansmi e s (A aque e al.2014). Th ough
his physiological mechanism, as ocy es locally adjus synap ic
unc ions and modula e neu onal ne wo k s a es, he eby shap-
ing a a ie y o beha io s (San ello, Toni, and Vol e a 2019;
Oli ei a and A aque2022). A he co ical le el, calcium- based
as ocy e signaling con ols senso y- e oked neu onal ne wo k
ac i i y (Lines e al.2020; Wang e al.2006; Miguel- Quesada
e al.2023) and modula es he gene a ion o slow- wa e oscilla-
o y pa e ns in ol ed in cogni i e pe o mance (Poskanze and
Yus e2016; Fellin e al.2009).
As ocy e–neu on in e ac ions unde go dele e ious ans o -
ma ions in CNS pa hologies wi h eme ging impac s on disease
symp oma ology. Combined calcium imaging and elec o-
physiological s udies ha e e ealed disease- speci ic as ocy e
al e a ions a ec ing b ain ci cui s ele an o cogni i e unc-
ion in mouse models o a ious neu odegene a i e diso de s
(Deleka e e al.2014; Nancla es e al.2023; Yu e al.2020; Shah
e al.2022). In he expe imen al au oimmune encephalomyeli-
is (EAE) mouse model o MS, as ocy es display abe an cal-
cium ac i i y causing exace ba ed glu ama e glio ansmission
and long- las ing al e a ions o exci a o y synap ic ansmission
associa ed wi h memo y de ici s (Ba aiba e al.2024; Habbas
e  al. 2015). These ex i o s udies showed cell- au onomous
unc ional de ici s media ed by local in apa enchymal in lam-
ma ion and suppo he no ion ha de egula ed as ocy e ne -
wo k ac i i y a ec s he b ain co ex in MS. Howe e , in i o
s udies assessing he eme gence o abe an as ocy e ac i i y
pa e ns in expe imen al MS a e lacking. He e, we applied ibe
pho ome y o he longi udinal analysis o he as ocy ic ne -
wo k in he b ain co ex o eely mo ing mice du ing he onse
and p og ession o EAE. We show ha he popula ion o co i-
cal as ocy es displays spon aneously occu ing calcium e en s
o enhanced du a ion a acu e EAE disease. Fu he mo e, bo h
spon aneous and senso y- e oked as ocy e calcium signals
showed educed ampli udes ha co ela ed o he se e i y o
clinical symp oma ology. These esul s poin o he in ol emen
o cell- au onomous and nonau onomous mechanisms unde ly-
ing as ocy e ne wo k dys unc ion in co ical MS wi h po en ial
implica ions in disease symp oma ology and p og ession.
2 | Me hods
2.1 | Mice
All expe imen s we e pe o med in acco dance wi h he
Guidelines o he Animal Ca e and Use and he Eu opean
Communi ies Council Di ec i e o Sep embe 22 h 2010
(2010/63/EU74). Expe imen s we e app o ed by he local e hical
commi ees o he Uni e si y o he Basque Coun y (app o al
numbe s 2017140, M202017144, and 2022245), he Uni e si y
o Bo deaux (app o al numbe A33063098), and he F ench
Minis y o Ag icul u e and Fo es y (au ho iza ion numbe
3306369). Nai e mice on a C57BL/6N backg ound (Jan ie ,
F ance; RRID: IMSR_JAX:000664) we e used. Cages we e en-
iched and mice we e main ained unde s anda d condi ions
( ood and wa e adlibi um; 12–12 h ligh –da k cycle; wo o i e
mice pe cage). Expe imen s we e pe o med du ing da k cycle
(ligh o a 8:00 a.m.). EAE was induced in emale mice based
on epidemiological e idence ha MS a ec s wo o ou imes
mo e women han men (Wal on e al.2020).
2.2 | Su ge y o AAV Adminis a ion and Fibe
Implan a ion
A o al o 38 mice (8–9 weeks; 19–22 g) we e anes he ized wi h
iso lu ane (4% o induc ion and 2.5% o main enance) and
placed on a hea ing pad o keep hei body empe a u e a
37°C. Eye dehyd a ion was p e en ed by opical applica ion o
oph halmic gel, and analgesia was achie ed by subcu aneous
(s.c.) injec ion o bup eno phine (Bup eca e, 0.05 mg/kg). The
skin abo e he skull was sha ed wi h a azo and disin ec ed
wi h modi ied 70% e hanol and be adine be o e an incision was
made. Mice we e placed in a s e eo axic appa a us (Da id Kop
Ins umen s) wi h a mouse adap o and la e al ea ba s.
Mice we e injec ed wi h ssAAV- 9/2- hGFAP- hHBbI/E-
GCaMP6 - bGHp(A) (ETH i al ec o acili y—ETH Zü ich;
ca . no. 275) o ibe pho ome y imaging o as ocy es. Vi us
i e s we e be ween 1010 and 1012 genomic copies pe millili e .
S e eo axic injec ions we e a ge ed o he mouse soma osenso y
co ex acco ding o he ollowing coo dina es ( om b egma):
an e io –pos e io −1.5, medial–la e al ±2.5, and do sal– en al
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3 o 10
−1.5. Vi al pa icles we e injec ed a 400–500 nL alone o in
combina ion a a maximum a e o 100 nL/min using a glass
pipe e a ached o a Nanoje III (D ummond, B oomall, USA).
Following i us deli e y, he sy inge was le in place o 10 min
be o e being slowly wi hd awn om he b ain. The op ical ibe
(400 μm diame e ) was placed 250 μm abo e he injec ion si e
du ing he same su gical session. Mice we e weighed daily, and
indi iduals ha ailed o e u n o hei p esu ge y body weigh
we e excluded om subsequen expe imen s. Mice we e used o
EAE induc ion 2 weeks a e su ge y.
2.3 | EAE Model
Mice we e a bi a ily assigned o con ol and EAE g oups.
