The Subventricular Zone Neurogenic Niche Provides Adult Born Functional Neurons to Repair Cortical Brain Injuries in Response to Diterpenoid Therapy

Pa dillo‑Díaze al.
S em Cell Resea ch & The apy (2025) 16:1
h ps://doi.o g/10.1186/s13287‑024‑04105‑4
RESEARCH Open Access
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S em Cell Resea ch & The apy
The sub en icula zone neu ogenic niche
p o ides adul bo n unc ional neu ons o epai
co ical b ain inju ies in esponse odi e penoid
he apy
Rica do Pa dillo‑Díaz1,2,3†, Pa icia Pé ez‑Ga cía1,2,3†, Ma ía O ego‑Domínguez1,2,7†,
Rica do Gómez‑Oli a2,3, No a Ma ínez‑Gómez1, Samuel Domínguez‑Ga cía2,3,8, F ancisco Ga cía‑Cóza 3,4,
Juan Ped o Muñoz‑Mi anda3,4, Rosa io He nández‑Galán5,6, Li ia Ca ascal1,3, Ca men Cas o2,3* and
Ped o Nunez‑Abades1,3*
Abs ac
In oduc ion Neu al s em cells om he sub en icula zone (SVZ) neu ogenic niche p o ide neu ons ha in e‑
g a e in he ol ac o y bulb ci cui y. Howe e , in esponse o co ical inju ies, he neu ogenic ac i i y o he SVZ
is signi ican ly al e ed, leading o inc eased numbe o neu oblas s wi h a modi ied mig a ion pa e n ha leads cells
owa ds he si e o inju y. Despi e he inc eased neu ogenesis and mig a ion, many newly gene a ed neu ons ail
o su i e o unc ionally in eg a e in o he co ical ci cui y. P o iding he inju ed a ea wi h he adequa e signaling
molecules may imp o e bo h mig a ion and unc ional in eg a ion o newly gene a ed neu ons.
Me hods In he e, we ha e s udied he e ec o a di e pene wi h he capaci y o induce neu egulin elease a p o‑
mo ing neu ogenesis in a mu ine model o co ical b ain inju y. Using g een luo escen p o ein exp essing ec‑
o s we ha e labeled SVZ cells and ha e s udied he mig a ion o newly gene a ed neu oblas s owa d he inju y
in esponse he ea men . In addi ion, using elec ophysiological eco dings we ha e s udied he di e en ia ion
o hese neu oblas s in o ma u e neu ons and hei unc ional in eg a ion in o he co ical ci cui y. We ha e s udied
hei elec ical p ope ies, hei mo phology and co ical loca ion.
Resul s We ha e ound ha EOF2 ea men o adul mice wi h mechanical co ical inju ies acili a es he deli e y
o neu oblas s in o hese inju ies. The newly gene a ed neu ons de elop ea u es o ully unc ional neu ons. Ou
esul s show ha he newly gene a ed neu ons ecei e elec ical inpu s, i e ac ion po en ials, and unde go com‑
ple e di e en ia ion in o neu ons ecapi ula ing he s ages ha dis inguish on ogenic di e en ia ion. These neu ons
de elop ea u es ep esen a i e o neu ons belonging he co ical laye in which hey a e si ua ed. We ha e also s ud‑
ied ha EOF2 acili a es neu egulin elease in SVZ cells, a signaling ac o ha p omo es neu onal di e en ia ion.
†Rica do Pa dillo‑Díaz, Pa icia Pé ez‑Ga cía and Ma ía O ego‑Domínguez
hese au ho s con ibu ed equally o his wo k.
*Co espondence:
Ca men Cas o
[email p o ec ed]
Ped o Nunez‑Abades
[email p o ec ed]
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
Neu egulin is exp essed in mic oglial cells ha each he inju y in esponse o he damage and i s elease is inc eased
by EOF2 ea men .
Conclusion P omo ing neu egulin elease ia di e pene ea men acili a es mig a ion o SVZ‑de i ed neu oblas s
o co ical inju ies s imula ing hei di e en ia ion in o ma u e unc ional neu ons, which ecei e elec ical inpu s
and de elop ea u es o co ical neu ons. These indings highligh he ole o di e penoids as a po en ial he apy
o epai co ical b ain inju ies.
Keywo ds Neu al s em cells, Sub en icula zone, Neu ogenesis, Co ical b ain inju y, Neu onal di e en ia ion, Newly
gene a ed neu ons, Mo pho unc ional p ope ies, Di e penoids, B ain epai he apy
In oduc ion
Co ical inju ies, caused by auma ic inciden s o neu-
ological diso de s, lead o he i e e sible loss o neu-
ons and esul in signi ican challenges o cogni i e and
mo o unc ions. Fo decades, he adul co ex was con-
side ed o ha e limi ed egene a i e po en ial. Howe e ,
ecen esea ch has shed ligh on he ole o sub en icu-
la zone (SVZ) neu ogenesis in co ical inju y egene a-
ion [1].
In esponse o co ical inju ies, SVZ neu ogenesis plays
a c ucial ole in acili a ing egene a ion. Inju y-induced
signals, including g ow h ac o s and in lamma o y
media o s [2], igge he ac i a ion and mobiliza ion o
neu al s em cells (NSCs) wi hin he SVZ [3]. These ac i-
a ed NSCs gi e ise o neu oblas s, which physiologi-
cally mig a e along he os al mig a o y s eam (RMS)
owa ds he ol ac o y bulb (OB), whe e hey di e en ia e
in o in e neu ons [4]. Howe e , unde ce ain condi ions
SVZ gene a ed neu oblas s may al e hei mig a ion
pa e n o con ibu e o co ical epai and egene a-
ion. P e ious epo s show ha in esponse o a co ical
inju y, neu al p ogeni o s om he SVZ mig a e ec opi-
cally o he inju ed a ea assis ed by blood essels and
eac i e as ocy es [5]. Upon eaching he lesion a ea,
in lamma o y signaling cues s imula e he di e en ia-
ion o hese p ogeni o s mainly in o as ocy es and, e y
ew ma u e neu ons [5, 6]. Thus, neu onal eplacemen
in inju ies is a a e e en and does no con ibu e o he
eco e y o he los neu ophysiological unc ions p ob-
ably because he small numbe o neu oblas s ha each
o a e gene a ed wi hin he inju ed a ea ail o ully di -
e en ia e, ecei e elec ical inpu and inco po a e in o
exis ing ci cui s. I is hen necessa y o de elop s a egies
aimed a en iching inju ies wi h newly gene a ed neu-
oblas s, which will di e en ia e in o ma u e, unc ional
neu ons.