Animals we e immunized in he lank by s.c. injec ion o
200 μg myelin oligodend ocy e glycop o ein (MOG30–55) pep ide
(MEVGWYRSPFSRVVHLYRNGK) (Pep ide Syn hesis Co e
Facili ies o he Pompeu Fab a Uni e si y, Ba celona, Spain)
in incomple e F eund's adju an supplemen ed wi h 8 mg/mL
Mycobac e ium ube culosis H37Ra (Di co Labo a o ies; ca . no.
231141). Pe ussis oxin (500 ng; Millipo e; ca . no. 516560) was
injec ed in ape i oneally on he day o immuniza ion and again
2 days la e . Body weigh and neu ological symp oma ology we e
eco ded daily and sco ed om 0 o 8 as ollows: 0, no de ec able
changes in muscle one and beha io ; 1, laccid ail; 2, pa alyzed
ail; 3, impai men o loss o muscle one in hindlimbs; 4, hind-
limb hemipa alysis; 5, comple e hindlimb pa alysis; 6, comple e
hindlimb pa alysis and loss o muscle one in o elimbs; 7, e -
aplegia; and 8, mo ibund. A o al o six EAE mice displaying
neu ological sco es < 1 we e excluded om he analysis.
2.4 | Fibe Pho ome y Imaging
F eely mo ing mice we e imaged a e 3 days o handling ha-
bi ua ion. On he day o eco ding, each mouse was placed in
a ec angula chambe , and i s beha io was eco ded using a
came a placed abo e he chambe . Baseline eco dings o spon-
aneous as ocy e ac i i y we e made o 15 min e e y 1–2 days,
s a ing 3 days be o e MOG adminis a ion. The calcium signal
e oked by senso y s imula ion o he ail was assessed a he end
o he baseline pe iod.
Con ol and EAE mice we e imaged in pa allel du ing he ime
cou se o h ee independen expe imen s, including simila
numbe s o mice pe expe imen al g oup. Co ical as ocy e
GCaMP6 was imaged in i o using 470 and 405 nm LEDs. The
emi ed luo escence is p opo ional o he calcium concen a-
ion o s imula ion a 470 nm (Ake boom e al.2013; Ohku a
e al.2012). The isosbes ic 405 nm s imula ion (UV ligh ) was
used in al e na ion wi h he blue ligh (470 nm) o analysis pu -
poses, as he luo escence emi ed a e his s imula ion is no
dependen on calcium (Lü cke e al.2010). The GCaMP6 luo-
escence om he as ocy es was collec ed wi h an sCMOS cam-
e a h ough an op ic ibe di ided in o wo sec ions: a sho ibe
implan ed in he b ain o he mouse and a long ibe (modi ied
pa ch co d), bo h connec ed h ough a e ule– e ule (1.25 mm)
connec ion. A MATLAB p og am (Ma labWo ks; RRID:
SCR_001622) was used o synch onize each image eco ding
made by he came a and he GCaMP6 ligh exci a ion was made
by he LEDs (470 and 405 nm). The wo wa eleng hs o 470 and
405 nm, a a powe o 0.1 mW, we e al e na ed a a equency o
20 Hz each (40 Hz al e na ed ligh s imula ions).
To calcula e luo escence speci ically due o calcium luc ua-
ions and o emo e bleaching and mo emen a i ac s, he isos-
bes ic 405 nm signal was sub ac ed om he 470 nm calcium
signal. Speci ically, no malized luo escence changes (∆F/F0)
we e calcula ed by sub ac ing he mean luo escence (2 min
sliding window a e age) om he luo escence eco ded by he
ibe a each ime poin and di iding his alue by he mean
luo escence ((F−Fmean)/Fmean) using a cus omized Ma lab so -
wa e. Subsequen ly, he calcium- independen isosbes ic signal
was sub ac ed om he aw signal emi ed a e he 470 nm
exci a ion o elimina e unspeci ic luo escence. The esul is
he global calcium signal (∆F/F (%) = ∆FCa−∆Fisos), which was
used as an es ima e o onic ac i i y o he as ocy es. Calcium
ansien s we e de ec ed on he il e ed ace (high il e ) using a
h eshold o iden i y hem (2 median absolu e de ia ion [MAD]
o he en i e ace). Du a ion and equency we e calcula ed o
he de ec ed ansien s. Ampli ude was de e mined as he MAD
o each s udied pe iod (Se a e al.2021; Ba aiba e al.2024).
2.5 | Fluo escence Immunohis ochemis y
Mice we e deeply anes he ized by in ape i oneal injec ion o
pen oba bi al (Exagon, Axience SAS, 400 mg/kg body weigh )
and ansca dially pe used wi h phospha e- bu e ed saline
(PBS, 0.1 M, pH 7.4) be o e being ixed wi h 4% o maldehyde
(Sigma, HT501128). B ains we e isola ed, incuba ed in he same
ixa i e solu ion o e nigh a 4°C, and subsequen ly main-
ained in PBS + 0.025% azide a 4°C un il use. F ee- loa ing
ozen co onal sec ions (40 μm) we e cu ou using a c yos a
(Leica Biosys ems CM1950S; RRID: SCR_018061), collec ed
in an i eeze solu ion, and conse ed a −20°C un il u he
use. Floa ing sec ions we e washed h ee imes o 5 min in
PBS (0.1 M, pH 7.4) and incuba ed wi h abbi an i- GFP (1:500;
In i ogen; RRID: AB_221569) and chicken an i- GFAP (1:500;
Abcam; RRID: AB_304558) o e nigh a 4°C in a blocking solu-
ion con aining 10% donkey se um and 0.3% T i on X- 100 in
PBS. The sec ions we e hen washed in PBS o 30 min a oom
empe a u e (RT), and p ima y an ibodies we e de ec ed by in-
cuba ion wi h donkey an i- abbi Alexa 647 (1:500; In i ogen;
RRID: AB_2536183) and donkey an i- chicken Rhodamine Red
(1:500; Jackson ImmunoResea ch; RRID: AB_2340371) o 2 h
a RT. Then, sec ions we e washed o 15 min in PBS, moun ed,
d ied, and moun ed on mic oscope slides in Fluo omoun - G
(In i ogen; ca . no. 00- 4958- 02).
Op ical images om issue sec ions p ocessed in pa allel we e
acqui ed in he same session using a 20× oil lens on a luo es-
cence mic oscope (Leica Mic osys ems CMS GmBbH, Type:
11504197). Image acquisi ion was ca ied ou using luo escence
in ensi y se ings a which he con ol sec ions wi hou p ima y
an ibodies ga e no signal. Immunolabeling was examined in
he issue a ea su ounding he p obe using open- sou ce image
analysis so wa e Fiji ImageJ (Schindelin e al.2012). GFP+ and
GFAP+ cells we e quan i ied by cell coun ing in wo op ical sec-
ions pe issue slice, and da a we e exp essed as he mean cell
numbe pe squa e millime e (mm2) o issue a ea.
14714159, 2025, 2, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/jnc.16305 by Uni e sidad Del Paã-S Vasco, Wiley Online Lib a y on [07/01/2026]. 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 o 10 Jou nal o Neu ochemis y, 2025
2.6 | S a is ical Analyses
No s a is ical me hods we e used o p ede e mine sample
size. The numbe o subjec s included in he s udy was de e -
mined based on p e ious s udies o a simila na u e (Ba aiba
e al.2024). No andomiza ion was pe o med o alloca e subjec s
o he s udy, and no blinding was pe o med. Summa y esul s
a e p esen ed as he mean o independen da a poin s ± SEM,
ep esen ing he numbe o animals es ed. Da ase s we e ini-
ially es ed o no mal dis ibu ion using he Shapi o–Wilk
es , and s a is ical analysis o he di e ences be ween g oups
was de e mined by a wo- ailed unpai ed S uden - es o
Mann–Whi ney es . Co ela ion analysis was pe o med using
Pea son's o Spea man's es . No es o ou lie s was conduc ed.
S a is ical analyses we e pe o med using G aphPad P ism so -
wa e ( e sion 10.1.2). Di e ences we e conside ed signi ican ly
di e en when p < 0.05.
3 | Resul s
3.1 | Co ical As ocy es Show De egula ed
Spon aneous Calcium Signals a Ea ly S ages o EAE
Disease
P e ious esul s in b ain slices show ha as ocy es o he
mouse soma osenso y co ex display spon aneous calcium
hype ac i i y a acu e EAE disease (Ba aiba e al.2024). To
deciphe he in i o ea u es o co ical as ocy e ne wo k
dys unc ion in MS, in his s udy we applied GCaMP6 - based
ibe pho ome y o he analysis o as ocy e ac i i y o e he
ime cou se o EAE. The as ocy e calcium signals o eely
beha ing EAE and non- immunized mice we e moni o ed in
pa allel o 3 days be o e MOG35–55 adminis a ion and e e y
2–3 days he ea e s a ing a 3 days pos - immuniza ion (dpi)
(Figu e1a). The i s clinical mani es a ions o EAE occu ed
a 10 dpi (sco e = 0.173 ± 0.072) wi h acu e disease peaking
a ound 18 dpi (sco e = 3.404 ± 0.424) and a enua ion o neu-
ological se e i y aking place a la e ime poin s (Figu e1b).
His ological analysis o pos mo em issues showed speci ic
exp ession o he calcium senso in as ocy es om con ol
and EAE mice a 21 dpi ollowing ch onic ibe pho ome y
imaging (Figu eS1).
As i s app oach o in es iga e he impac o au oimmune in-
lamma ion on he calcium ac i i y o co ical as ocy es, we
sough o possible di e ences in na u ally occu ing calcium
luc ua ions, which e lec he in eg a ion o bo h ac i i y-
dependen and - independen cellula signals (Wang e al.2006;
Zu Nieden and Dei me 2006), in EAE mice as compa ed o
con ol animals. Consis en wi h p e ious ex i o epo s o ex-
ace ba ed as ocy e ac i i y du ing EAE (Ba aiba e al.2024),
imaging o GCaMP6 exp essing as ocy es in eely beha ing
mice shows a sus ained inc ease in he du a ion o spon a-
neous calcium signals du ing acu e disease (Figu e1c–e). The
equency o as ocy e calcium oscilla ions was concomi an ly
educed a ea lies s ages o acu e EAE (Figu e1c,d, ). Fu he
analysis showed ha spon aneous as ocy e calcium ac i i y
displays e en s o educed ampli ude in EAE mice as com-
pa ed o con ol animals eco ded in pa allel (Figu e1c,d,g).
Rema kably, de egula ion o as ocy e calcium signals in e ms
o du a ion, equency, and ampli ude eme ged a ea ly s ages
o acu e disease (12 dpi), hus encompassing he onse o EAE
symp oma ology (Figu e1b). Hence, EAE causes ea ly impai -
men s o spon aneously occu ing as ocy e ne wo k ac i i y in
he b ain co ex o eely mo ing mice.
We nex in es iga ed he possible ela ionship be ween de egu-
la ed spon aneous calcium ac i i y in co ical as ocy es and dis-
ease sco es, which e lec s spinal co d axon loss associa ed wi h
he eme gence o in lamma o y demyelina ing lesions du ing
EAE p og ession (Wujek e al.2002; Liu e al.2008). Changes in
he du a ion and equency o as ocy e calcium signals did no
co ela e wi h he se e i y o clinical disabili y a any o he ime
poin s es ed (Figu e2a,b). Howe e , we ound a nega i e co -
ela ion be ween he ampli ude de ici s a ec ing spon aneously
occu ing as ocy e calcium signals and neu ological disabili y
in EAE mice ha eached s a is ical signi icance a 12 and 14 dpi
(Figu e2c). These obse a ions sugges ha he neu odegene a-
i e and in lamma o y mechanisms ha de e mine he se e i y
o disease symp oma ology in EAE mice a e ansla ed in o a
diminished ampli ude o spon aneous as ocy e calcium signals
a he co ical ne wo k le el.