In he s udy o neu oblas mig a ion is essen ial he ole
o p o eins kinases (PK) such as PKA o PKC [7, 8]. In
ela ion o his, p e ious epo s ha e e ealed ha he
ea men o mouse co ical inju ies wi h a no el PKC
ac i a ing di e penoid esul ed in neu oblas en ich-
men and in hei di e en ia ion in o ma u e neu ons [9],
sugges ing a ole o his molecule a p omo ing eplace-
men o unc ional neu ons in co ical inju ies. The
mechanism o ac ion o his di e penoid is based on i s
capaci y o modi y he concen a ions o signaling mol-
ecules ha egula e neu ogenesis in esponse o an inju y
[9]. Chemo ac ic and in lamma o y cues eleased by
mic oglial cells and as ocy es seem o play an impo an
ole in de ining he di e en ia ion o SVZ p ogeni o s
in o glial cells in esponse o an inju y, in p ejudice o
neu onal di e en ia ion [10, 11]. In esponse o an inju y,
SVZ NSC gi e ise o a subpopula ion o eac i e as o-
cy es in he co ex ha con ibu e o as ogliosis and sca
o ma ion [12]. Wi hin he inju y en i onmen and he
SVZ, g ow h ac o s ha p omo e p oli e a ion and glial
di e en ia ion a e highly exp essed such as ans o m-
ing g ow h ac o alpha (TGFα) [6, 13] and hey need
o be coun e balanced wi h signals ha p omo e di e -
en ia ion such as neu egulins o allow egene a ion and
eplacemen o he los neu ons. In e es ingly, e idences
show ha in esponse o di e penoid EOF2, which ac i-
a es no el PKC ac i i y and neu egulin elease, hese
signaling cues [9] may be al e ed o p omo e he p ema-
u e di e en ia ion o neu oblas s and hei mig a ion
owa d he inju ed a ea [9] sugges ing a ole o neu egu-
lin 1 (NRG1) and no el PKC in neu onal eplacemen in
co ical inju ies [14, 15].
Despi e he e idence showing he ac i a ion o SVZ
neu ogenesis du ing co ical inju y epai and i s po en-
ial o eplace damaged neu ons, li le is known abou
whe he he gene a ed neu oblas s can di e en ia e in o
ma u e neu ons a he inju y si e and become unc ional
co ical neu ons. Ea lie s udies ha e ocused on his
phenomenon by examining he di e en ia ion o neu o-
blas s wi hin he s ia um in esponse o ischemia [16].
Howe e , u he in es iga ions a e equi ed o de e -
mine he ole ha SVZ neu ogenesis may play in he
eplacemen o unc ional co ical neu ons in inju ies.
We ha e analyzed in he e whe he he ea men
wi h EOF2, s imula es he ma u a ion and in eg a ion
o newly gene a ed neu ons wi hin he co ical inju ed
issue. Using ZsG een luo escen p o ein exp ess-
ing len i i al ec o s, we labeled cells o he SVZ be o e
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Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
pe o ming inju ies in he p ima y mo o co ex o adul
mice. We ha e s udied he mig a ion o ZsG een-labeled
(ZsG een+) neu oblas s owa d he pe ilesional a ea as
well as he ime cou se o he unc ional di e en ia ion
o hese newly gene a ed neu ons. We ha e analyzed he
unc ional p ope ies o newly gene a ed neu oblas s
and neu ons o e he cou se o 7 o 90days pos inju y
(dpi). Passi e and ac i e memb ane p ope ies, exci a o y
inpu s and epe i i e i ing p ope ies we e cha ac e ized
by elec ophysiological eco dings in ZsG een+ cells and
compa ed wi h hose o unlabeled py amidal ma u e neu-
ons o he laye V o he mo o co ex. Finally, in o de
o s udy he signaling cues esponsible o his e ec ,
we ha e analyzed he ime-cou se o neu egulin exp es-
sion pos -inju y in he SVZ and co ex. We show ha he
exp ession o NRG1 occu s mainly in mic oglial cells and
o a lesse ex en in as ocy es. We ha e acili a ed NRG1
elease using pha macological compound EOF2, which
in addi ion s imula es neu oblas di e en ia ion, on he
gene a ion o new neu ons in esponse o a mechanical
co ical b ain inju y.
Me hods
Animal subjec s
CD1 mice o bo h sexes we e used h oughou his s udy.
Animals we e housed unde con olled condi ions o
empe a u e (21–23°C) and ligh (LD 12:12) wi h ee
access o ood (AO4 s anda d main enance die , SAFE,
Épinay-su -O ge, F ance) and wa e . Ca e and handling
o animals we e pe o med acco ding o he Guidelines
o he Eu opean Union Council (2010/63/EU), and he
Spanish egula ions (65/2012 and RD53/2013) o he use
o labo a o y animals.
The numbe o animals used in each expe imen was
de e mined based on p e ious s udies [17–19]. Adul
male mice we e andomized du ing he i s week a e
bi h by c oss- os e ing and used when hey became wo
mon hs old. The p o ocol used has been au ho ized by
he E hics Commi ee o he “Conseje ía de Ag icul u a,
Ganade ía, Pesca y Desa ollo Sos enible de la Jun a de
Andalucía”, in Spain wi h he p o ocol en i led “Neu al
Replacemen hE apies in wo Models o b ain damage:
owa ds Innding New D ugs (REMIND)” app o al num-
be 04/03/2020/033. All s udies in ol ing animals a e
epo ed in acco dance wi h he ARRIVE guidelines 2.0
o epo ing expe imen s in ol ing animals [20, 21]. The
numbe o animals used in each expe imen is indica ed
in he igu e legends.
Design o  hes udy
Mice used in he s udy we e lesioned in he p ima y
mo o co ex by gene a ing con olled mechanical inju-
ies while anes he ized by a cock ail o ke amine (100mg/
kg) and xylazine (20mg/kg). Fo he mig a ion s udies,
mice we e injec ed wi h len i i al ec o s p io o he
inju y in he same su gical ac . Once inju ed, ehicle con-
sis ing on saline solu ion was used in con ol animals and
di e pene EOF2 was used as ea men . Ei he ehicle o
EOF2 we e adminis e ed daily by in anasal in usions
o ei he 14, 28, o 56days as we desc ibe in he pa a-
g aphs below. Upon he comple ion o he ea men s,
mice we e anes he ized wi h a cock ail o ke amine (100
mg/kg)and xylazine (20 mg/kg)and ce eb ospinal luid
(CSF) was ex ac ed as explained below, hen a dose o
Dole hal® (Ven oquinol, Lu e, F ance) con aining a le hal
50mg dose o pen oba bi al o eu hanized he animals
was applied ollowed by ei he b ain pe usion wi h pa a-
o maldehyde ( o his ological s udies) o b ain ex ac-
ion ( o molecula biology and s udies). See desc ip ion
o he di e en p ocedu es below. In he case o elec o-
physiological s udies mice we e anes he ized wi h a le hal
dose o anes he ic p e ious o pe usion wi h a i icial
CSF.
In all in i o expe imen s, he expe imen al g oups
used we e saline ea ed (con ol) o EOF2 ea ed mice
(EOF2). In pos mo em s udies, he expe imen al uni
was he single animal and sample size o each expe i-
men is indica ed in he igu e legends. In elec ophysi-
ological s udies he expe imen al uni was he single cell
and he sample size is indica ed in Addi onal ile 1: Tables
S4-S7.
Injec ion o ZsG een exp essing len i i al ec o s
in heSVZ andunila e al mechanical co ical b ain lesions
Con olled unila e al mechanical co ical b ain inju-
ies we e pe o med in he p ima y mo o co ex o he
igh b ain hemisphe e o anes he ized mice. Mice we e
anes he ized using an anes he ic mix u e composed
o ke amine (100 mg/mL) and xylazine (20 mg/Kg) in
s e ile physiological saline. Using a s e eo axic ame
(Ha a d Appa a us), a longi udinal incision was made
in he skin o he head o expose he skull and p oceed
o injec he ZsG een exp essing len i i al ec o . A
B egma/−0.8 mm, a small c anio omy was pe o med
using a hand d ill and a 0.9 mm d ill bi (Meisinge ,
Neuss, Ge many). Then a Hamil on Gas igh sy inge
(Hamil on Company, NV, USA) was in oduced a ached
o he s e eo axic de ice h ough he incision made, and 1
µL o he ZsG een i us was injec ed in o he la e al en-
icle a a a e o 0.1 µL/min. The len i i al cons uc was
p oduced by us as desc ibed p e iously [14]. HEK Len i-
XTM 293T we e used as packaging cell lines o p oduce
len i i al supe na an as p e iously desc ibed [22]. Cells
we e co- ans ec ed wi h he pHRSincPPT- SEW ans-
e ec o exp essing he g een luo escen p o ein Zs-
G een, oge he wi h plasmids pCMV∆R8.91, coding o
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Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
HIV-1 GAG / POL p o eins and pMD2.G o pseudo yp-
ing wi h he Vesicula S oma i is Vi us G p o ein (VSVG).
Cells we e ans ec ed in Op iMEM™ medium (The mo
Fishe Scien i ic Inc., Ca lsbad, CA, USA) by polye hyl-
enimine (PEI)-media ed ans ec ion [23] and a e one
hou he medium was eplaced by DMEM supplemen ed
wi h 10% e al cal se um (GIBCO; www. he m o ish e .
com/ gibco). Supe na an s we e collec ed a 48 and 72h,
cen i uged a 2100g o 5min o emo e cell deb is and
subjec ed o wo concen a ion ounds using Len i-X™
Concen a o (Clon ech;Moun ain View, CA, USA) o
ob ain a clean high- i e i us-con aining pelle . B ie ly,
i al supe na an s we e incuba ed o 30min a 4°C wi h
3 olumes o Len i-X concen a o eagen , cen i uged a
1500 × g o 50min and he pelle esuspended in 1mL
PBS. Len i-X was u he added and upon 30min incu-
ba ion a 4ºC and an addi ional cen i uga ion, he pelle
was snap ozen in liquid ni ogen and s o ed a −80°C
un il use. Vi al i e s we e de e mined, by e alua ing hei
e iciency in ansducing Ju ka cells by means o a Cy o-
lex™ low cy ome e (Beckman, Indianapolis, IN) 48h
a e ansduc ion. Vi al i e s we e always abo e 2 × 105
ansducing uni s (TU) pe mL.
In he same su gical ac , mice we e unila e ally
lesioned in he igh hemisphe e o he p ima y mo o
co ex. They we e c anio omized wi h a manual d ill a
+1.5mm os al and -1.1mm la e al o B egma. The e-
a e , a con olled mechanical lesion was pe o med in
he unde lying p ima y mo o co ex using a manual
d ill (0.9mm diame e ). This d ill was allowed o pen-
e a e 1mm below he bone su ace. Mice we e inju ed
and placed in o a con olled cage du ing he equi ed
dpi ha depended on he ea men and expe imen al
design. Lesions we e pe o med unila e ally; he inju ed
hemisphe e was conside ed he ipsila e al side, while
he in ac hemisphe e was conside ed he con ala e al
hemisphe e and was used as a con ol. A e pe o ming
he inju y, c anio omies we e sealed by su gical cemen
(Fishe Scien i ic) and he incision made in he skin was
su u ed. Subsequen analgesic and asep ic measu es we e
aken o ensu e animals wel a e. This p ocedu e was p e-
iously s ablished by ou esea ch g oup and has been
used elsewhe e [6, 9, 14, 24].
In anasal adminis a ion o EOF2
EOF2 (CAS numbe 2230806–06–9) was p oduced by us
as p e iously desc ibed [9] and was deli e ed in anasally
while he animal was placed in a s anding posi ion wi h
an ex ended neck as p e iously desc ibed [25]. Eigh een
mic oli e s o each solu ion (5 μM EOF2 in saline, o
saline as ehicle) was deli e ed o e bo h nasal ca i ies
al e na ing 3 μL/each using a mic opipe e. Mouse was
main ained in his posi ion o 10 addi ional seconds o
ensu e all luid was inhaled. In all expe imen s, mice we e
coded and ea men ( ehicle o EOF2) was assigned an-
domly o code numbe s and applied. The ea men was
adminis e ed daily un il 90 dpi.