3.2 | Impai ed Senso y- E oked As ocy e Calcium
Responses o Co ical As ocy es Co ela e Wi h
Disease Se e i y
The popula ion o co ical as ocy es esponds o senso y s im-
ula ion in i o wi h a high deg ee o ne wo k eliabili y (Lines
e al.2020; Wang e al.2006). In u n, as ocy e calcium sig-
naling egula es neu onal ne wo k ac i i y and senso y- e oked
beha io s (Lines e  al. 2020; Miguel- Quesada e  al. 2023).
Thus, o u he in es iga e he ela ionship be ween co ical
as ocy e dys unc ion and senso y in o ma ion p ocessing in
MS, we nex add essed changes in as ocy ic calcium signals
e oked by senso y s imula ion o he ail in EAE mice. Unde
ou expe imen al se ings, suspension by he ail induced eli-
able calcium esponses in co ical as ocy es om nai e mice
ha emained s able in size du ing he ch onic eco ding pe iod
(Figu e3a,b). EAE induc ion was associa ed wi h a signi ican ly
dec eased ampli ude, bu no du a ion, o as ocy ic calcium e-
sponses e oked by ail- holding ha pa alleled symp om onse
(Figu e 3a–c). Reminiscen o ou obse a ions conce ning
spon aneously occu ing calcium oscilla ions in eely beha -
ing mice, we obse ed an in e se co ela ion be ween he am-
pli ude o as ocy e calcium ansien s e oked by ail- holding
and neu ological disabili y sco e alues du ing EAE p og ession
ha eached s a is ical signi icance a 17 and 19 dpi (Figu e3d).
Thus, EAE causes hypo esponsi eness o co ical as ocy es o
senso y s imula ion associa ed wi h clinical disabili y a acu e
disease.
4 | Discussion
Neu oin lamma o y mechanisms a ec ing he b ain co ex play
a majo ole in ele an clinical aspec s o MS wi h bo h physical
and cogni i e ebounds. Reac i e as ocy es p oduce abe an
signals ha lead o synap ic and beha io al al e a ions du ing
au oimmune co ical in lamma ion (Ba aiba e  al. 2024;
14714159, 2025, 2, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/jnc.16305 by Uni e sidad Del Paã-S Vasco, Wiley Online Lib a y on [07/01/2026]. 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
5 o 10
Habbas e al.2015). Howe e , he unc ional implica ions o co -
ical MS a he as ocy e ne wo k le el emain la gely de ined.
In his s udy, we asked whe he he popula ion o co ical as o-
cy es displays ac i i y de ici s in MS by applying ibe pho om-
e y imaging in eely beha ing mice o he analysis o calcium
signals in he EAE model o au oimmune demyelina ion. Ou
esul s link abe an as ocy e ne wo k unc ion in he b ain
co ex o neu ological de ici s and shed ligh on he mechanism
unde lying co ical dys unc ion in MS.
Al hough p e ious ex i o s udies ha e epo ed on abe -
an calcium signals o co ical as ocy es in he EAE model
(Ba aiba e al.2024), he e we p o ide he i s quan i ica ion
o spon aneous and pe iphe ally e oked as ocy e ne wo k
FIGURE 1 | Co ical as ocy es show dys egula ed spon aneous calcium ac i i y a ea ly EAE s ages. (a) Expe imen al app oach o in i o ime-
cou se analysis o as ocy ic calcium ac i i y du ing EAE. Fibe pho ome y imaging was pe o med in con ol and EAE mice o 3 consecu i e days
be o e EAE induc ion by immuniza ion wi h MOG30- 55 and a 3, 5, 7, 10, 12, 14, 17, 19, and 21 days pos immuniza ion (dpi). (b) Neu ological sco e o
mice included in he in i o analysis o co ical as ocy e calcium signals du ing EAE ime cou se. (c) Rep esen a i e aces o spon aneous ac i i y
in he popula ion o co ical as ocy es du ing he p eimmuniza ion phase (−1 dpi) ( op) and du ing acu e EAE disease (14 dpi) (bo om). Do s co e-
spond o ansien s de ec ed abo e he h eshold (median + 2*MAD). (d) Time cou se o no malized du a ion, equency, and ampli ude o co ical
as ocy e calcium signals eco ded du ing EAE p og ession. Raw da a eco ded om EAE mice we e exp essed ela i e o alues om con ol ani-
mals eco ded in pa allel du ing EAE p og ession (n = 13–19 mice). (e–g) Compa ison be ween spon aneous as ocy e calcium esponses in eely be-
ha ing naï e and EAE mice a di e en ime poin s o acu e disease p og ession. Da a we e analyzed by wo- ailed unpai ed - es o Mann–Whi ney
es . (e) Du a ion: 12 dpi ( , d = 3.948, 30; p = 0.00044), 14 dpi ( , d = 2.865, 30; p = 0.00754), 17 dpi ( , d = 3.284, 30; p = 0.00260), 19 dpi ( , d = 2.232,
30; p = 0.03325), and 21 dpi (U = 117; p = 0.82055). ( ) E en s pe minu e: 12 dpi ( , d = 4.725, 30; p = 0.00005), 14 dpi ( , d = 3.254, 30; p = 0.00282), 17
dpi (U = 62; p = 0.01765), 19 dpi ( , d = 1.232, 30; p = 0.2274), and 21 dpi (U = 91; p = 0.21590). (g) Ampli ude: 12 dpi (U = 44; p = 0.00165), 14 dpi (U = 26;
p = 0.00006), 17 dpi (U = 38; p = 0.00062), 19 dpi ( , d = 4.520, 30; p = 0.00009), and 21 dpi ( , d = 4.481, 30; p = 0.00010).