B ain p ocessing o immunohis ochemis y s udies
A he end o he ea men , b ains we e pe used wi h
pa a o maldehyde (PFA) and sliced using a c yo ome in o
30μm sec ions. Immunohis ochemis y was pe o med
as p e iously desc ibed [18, 24, 26]. See an ibodies in
Addi onal ile 1: ables S1-S3. The ma ke s used o de ec
he di e en cell ypes we e doubleco in (DCX) o
de ec neu oblas s, glial ib illa y acidic p o ein (GFAP)
o de ec as ocy es, ionized calcium binding adap o
molecule 1(Iba1) o de ec mic oglial cells, neu oepi he-
lial s em cell p o ein (nes in) o de ec neu al s em cells
and p ogeni o cells and he neu onal nuclea p o ein
(NeuN) o de ec ma u e neu ons.
B ain slices ob en ion o elec ophysiological s udies
B ain slices we e acqui ed om p e iously ea ed
mice by anes he izing hem and pe using wi h a modi-
ied a i icial CSF (ACSF) o cu ing solu ion. Follow-
ing pe usion, he b ain was swi ly ex ac ed, and a e
emo al o he ce ebellum and os al elencephalon,
co onal slices o 300µm hickness we e ob ained using
a ib a ome (Leica VT1000S, Leica Biosys ems, Uni ed
Kingdom). These slices we e hen incuba ed in a chambe
con aining cu ing solu ion a 34º C o 10min, ollowed
by ans e o ano he chambe illed wi h eco ding solu-
ion a oom empe a u e o a leas 1h be o e u he
use. The composi ion o he di e en ACSF used was as
ollows (da a in mM): i) Reco ding solu ion: 126 NaCl, 2
KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 MgCl2,
and 2 CaCl2; ii) Cu ing solu ion: 92 NMDG, 2.5 KCl, 1.2
NaH2PO4, 30 NaHCO3, 20 HEPES, 25 Glucose, 4 MgCl2,
0.1 CaCl2. Bo h solu ions we e bubbled wi h 95% O2–5%
CO2 (pH 7.4, adjus ed wi h HCl; 295–305 mOsmol/kg).
Whole‑Cell pa ch clamp eco dings andanalysis
A e one hou o incuba ion in he holding chambe , he
slices we e ans e ed o he eco ding chambe o he
mic oscope. To isualize he cells, a Nikon Eclipse FN1
mic oscope equipped wi h in a ed di e en ial in e e -
ence con as (IR-DIC) op ics, a 40 × wa e imme sion
objec i e and an in a ed came a WAT-902H2 is used. In
he holding chambe , he slices we e cons an ly pe used
wi h ACSF ae a ed a oom empe a u e and a a a e o
1mL/min using a pe is al ic pump (Ha a d Appa a us
MPII, Hollis on, MA, USA). The mic opipe es used o
pe o m he pa ch-clamp we e ob ained om bo osili-
ca e glass capilla ies (od 1mm, id 0.58mm, leng h 10cm,
Su e ) s e ched wi h a e ical pulle (PC-10, Na ishige,
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Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
Tokyo, Japan) adjus ed o ge a esis ance be ween 3 and
6 MΩ. The mic opipe es we e illed wi h a K-glucona e
based solu ion wi h he ollowing composi ion (in mM):
120K-Glucona e, 10 KCl, 10 phosphoc ea ine disodium
sal , 2Mg-ATP, 0.3 Na-GTP, 0.1 EGTA, 10 HEPES. pH
was adjus ed o 7.3 using KOH and he osmolali y o 285
mOsmol/kg wi h suc ose wi h he help o an osmome e
(Osmoma 300, gono ec). To pe o m he eco dings,
he mic opipe es we e placed using a mic omanipula-
o (MP-225, Su e Ins umen , CA, Uni ed S a es) in
he inju ed a ea. In his a ea, ZsG een labelled cells we e
iden i ied using he luo escence mic oscopy sys em cou-
pled o he Nikon mic oscope (see Addi onal ile 1: Fig.
S1A). To achie e he whole-cell pa ch-clamp con igu a-
ion, we use an ampli ie (Mul iClamp 700B) and a Digi-
da a 1550 analog- o-digi al con e e (Axon Ins umen s,
Molecula De ices, Sunny ale, CA, Uni ed S a es).
Reco dings we e acqui ed wi h he pCLAMP 10.4 so -
wa e (Molecula De ices), lowpass Bessel- il e ed a
3kHz and he da a we e digi ized a 20kHz. Fo he da a
analysis, Clamp i 10.4 so wa e (Molecula De ices).
Se ies esis ances was ypically 10–20 MΩ, and he
expe imen s we e disca ded i highe han 25 MΩ. Liquid
junc ion po en ials we e compensa ed au oma ically.
Cu en clamp s udies
In his s udy, bo h passi e and ac i e memb ane p op-
e ies o he cells we e s udied as p e iously desc ibed
[27, 28]. In b ie , he es ing memb ane po en ial was
calcula ed by sub ac ing in acellula om ex acellula
po en ial a e he emo al o he eco ding elec ode.
Inpu esis ance was measu ed h ough he injec ion o
hype pola izing and depola izing squa e cu en pulses
(500ms, 1Hz) wi h 10 pA inc emen s be ween each one,
and hen calcula ed as he slope o he cu en – ol age
ela ionship, ollowing Ohm’s Law. Rheobase, de ined as
he minimum in ensi y o cu en necessa y o p o oke
an ac ion po en ial, was de e mined by applying squa e
pulses o 100ms, 1Hz, wi h 10 pA inc emen s. Vol age
h eshold and depola iza ion ol age we e compu ed el-
a i e o he es ing memb ane po en ial. To asce ain he
spike h eshold, ac ion po en ial eco dings we e di e -
en ia ed, wi h he spike onse iden i ied as he memb ane
po en ial a which he i s de i a i e exceeded 10V/s
[29, 30].
Ac ion po en ial ampli ude and du a ion we e calcu-
la ed based on peak ol age and wid h a hal ampli ude.
Repe i i e i ing p ope ies we e assessed by applying
depola izing cu en s eps (1s, 0.5Hz) wi h 20–50 pA
inc emen s. Maximum i ing equency was de ined as
he highes numbe o spikes achie ed du ing epe i i e
discha ge, ega dless o cu en in ensi y, while equency
gain was de e mined as he slope o he ela ionship
be ween i ing equency and applied cu en . Cancella-
ion cu en ep esen ed he in ensi y a which he neu-
on ceased i ing du ing maximal discha ge.
Vol age clamp s udies
Vol age-dependen cu en s we e elici ed by 50 ms
squa e depola izing pulses anging om −60 o + 40mV,
in 10 mV s eps. No leak subs ac ion was pe o med.