0510 15 20
0
1
2
3
4
5
6
Dpi
Mean clinical sco e
12 14 17 19 21
0
1
2
3
4
Days pos -immuniza ion
E en s pe minu e
Con ol
EAE
*** **
*
n.s. n.s.
12 14 17 19 21
0
1
2
3
4
5
Days pos -immuniza ion
Ampli ude (ΔF/F
0
)
Con ol
EAE
**
***
****** ***
S imula ion
Fibe pho ome y imaging
2 weeks
ca
P e-EAE
EAE
Day -1
Day -2
Day -3
…
Dpi 3
Dpi 5
…
Dpi 21
EAE
GFAP GCaMP6
15 min
Spon aneous
de
b
g
12 14 17 19 21
0
10
20
30
40
50
Days pos -immuniza ion
Du a ion (s)
Con ol
EAE
***
**
**
** *
0510 15 20
0
1
2
3
4
Days pos -immuniza ion
Spon aneous as ocy e
calcium signals
F equency
Du a ion
Ampli ude
30 s
2.5 (ΔF/F0)
14714159, 2025, 2, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/jnc.16305 by Uni e sidad Del Paã-S Vasco, Wiley Online Lib a y on [07/01/2026]. 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

6 o 10 Jou nal o Neu ochemis y, 2025
ac i i ies du ing he eme gence o disease symp oma ology.
As ocy es o he soma osenso y co ex displayed na u ally
occu ing calcium signals o inc eased du a ion bu educed
ampli ude and e en equency du ing acu e disease. These
obse a ions highligh he complexi y o abe an as ocy e
ne wo k ac i i y in eely beha ing EAE mice and sugges
ha he dys unc ional p ope ies o as ocy ic calcium signals
in he b ain co ex may eme ge om di e se physiopa holog-
ical mechanisms. In his ega d, ou ecen s udy in co ical
slices showed ha EAE p omo es as ocy e calcium hype ac-
i i y consis ing o spon aneous e en s o enhanced du a ion
and equency ha migh po en ially ansla e in o in i o sig-
nals wi h an ex ended du a ion du ing acu e disease (Ba aiba
e al.2024). A a mechanis ic le el, calcium hype exci abili y
o co ical as ocy es eco ded ex i o was independen o neu-
onal ac i i y and eme ged om eac i i y- ela ed adap a ions
du ing au oimmune in lamma ion (Ba aiba e al.2024). In
he con ex o hese p e ious esul s, ou in i o obse a ions
sugges ha he dys unc ional spon aneous as ocy e ne wo k
ac i i y in he soma osenso y co ex esul s, in pa , om in-
insic mechanisms associa ed wi h he pa hogenic ans o -
ma ion o as oglial cells in he local in lamma o y milieu.
The p esen obse a ion ha as ocy es o he soma osenso y
co ex display spon aneous calcium e en s o educed ampli-
ude when eco ded in i o is, howe e , in appa en con as o
ex i o slice imaging esul s o as ocy e hype ac i i y a acu e
FIGURE 2 | Dys egula ion o spon aneous as ocy e calcium signals in he EAE co ex: Co ela ion o neu ological de ici s. Co ela ion analysis
be ween he du a ion (a), equency (b), and ampli ude (c) o spon aneous as ocy e calcium signals in he mouse soma osenso y co ex and clinical
disabili y sco es a di e en ime poin s o EAE disease p og ession (n = 13 mice). Raw calcium imaging da a om EAE mice we e no malized o
alues eco ded du ing he p esymp oma ic phase and plo ed agains neu ological sco es. Pea son's o Spea man's co ela ion coe icien s and p
alues a e indica ed in each do plo .
02
46
0
1
2
3
4
5
Neu ological sco e
E en s pe minu e
21 dpi
=0.2017
p=0.5059
a
b
c
0246
0
1
2
3
417 dpi
=-0.5450
p=0.0570
Neu ological sco e
Ampli ude
0246
0
1
2
3
4
5
Neu ological sco e
Du a ion
19 dpi
=-0.0504
p=0.8713
0123
0
1
2
3
412 dpi
=-0.5705
p=0.0418
Neu ological sco e
Ampli ude
0246
0
1
2
3
4
Neu ological sco e
Ampli ude
19 dpi
=-0.5446
p=0.0543
02
46
0
1
2
3
4
Neu ological sco e
Ampli ude
21 dpi
=-0.5290
p=0.0630
0123
0
1
2
3
4
512 dpi
=-0.1981
p=0.5136
Neu ological sco e
E en spe minu e
0246
0
1
2
3
4
5
Neu ological sco e
E en spe minu e
19 dpi
=0.0707
p=0.8185
0246
0
1
2
3
4
517 dpi
=0.3126
p=0.2964
Neu ological sco e
E en s pe minu e
0246
0
1
2
3
414 dpi
=-0.6851
p=0.0120
Neu ological sco e
Ampli ude
0246
0
1
2
3
4
514 dpi
=-0.1934
p=0.5270
Neu ological sco e
E en spe minu e
0123
0
1
2
3
4
512 dpi
=-0.0650
p=0.8337
Neu ological sco e
Du a ion
0246
0
1
2
3
4
514 dpi
=0.2093
p=0.4925
Neu ological sco e
Du a ion
0246
0
1
2
3
4
517 dpi
=-0.2265
p=0.4569
Neu ological sco e
Du a ion
02
46
0
1
2
3
4
5
Neu ological sco e
Du a ion
21 dpi
=-0.1740
p=0.5670
14714159, 2025, 2, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/jnc.16305 by Uni e sidad Del Paã-S Vasco, Wiley Online Lib a y on [07/01/2026]. 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
7 o 10
EAE disease (Ba aiba e al.2024). Disc epancies conce ning
spon aneous ac i i y measu ed using ibe pho ome y and
wo pho on slice imaging o as ocy es in he soma osenso y
co ex o EAE mice migh be ela ed o dispa i ies be ween
bo h expe imen al p ocedu es, such as ibe implan a ion, and
he use o TTX du ing ex i o analysis (Ba aiba e al.2024).
An addi ional con ounding ac o is ha ibe pho ome y e-
co dings ep esen he added signal om a popula ion o as-
ocy es—including somas and p ocesses—whe eas ex i o
slice imaging moni o s indi idual cells and p ocesses. The
signal measu ed in i o, equi alen o he popula ion a e -
age, could esul in il e ing some e en s, o example, hose
e y slow o wi h ela i ely low ampli udes. Fu he s udies
a e needed o deciphe how de egula ed calcium signaling a
he single cell le el con ibu es o complex changes in he spa-
ial and empo al p ope ies o as ocy e popula ion ac i i y
du ing au oimmune in lamma ion.
The calcium dynamics o co ical as ocy es in he in ac b ain
a e modula ed by local and pe iphe ally e oked neu onal ne -
wo k ac i i ies ia neu on- o- as ocy e communica ion (Hi ase
e al.2004; Wang e al.2006; Lines e al.2020). In his s udy,
we show ha educ ions in he ampli ude o spon aneous as o-
cy e signals du ing acu e EAE symp oma ology a e mi o ed by
de ici s in pe iphe ally e oked calcium esponses, which e lec
he ac i a ion o co ical neu ons du ing in o ma ion p ocessing
(Lines e al.2020; Wang e al.2006). Al hough cau ion is needed
when a emp ing o compa e da a om ex i o and in i o ap-
p oaches, his obse a ion si s well wi h p e ious indings ha
as ocy es in he EAE co ex demons a e dec eased calcium sig-
naling o neu o ansmi e ecep o agonis s and chemogene ic
ac i a ion o Gq and Gi p o eins despi e being spon aneously
hype ac i e (Ba aiba e al.2024). Bo h mechanisms o as o-
cy e dys unc ion a e no mu ually exclusi e and may ac inde-
penden ly, syne gis ically, o in opposi ion o modula e speci ic
pa e ns o as ocy e calcium ac i i y in i o, hus p o iding an
explana ion o he appa en ly con adic o y indings ega ding
he modula ion o spon aneous calcium ac i i y in e ms o du-
a ion and ampli ude du ing EAE. Beyond mechanis ic impli-
ca ions, he cell- au onomous hypo esponsi eness o as ocy es
o acu e pha macological s imuli poin s o in insic de ici s in
as ocy e eedbacks o neu onal ac i i y a he co ical le el ha
would be ansla ed in o a educed calcium popula ion ac i i y
in eely mo ing mice.
FIGURE 3 | Impai ed as ocy e calcium esponses o senso y s imula ion in he EAE co ex co ela e o disease se e i y. (a) Rep esen a i e aces
showing calcium esponses o co ical as ocy es e oked by ail- holding in con ol ( op) and EAE (17 dpi) (bo om) mice. (b) No malized ampli ude
and du a ion o e oked co ical as ocy e calcium esponses du ing EAE p og ession. Raw da a eco ded om EAE mice we e exp essed ela i e o
alues om con ol animals eco ded in pa allel du ing EAE p og ession (n = 13–19 mice). (c) Ampli ude o calcium ansien s e oked by senso y
s imula ion o he ail in immunized mice a di e en ime poin s o disease p og ession and con ol animals eco ded in pa allel. Da a we e analyzed
by wo- ailed unpai ed - es o Mann–Whi ney es : 12 dpi (U = 37; p = 0.00052), 14 dpi (U = 34; p = 0.00030), 17 dpi ( , d = 4.193, 30; p = 0.00022), 19
dpi ( , d = 3.583, 30; p = 0.00118), and 21 dpi ( , d = 3.872, 30; p = 0.00054). (d) Co ela ion analysis be ween he ampli ude o senso y- e oked calcium
esponses in co ical as ocy es and clinical se e i y du ing EAE. Raw da a om EAE mice we e no malized o alues eco ded du ing he p esymp-
oma ic phase and plo ed agains neu ological sco es. Pea son's o Spea man's co ela ion coe icien s and p alues a e indica ed in each do plo .