Cu en ampli udes we e measu ed in he peak o
inwa d cu en s and a he end o he pulse o ou wa d
cu en s. Conduc ances we e calcula ed as cho d con-
duc ance [31]. Thus, G = I/(V-VE), being G, conduc ance,
I, he measu ed cu en , V, command ol age and VE he
heo ical Ne ns po en ial o po assium (ou wa d cu -
en s) o sodium (inwa d cu en s). Te odo oxin (TTX)
(Toc is), Te ae hylammonium (TEA), (Sigma-Ald ich)
and 4-AP (Sigma-Ald ich) we e dilu ed in he ba h solu-
ion o blocking he di e en conduc ances.
To in es iga e spon aneous pos synap ic cu en s
(sPSC), 60s con inuous eco dings we e conduc ed ol-
lowing p e iously desc ibed me hods [32]. The holding
po en ial was clamped a 0mV o spon aneous inhibi-
o y pos synap ic cu en s (sIPSCs) o −60mV o spon-
aneous exci a o y pos synap ic cu en s (sEPSCs).
Synap ic e en s we e de ec ed and analyzed using EasyE-
lec ophysiology so wa e. A empla e was c ea ed by i -
ing a unc ion o a single e en , and subsequen e en s
we e ex ac ed using a ying de ec ion h esholds. False
posi i es we e manually emo ed, and eal e en s we e
i ed wi h a biexponen ial unc ion o de e mine pe -
cen age appea ance, equency (e en s pe minu e), and
ampli ude (baseline o peak). To assess he na u e o syn-
ap ic e en s, CNQX (50μM), APV (20μM), and SR95531
(gabazine, 20μM) we e used, all o which we e pu chased
om Toc is. The p o ocol in ol ed ini ially supe using
each slice wi h no mal ACSF o con ol eco dings, ol-
lowed by ACSF con aining he d ugs o eco d ol age
esponses.
Mo phological s udy o  henewly gene a ed neu ons
To ca y ou he mo phome ic s udy o he new gene -
a ed cells, dye- illing echnique was used. Fo his pu -
pose, ion opho e ic injec ion o 0.2% neu obio in (Vec o
Labo a o ies, Bu lingame, CA, USA) con ained in he
in e nal pipe e solu ion was ca ied ou by applying
cu en s eps o 400 pA o 500ms a 0.5Hz o 20min
[33]. Slices con aining labeled cells we e deposi ed in
a 4% pa a o maldehyde solu ion a 4º C o e nigh and
hen ans e ed o 30% suc ose in phospha e bu e a 4º
C o main ain hem. The ea e , dye- illed neu ons we e
e ealed wi h a goa -polyclonal an ibio in Texas Red con-
juga ed an ibody (1:900) om Rockland (Pennsyl ania,
USA). A Zeiss LSM 900 Ai yscan 2 con ocal mic oscope

Page 6 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
was used o he isualiza ion and econs uc ion o he
labeled cells. S acks o 30–70 pho og aphs we e pe -
o med, using a 1µm in e al. Images we e p ocessed
wi h ZEN 3.2 Blue Edi ion so wa e and he cells we e
econs uc ed using he Neu olucida 360 e sion 2020
3.1 sys em (Mic oB igh Field, Willis on, VT, Uni ed
S a es).
Analysis we e made acco ding o p e ious wo ks [33,
34]. The quan i a i e mo phome ic da a p esen ed in
his s udy we e gene a ed using Neu oexplo e so wa e.
Each dend i ic segmen was sys ema ically assigned an
o de in a cen i ugal manne , om he soma o he e -
minal segmen s (see Addi onal ile 1: Fig. S1). A node,
o in e sec ion, was de ined as any bi u ca ion along he
dend i e. Fu he mo e, a segmen ep esen ed he po -
ion o he dend i e linking ei he he soma o an in e -
sec ion o wo in e sec ions, while a e minal segmen
deno ed he pa connec ing he las b anching poin o
he dend i e’s e minal ending. This s udy ocused on
h ee key aspec s o neu ons. Fi s ly, neu on su ace a ea
was examined, wi h o al dend i ic su ace a ea de ined as
he sum o each dend i e’s su ace a ea and o al su ace
a ea calcula ed as he sum o soma ic and dend i ic a eas.
Secondly, dend i ic leng h was in es iga ed, speci ically
a ge ing o al dend i ic leng h, which encompasses he
sum o indi idual dend i e leng hs. Las ly, neu onal com-
plexi y was analyzed. B anch o de was sco ed o meas-
u e dend i ic complexi y, ep esen ing he highes o de
eached in each neu on. Addi ional complexi y meas-
u es included he o al numbe o segmen s, ep esen -
ing he en i e y o segmen s wi hin a neu on; he numbe
o nodes, indica ing he numbe o in e sec ions; and he
numbe o e minals, de ined as he sum o e minal seg-
men s wi hin a neu on. Sholl diag ams we e cons uc ed
o he econs uc ed cells. Neu ons we e o ien ed along
do sal and la e al axes, wi h he soma posi ioned a he
cen e o concen ic ci cles p og essi ely inc easing by
50µm in adius. The numbe o dend i es in e sec ing
each ci cle was eco ded as pa o he analysis.
The expe imen al animal g oups o elec ophysiologi-
cal eco dings and mo phome ic analysis we e as ol-
lows: G oup 1 consis ed o animals analyzed a 7–14 dpi,
G oup 2 a 15–28 dpi, G oup 3 a 29–56 dpi, G oup 4
a 57–90 dpi, and G oup 5, o con ol g oup, comp ised
py amidal neu ons om laye V eco ded om he con-
ala e al side o he inju y in 3-mon h-old animals.
RNA isola ion, e e se ansc ip ion and eal‑ ime
quan i a i e PCR
Fo RT-qPCR analysis, RNA was isola ed om he SVZ;
in ac SVZ we e p ocessed o RNA ex ac ion using
he TRIzol (Ca . 15,596,026, In i ogen, Ca lsbad, CA,
USA), sepa a ion me hod, ollowing he manu ac u e ’s
ins uc ions and esuspended in pu i ied nuclease- ee
wa e . RNA was quan i ied using a BioTek’s Syne gy
Mx luo ime e (BioTek Ins umen s, Inc, Winooski,
VT, USA). cDNA was p epa ed om 500ng RNA using
iSc ip TM cDNA Syn hesis Ki (Ca .1708890, Bio-Rad
Labo a o ies Inc, He cules, CA, USA) on a Techne
Genius he mal cycle (Techne L d., Camb idge, UK).