a
d
Con ol
EAE
2.5 (ΔF/F0)
30 s
bc
12 14 17 19 21
0
5
10
15
20
Days pos -immuniza ion
Ampli ude (ΔF/F
0
)
Con ol
EAE
***
***
*****
***
0510 15 20
0
1
2
3
4
Dpi
E oked as ocy ic
Ca
2+
ansien s
Du a ion
Ampli ude
0246
0
1
2
3
417 dpi
= - 0.6058
p = 0.0311
Neu ological sco e
Ampli ude (ΔF/F
0
)
0123
0
1
2
3
412 dpi
= - 0.3849
p = 0.1936
Neu ological sco e
Ampli ude (ΔF/F
0
)
0246
0
1
2
3
414 dpi
= - 0.5148
p = 0.0718
Neu ological sco e
Ampli ude (ΔF/F
0
)
0246
0
1
2
3
4
Neu ological sco e
Ampli ude (ΔF/F
0
)
21 dpi
= - 0.5188
p = 0.0693
0246
0
1
2
3
4
Neu ological sco e
Ampli ude (ΔF/F
0
)
19 dpi
= - 0.5714
p = 0.0413
14714159, 2025, 2, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/jnc.16305 by Uni e sidad Del Paã-S Vasco, Wiley Online Lib a y on [07/01/2026]. 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
8 o 10 Jou nal o Neu ochemis y, 2025
P esen esul s un eil in e es ing aspec s o in i o as ocy e
pa hology in he MS co ex wi h po en ial implica ions o dis-
ease symp oma ology. Fi s , as ocy e ne wo k ac i i y de ici s
encompass he appea ance o neu ological disabili y a acu e
EAE disease. Second, impai men s o spon aneous ac i i y and
pe iphe ally e oked as ocy e esponses in e ms o ampli ude o
he calcium signals co ela e o he se e i y o clinical sco es a
di e en ime poin s o disease p og ession. Al hough a causal
ela ionship be ween neu opa hology and as ocy e dys unc ion
in he soma osenso y co ex canno be i mly es ablished based
on ou co ela ion analysis, hese obse a ions suppo he pos-
sibili y ha he educ ions in co ical as ocy e ne wo k ac i i y
and neu ological disabili y sco es e lec common pa hological
mechanisms. The clinical de ici s ha cha ac e ize EAE man-
i es as an ascending spas ici y and pa alysis gene ally g aded
on a poin scale based on he isual sco ing o mo o signs, al-
hough disease sco es mos likely e lec a combina ion o senso-
imo o abno mali ies p og essi ely a ec ing he ail and limbs
(Thibaul , Cal ino, and Peze  2011; Po e e  al. 2016; Segal
e al.2020). Al hough a numbe o s udies ha e highligh ed in-
lamma o y mechanisms and unc ional de ici s ha a ec he
soma osenso y co ex du ing EAE (Po e e al.2016; Ba aiba
e al.2024; Yang e al.2013), he ascending pa alysis ha yp-
i ies disease p og ession seems o eme ge p edominan ly om
he loss o spinal co d axons associa ed wi h he occu ence o
in lamma o y, demyelina ing lesions in whi e ma e acks,
which mimics he degene a ion o senso y and co icospinal
ac s in MS pa ien s (Wujek e al.2002; Liu e al.2008; DeLuca,
Ebe s, and Esi i2004). Indeed, he ex en o spinal co d axo-
nal degene a ion co ela es closely o neu ological disabili y
bo h in pa ien s wi h MS and in he EAE model o he disease
(Liu e al.2008; Bja ma e al.2000). On he o he hand, he
p ima y soma osenso y co ex compu es senso y in o ma ion
om di e en modali ies ia halamic inpu s o laye IV and
e en ually ansmi s ou pu signals o he spinal co d h ough
he co icospinal ac , which o igina es om he py amidal
neu ons in laye V and cons i u es he majo co ical ou pu o
con ol mo o beha io and senso y ascending eedback inpu s
(Macías e al. 2022; Mo eno- López e al. 2016; Lemon 2008).
Finally, i is well es ablished ha as ocy e ne wo k signals in
he soma osenso y co ical ci cui s a e posi i ely modula ed
by pe iphe al s imuli ha elici neu onal ac i i y in a s imulus-
dependen manne and hus ollow he in o ma ion encoded by
neu onal inpu s o he co ex (Miguel- Quesada e al.2023; Lines
e al.2020; Zhao, Wang, and Wang2012). Thus, in he con ex
o hese p e ious obse a ions, ou p esen esul s sugges ha
de ici s in he ansmission o pe iphe ally e oked neu onal
signals eme ging, a leas in pa , om spinal co d pa hology,
may con ibu e o as ocy e ne wo k ac i i y impai men s in he
EAE co ex. I should be no ed, howe e , ha in his s udy we
pe o med ibe pho ome y imaging o as ocy e ac i i y in he
whiske - ela ed p ima y soma osenso y co ex while applying
senso y s imula ion in he mouse ail. This mechanical manip-
ula ion induces an indi ec senso y s imula ion e ec wi hin
he soma osenso y ba el co ex linked o he ac i a ion o di -
e en b ain s uc u es as neu omodula o y sys ems (A ono
e al.2010) a he han a di ec senso y inpu om he pe iph-
e y o he co ex. Fu he mo e, suspension by he ail migh
po en ially ac i a e as ocy es wi hin soma osenso y co ical
ci cui s due o s ess- ela ed deli e y o ace ylcholine and/o
no epineph ine (Rasmussen e al.2023; Gau e al.2024; Wang
e al.2023), hus adding u he complexi y o he in e p e a ion
o ou p esen esul s. Fu u e s udies combining senso y s imu-
la ion o inc easing in ensi ies wi h ac i i y eadou s o whiske
and/o limb ep esen a ion o he mouse soma osenso y co -
ex may help cla i y he mechanis ic implica ions o as ocy e
dys unc ion in he pa hological modula ion- speci ic senso y
modali ies du ing EAE (Ziegle e  al. 2023; Miguel- Quesada
e al.2023).