The 15μl RT-qPCR eac ion mix con ained 7.5μl 2X
iTaq Uni e sal SYBR G een Supe mix (Ca . 1,725,122,
Bio-Rad Labo a o ies Inc, He cules, CA, USA), 10nmol
o bo h he o wa d and he e e se p ime s, and 1μl
o he sample. The PCR he mal p o ile included 40
cycles o dena u a ion a 95°C o 10s, an annealing
empe a u e acco ding o each se o p ime s o 15s,
and ex ension a 72°C o 20s, ollowed by a mel ing
cu e analysis. Each sample was analyzed in iplica e.
The mRNA le el o RNA18S was used as in e nal con-
ol. Rela i e quan i ica ion alues o mRNA exp ession
we e calcula ed as 2 (Li ak Me hod). Oligonucleo-
ides used in his s udy we e designed by BLAST and
we e ob ained om Me ck (Mad id, Spain). P ime
sequences (5 −3 ) o de ec ing exp ession o mouse
mRNA we e he ollowing: o NRG1, FW: CGC TGT
TCT GGT CTC ATC CG, RW: GCG GTG GAG TGG AGT
GTA AG; o E bB4, FW: TAC CTC CTC CCA TCT ACA
CATCC, RW: CCT CTG GTA TGG TGC TGG TTG; o
PKCδ FW: GAG GCC TTG AAC CAA GTG ACCC and
RW: CTT GCC A TAG GTC CAG TTG TTG.
PKC kinase ac i i y assay
Mice we e inju ed as p e iously desc ibed and sac-
i iced 7, and 14 dpi. Then b ains we e emo ed and
he issue co esponding o he SVZ and he pe ile-
sional co ex a ea was used in he assay. Tissue was
mechanically disagg ega ed and homogenized in PBS
bu e ollowed by a s ep o ul asound ea men and
he homogena e cen i uged 10,000 × g o 15 min.
Then, he p o ein con en was measu e in he homoge-
na es using he BCA me hod (The moFishe Scien-
i ic, Rock o d, IL, USA), and 1.5μg o c ude p o ein
o each homogena e was used pe assay. The amoun
o PKC kinase ac i i y was measu ed in each sample
using he PKC Kinase Ac i i y Assay Ki (Abcam, Cam-
b idge, U.K.; ca . No. ab139437), ollowing he manu-
ac u e ’s ins uc ions. Posi i e con ols (20–60 ng
o pu i ied ac i e PKC supplied by he ki ) and blanks
(diluen only) we e included in each independen de e -
mina ion. Blanks we e sub ac ed om measu emen s
be o e compa isons we e made. Ac i i y was calcula ed
ela ed o he alue o he con ala e al side.
Page 7 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
SVZ cell isola ion andcul u e
NPCs we e ob ained om he SVZ o 7-day pos na-
al mice ollowing he same p ocedu e desc ibed in
[19]. Neu osphe e cul u es we e main ained in de ined
medium (DM) composed o Dulbecco’s modi ied Eagle’s
medium/F12 medium (1:1 ol/ ol) plus 1 mg/L gen-
amicin (GIBCO) and he B27 supplemen (In i ogen,
Ca lsbad, CA). EGF (20ng/mL) and bFGF (10ng/mL;
bo h om Pep oTech, F ank u , Ge many) we e added
o DM o cul u e expansion.
Neu osphe e di e en ia ion assay
Neu osphe e cells ob ained om cul u es ha had gone
h ough h ee consecu i e passages we e cen i uged,
esuspended in de ined medium wi hou g ow h ac-
o s, and seeded. NRG1 soluble ligand (R&D Sys ems)
was added a di e en concen a ions (1, 5 o 10ng/mL)
and cells we e main ained o 72h be o e being ixed o
immunocy ochemis y. The di e en cells pheno ypes
ob ained as a esul o di e en ia ion we e de ec ed by
he p esence o he ma ke be a-III- ubulin (neu ons and
neu oblas s) o GFAP (as ocy es). Each expe imen was
pe o med using iplica e samples. The measu emen s
a e he a e age o h ee independen expe imen s. The
in i o model o s udy di e en ia ion had been success-
ully used in p e ious epo s [35–37].
Cloning o human TGFα andNRG1 cDNA used oeGFP
andmChe y
Full-leng h cDNA encoding he memb ane-bound iso-
o m o human p o-neu egulin-1 β1- ype (NRG1, NCBI
e e ence sequence: NP_039250.2) wi h mChe y cDNA
inse ed be ween nucleo ides 93 and 94 o NRG1 open
eading ame was cloned in o pEGFP-N1 o add EGFP
cDNA o he 3′ end. Cons uc was syn hesized by Gene-
Cus (Boynes, F ance) o gene a e he mChe y-NRG1-
GFP cons uc .
Time‑lapse expe imen s and luo escence analysis
o  ecombinan mChe y‑NRG1‑eGFP p o ein in hecul u e
medium o NPC
NPC we e pla ed in μ–dishes (35mm high; Ibidi) and
ans ec ed wi h mChe y-NRG1-eGFP cons uc . A e
o e nigh incuba ion, cells we e le o 30min in se um-
ee Fluo ob i e DMEM (The mo Fishe Scien i ic) and
used ei he in ime-lapse expe imen s. Cells we e ea ed
wi h EOF2 compound (5 µM) and images we e aken
e e y 2min. Images o 10 independen cells pe condi-
ion we e analyzed. Measu emen s a e he a e age o
h ee independen expe imen s.
HEK293 cul u e, cloning and ans ec ion
HEK293T ob ained om ATCC (Manassas, VA, USA)
we e cul u ed and ans ec ed as p e iously dec ibed
[14]. A e an o e nigh incuba ion, cells we e le o
30min in se um- ee Fluo ob i e DMEM (The mo Fishe
Scien i ic) and used ei he in luo escence expe imen s.
Fluo escence analysis o mChe y‑ used NRG1
in hecul u e medium o HEK293
HEK293T we e pla ed in µ–dishes (35mm high, Ibidi,
Munich, Ge many). Cells we e ea ed wi h EOF2 o
30 o 180min. Fo luo escence measu emen s, 200,000
HEK293T cells we e pla ed in 1mL o medium Cos a ®
12-well cell cul u e mic opla e and luo escence in he
cul u e medium was measu ed as desc ibed in he legend
o  igu e10.