Senso y pe cep ion, locomo ion, and a ousal a e di ec ly as-
socia ed wi h inc eases in co ical as ocy ic calcium ac i i y
in i o (Boja skai e e al.2020; Wang e al.2006, 2023; Pauke
e al.2014; Rasmussen e al.2023; Gau e al.2024). Howe e ,
he consequences o as ocy e ne wo k ac i i y on co ical ou -
pu unc ion ha e been bes s udied in he con ex o senso y
in o ma ion p ocessing. Recen s udies in he ield demons a e
ha as ocy e esponses o pe iphe ally e oked neu onal ac i -
i y modula e he dynamic ange o which co ical ne wo ks e-
spond o senso y s imuli o con ol beha io (Lines e al.2020;
Miguel- Quesada e al.2023; Wang e al.2023). The co olla y o
hese obse a ions is ha as ocy e ne wo k dys unc ion may
signi ican ly con ibu e o senso y symp oms and po en ially
o he clinically ele an aspec s ela ed o co ical dys unc ion
in neu ological diso de s. Al hough in i o s udies assessing
he implica ions o as ocy e- o- neu on communica ion de i-
ci s in neu oin lamma o y and neu odegene a i e pa hologies
a e s ill sca ce, a ailable e idence suppo s he hypo hesis ha
abe an as ocy e calcium signaling in he b ain co ex causes
neu onal ne wo k dys unc ion and beha io al impai men s in
Alzheime 's disease (Lines e al.2022; Åbjø sb å en e al.2022;
Shah e  al. 2022). In his con ex , i seems plausible ha he
unc ional dis u bances o co ical as ocy es dys egula e neu-
on ne wo k ope a ion, leading o senso imo o pa hology and
cogni i e impai men s in MS pa ien s (Ma goni e  al. 2023;
Madsen e al.2022). We need o ema k, howe e , ha in his
s udy we did no moni o neu onal ac i i y and ou esul s do
no p o ide de ini i e e idence linking de egula ion o as ocy e
calcium ac i i y o de ici s in co ical neu on ne wo k espon-
si eness du ing senso y in o ma ion p ocessing. None heless,
ecen elec ophysiological esul s in he EAE mouse model
demons a e ha abe an as ocy e calcium signaling is associ-
a ed wi h exace ba ed glu ama e- media ed glio ansmission and
wi h synap ic plas ici y de ici s a ec ing laye V py amidal neu-
ons o he soma osenso y co ex du ing acu e disease (Ba aiba
e  al. 2024). Toge he wi h hese p e ious obse a ions, ou
p esen indings s ongly sugges ha de ec s in he as ocy e
calcium popula ion ac i i y may indeed ansla e in o abe an
co ical neu onal ne wo k unc ion du ing EAE. Fu u e in i o
s udies combining elec ophysiological neu onal eco dings in
esponse o senso y s imula ion while simul aneously moni o -
ing and/o manipula ing as ocy e calcium ac i i y will help
iden i y mechanis ic associa ions be ween de ici s in as ocy e
and neu onal ne wo k esponsi eness in co ical MS, as well as
hei impac on disease symp oma ology.
In conclusion, he p esen s udy p o ides se e al key indings on
he ole o as ocy es in MS co ical dys unc ion, which is one
o he ea lies and mos consis en indings in pa ien s. Ou e-
sul s demons a e ha na u ally occu ing and e oked as ocy e
popula ion ac i i ies a e al e ed in he co ex o EAE mice a
ea ly s ages o disabili y and link as ocy e ne wo k dys unc ion
14714159, 2025, 2, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/jnc.16305 by Uni e sidad Del Paã-S Vasco, Wiley Online Lib a y on [07/01/2026]. 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
9 o 10
a he co ical le el o neu ological de ici s eme ging om au o-
immune demyelina ion. In he con ex o p e ious s udies, hese
indings indica e ha abe an as ocy e ac i i y is a consis en
obse a ion in co ical MS ha eme ges in pa om de ici s in
popula ion esponses o pe iphe al inpu s. These esul s suppo
he exis ence o a dis up ed as ocy e–neu onal ne wo k in e -
play, con ibu ing o co ical dys unc ion and associa ed senso-
imo o symp oma ology in MS pa ien s.
Au ho Con ibu ions
A. Mo eno- Ga cía: o mal analysis, in es iga ion. R. Se a : o mal
analysis, in es iga ion, me hodology, so wa e, supe ision, w i ing –
e iew and edi ing. F. Julio- Kalajzic: in es iga ion. A. Be nal- Chico:
in es iga ion. A. M. Ba aiba : w i ing – o iginal d a , w i ing – e-
iew and edi ing. C. Ma u e: in es iga ion. G. Ma sicano: concep u-
aliza ion, da a cu a ion, unding acquisi ion, p ojec adminis a ion,
esou ces, supe ision, w i ing – e iew and edi ing. S. Ma o: concep-
ualiza ion, da a cu a ion, unding acquisi ion, p ojec adminis a ion,
esou ces, supe ision, isualiza ion, w i ing – o iginal d a , w i ing
– e iew and edi ing.
Acknowledgemen s
We would like o hank he pe sonnel o he Animal Facili ies o he
Uni e si y o he Basque Coun y and Neu ocen e Magendie o mouse
ca e. We also hank all membe s o Ma o's and Ma sicano's labs o use-
ul discussions and ad ice. This wo k was unded by he Ins i u o de
Salud Ca los III (PI21/00629, o S.M.) and co ounded by he Eu opean
Union, Basque Go e nmen (PIBA_2023_1_0046; 2023111031;
IT1473- 22, o S.M.; CannaMe HD, o S.M. and G.M.; IT1203- 19, o
C.M.), ARSEP Founda ion (ARSEP- 1310 o S.M. and G.M.), INSERM
( o G.M.), he Eu opean Resea ch Council (MiCaB a, ERC- 2017-
AdG- 786467, o G.M.), Fonda ion pou la Reche che Medicale (FRM,
DRM20101220445 o G.M.), Region Aqui aine (CanB ain, AAP2022A-
2021- 16763610 and - 17219710 o G.M.); F ench S a e/Agence Na ionale
de la Reche che (HippObese, ANR- 23- ce14- 0004- 03; ERA- Ne Neu on
CanShank, ANR- 21- NEU2- 0001- 04, o G.M.), La Caixa Resea ch
Heal h 2023 (PsychoCannabis, HR23- 00793, o G.M.), Minis e io
de Ciencia e Inno acion (PID2019- 109724RB- 100 o C.M.), and he
Pos doc o al P og am o he Basque Go e nmen ( o A.M.B.). The ca -
oon in Figu e1a was c ea ed using BioRe nde . com.
Con lic s o In e es
The au ho s decla e no con lic s o in e es .
Da a A ailabili y S a emen
All da a a e a ailable in he main ex and he Suppo ing In o ma ion,
o om he co esponding au ho on easonable eques .
Pee Re iew
The pee e iew his o y o his a icle is a ailable a h ps:// www. webo
scien ce. com/ api/ ga ew ay/ wos/ pee - e iew/ 10. 1111/ jnc. 16305 .
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