S a is ical analysis
The da a and s a is ical analysis comply wi h he ecom-
menda ions on expe imen al design and analysis in pha -
macology [38]. All s a is ical analyses we e conduc ed on
aw da a. Resul s a e p esen ed as he mean ± s anda d
e o o he mean (SEM), whe e ’n’ deno es he num-
be o cells o animals included. S a is ical calcula ions
we e pe o med using G aphPad P ism so wa e. Ini-
ially, he no mali y o he da a dis ibu ion was assessed
using he Shapi o–Wilk es . Fo compa isons o means
be ween g oups, a epea ed measu es analysis o a i-
ance (ANOVA) was applied. I signi ican di e ences
we e de ec ed, he Tukey es was used o pai wise com-
pa isons be ween g oups. When compa ing only wo
unpai ed expe imen al g oups, he S uden ’s - es was
employed. To de e mine s a is ically signi ican di e -
ences in he pe cen age o cells i ing ac ion po en ials,
epe i i e discha ge, o synap ic inpu s be ween g oups,
he Chi-squa e es o independence was used. I he
expec ed equencies we e small, Fishe ’s exac es was
u ilized. A 95% con idence in e al was applied in all
analyses, and g oups we e conside ed s a is ically di e -
en i p ≤ 0.05. Unless indica ed,in all igu es and in ables
included inAddi onal ile 1, as e isk (*) indica es s a is i-
cal di e ences be ween g oups, and c osses (†) indica e
di e ences be ween he a ious g oups and he 57–90
dpi g oup.
Resul s
To assess he abili y o EOF2 o enhance neu oblas
en ichmen in b ain inju ies and hei di e en ia ion in o
ma u e neu ons, len i i al ec o s exp essing ZsG een
we e injec ed in o he la e al en icle o adul mice, ol-
lowed by a con olled mo o co ex inju y. Mice ecei ed
Page 8 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
in anasal EOF2 o saline (con ol) o 3, 14, o 28days
pos -inju y, and he SVZ and pe ilesional a eas we e
analyzed o newly gene a ed neu oblas s, iden i ied as
DCX+ cells exp essing ZsG een.
Neu oblas en ichmen inmechanical co ical inju ies
ea ed wi hEOF2
As shown in Fig.1, in con ol animals a 3 dpi (Fig.1
A) he numbe o DCX+ neu oblas s ha had inco po-
a ed ZsG een in he SVZ was highe in he ipsila e al
SVZ han in he con ala e al SVZ. In e es ingly, ea -
men o mice wi h EOF2 inc eased he o al numbe o
neu oblas s in bo h he ipsila e al and he con ala e al
SVZ in compa ison wi h he con ol. Also, he numbe
o ZsG een+ neu oblas s in he ipsila e al SVZ o EOF2
ea ed mice was highe han in he con ala e al SVZ.
Howe e , a 14 dpi (Fig.1 B), he di e ence in he numbe
o ZsG een+/DCX+ cells in he ipsila e al SVZ o con ol
animals was no obse ed whe eas his di e ence was
obse ed in EOF2 ea ed mice, in which a highe num-
be o ZsG een+/DCX+ cells was obse ed in bo h he
ipsila e al SVZ and he con ala e al compa ed o con-
ol mice. These esul s indica ed ha in esponse o he
inju y he SVZ inc emen ed he numbe o neu oblas s
and he ea men wi h EOF2 magni ied his esponse.
Mig a ion o SVZ neu oblas s owa ds heinju y
s imula ed byEOF2
In o de o s udy whe he EOF2 ea men exe ed an
e ec on he mig a ion o neu oblas s om he SVZ o
he OB in inju ed mice, we also analyzed he numbe
o neu oblas s ound in he OB ha we e labeled wi h
ZsG een. As shown in Addi onal ile 1: Fig.S2, a 14 dpi,
he numbe o ZsG een+/DCX+ neu oblas s ound in he
ipsila e al OB co e o EOF2 ea ed mice was highe han
ha o he con ala e al side (Addi onal ile 1: Fig. S2 D-d,
G). Such di e ence was no obse ed in con ol mice
(Addi onal ile 1: Fig. S2 B, G). A 28 dpi, he numbe o
ZsG een+/DCX+ neu oblas s ound in he ipsila e al OB
co e o EOF2 ea ed mice was s ill highe han ha o he
con ala e al side and highe han ha o he ipsila e al
OB co e o con ol mice (Addi onal ile 1: Fig. S2 C, E, G).
Iden ical di e ences we e obse ed in he g anula cell
laye (Addi onal ile 1: Fig. S2 A, F). These esul s indi-
ca ed ha no e ec o he inju y on neu oblas mig a ion
o he OB was obse ed nei he a 14 no a 28 dpi. How-
e e , in he p esence o EOF2, mig a ion o neu oblas s o
he OB is acili a ed.
P e ious s udies ha e shown ha neu oblas s indeed
al e hei mig a ion pa e ns and mo e owa ds a eas
o co ical inju y. In esponse o auma ic b ain inju-
ies, neu oblas s mig a e om he SVZ o he inju y
si e, in luenced by a ious signaling molecules and en i-
onmen al cues [39] . Addi ionally, i has been obse ed
ha neu oblas s mo e owa d b ain lesions in di e en
models o co ical inju y, suppo ing he idea o al e ed
mig a ion pa e ns in esponse o such inju ies [13].
Thus, in ligh o he esul s ha indica ed a acili a ion
o neu oblas mig a ion induced by EOF2, i was nex
analyzed whe he mig a ion o neu oblas s o he pe -
ilesional a ea was also acili a ed by he ea men . Mice
we e gi en B dU injec ions o h ee consecu i e days.
Then, h ee days a e he las dose o B dU, an in ace -
eb o en icula injec ion o he ZsG een len i i al ec o
was gi en o each mouse be o e inju ies we e pe o med
as indica ed abo e. Mice we e hen ea ed o 1, 3, 5, 7,
10 and 14days wi h EOF2 and sac i iced on he las day
o ea men . In hese mice i was analyzed he p esence
o ZsG een+/DCX+ (Fig.2) and B dU+/DCX+ (Addi onal
ile 1: Fig. S3) neu oblas s in he a ea included be ween
he SVZ, and he inju ed co ex. As shown in Fig.2, no
DCX+ cells a e obse ed lea ing he SVZ a 3 dpi (Fig.2
A), whe eas as soon as 5 dpi DCX/ZsG een+ cells can
be obse ed lea ing he SVZ and c ossing he co pus
callosum owa d he inju y (Fig.2 B,b). Then a 7 dpi
DCX+/ZsG een+ cells we e iden i ied ha had c ossed
he co pus callosum and we e loca ed close o he inju y
(Fig.2 C,c). The numbe o DCX+/ZsG een+ cells ound
(See igu e on nex page.)
Fig. 1 The SVZ esponds o a co ical inju y by inc easing DCX+/ZsG een+ and his esponse is enhanced by he EOF2 ea men . A len i i al ec o
exp essing ZsG een was injec ed in he la e al en icle ipsila e al o he lesion o ma k SVZ cells in he same su gical p ocedu e o he inju y.
Immunohis ochemis y was pe o med o he de ec ion o he neu oblas ma ke DCX and ZsG een (shown in magen a o enhance i s
isibili y). LV = La e al Ven icle; scale ba s ep esen 100 µm. A (Le ) Rep esen a i e con ocal images o he sub en icula zone (SVZ) o adul
mice 3 days pos ‑inju y (dpi), ea ed in anasally wi h ehicle (uppe panel) o EOF2 (lowe panel). (Righ ) Quan i ica ion o DCX+/ZsG een+
cells/mm3 in he con ala e al and ipsila e al SVZ ela ed o a co ical b ain inju y in mice ea ed wi h EOF2 o ehicle (con ol) 3 dpi. Da a show
he mean ± SEM o 6 animals pe g oup. S a is ical analysis: * p = 0.0007 con ol con ala e al s. con ol ipsila e al; p = 0.0301 EOF2 con ala e al s.
EOF2 ipsila e al; p = 0.0003 con ol ipsila e al s. EOF2 ipsila e al; p = 0.0045 con ol con ala e al s. EOF2 con ala e al in wo‑way ANOVA. B (Le )
Rep esen a i e con ocal images o he sub en icula zone (SVZ) o adul mice 14 days pos ‑inju y (dpi), ea ed in anasally wi h ehicle (uppe
panel) o EOF2 (lowe panel). (Righ ) Quan i ica ion o DCX+/ZsG een+ cells/mm3 in he con ala e al and ipsila e al SVZ ela ed o a co ical b ain
inju y in mice ea ed wi h ehicle (con ol) o EOF2 14 dpi. Da a show he mean ± SEM. S a is ical analysis: * p = 0.0049 con ol con ala e al s. EOF2
con ala e al; p = 0.0228 EOF2 con ala e al s. EOF2 ipsila e al; p = 0.0172 con ol ipsila e al s. EOF2 ipsila e al in wo‑way ANOVA
Page 9 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
Con olEOF2
0
10000
20000
30000
DCX
+
/ZSG een
+
cells/mm
3
*
*
*
3 DPI
Con ala e al
Ipsila e al
Con olEOF2
0
10000
20000
30000
DCX
+
/ZSG een
+
cells/mm
3
*
*
*
*
EOF2
LV
LV
Con ol
100 μm
DCX
ZsG een
SVZ ipsila e alSVZ con ala e al
A
14 DPI
Con ala e al
Ipsila e al
EOF2
LV
Con ol
100 μm
DCX
ZsG een
SVZ ipsila e alSVZ con ala e al
B
LV
LV
Fig. 1 (See legend on p e ious page.)
Page 16 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
Fig.8 A illus a es 2 ypical neu ons localized in laye 2
and a neu on wi hin he landma k o laye V. When we
seg ega ed he new ZsG een+ neu ons in o wo g oups,
hose ha we e ins alled in he ou mos supe icial lay-
e s (I-IV), and hose ha we e ins alled in he wo deep-
es laye s (V-VI) o he co ex, g ea di e ences we e
obse ed in hei mo pho unc ional cha ac e is ics.
The new neu ons in laye V-VI p esen ed a signi ican ly
g ea e o al su ace a ea, o al dend i ic leng h, num-
be o o al dend i ic segmen s and numbe o e minal
endings (Fig.8 B-E and Addi onal ile 1: TableS7) han
he new neu ons ha a e ins alled in he mos supe icial
laye s. I is also s iking ha he mos supe icial neu-
ons ha e smalle alues o heobase and a highe alues
o inpu esis ance and mean i ing equency o ac ion
po en ials ( heobase 90 s 165 pA, inpu esis ance 163
s 121 MΩ and mean i ing equency 433 s 55 AP/nA)
han hose ZsG een+ neu ons ha a e ins alled in he
deepe laye s. This da a would suppo he conclusion
ha new gene a ed neu ons de eloped a di e en pheno-
ype depending on he laye whe e hey a e included.
Exp ession and elease o neu egulin
wi hin hepe ilesional a ea iss imula ed byEOF2
As a inal a emp , we aimed a elucida ing he mecha-
nism by which EOF2 exe s i s e ec . EOF2 is a di e pene
wi h he capaci y o ac i a e no el PKC. The ac i a ion
o no el PKC leads o he elease o neu egulins and
compe es wi h he elease o o he g ow h ac o s such
as TGFα o HB-EGF ha a e eleased in esponse o
classical PKC ac i a ion [40, 41]. Since NRG1 exe s an
e ec as a cell a ac an [42], we s a ed by analyzing he
exp ession o NRG1 and i s ecep o he E bB4 ecep o
in he SVZ and wi hin he inju ed co ex a 7 and 14 dpi.
As shown in Fig.9, a wo- old inc ease in he exp ession
o NRG1 was ound wi hin he ipsila e al inju ed co -
ex compa ed o he con ala e al o he sham-ope a ed
mice (Fig.9 A, C). The exp ession le els o NRG1 in he
inju ed co ex had e u ned o basal le els 14 dpi (Fig.9
A, C). No inc ease in he E bB4 ecep o was obse ed
a 7dpi (Fig.9 A, C), on he con a y a non-s a is ically
signi ican educ ion on E bB4 exp ession was ound
a 7dpi ha was no obse ed a 14 dpi (Fig.9 A, C).
Rega ding he SVZ, NRG1 exp ession inc eased by 1.5-
old a 7 dpi e u ning o basal le els a 14 dpi. In e es -
ingly, E bB4 exp ession in he SVZ was no al e ed a 7
dpi, howe e i had inc eased by wo old a 14 dpi (Fig.9
A, D). Immunohis ochemis y s udies show ha he
numbe o cells ha exp essed NRG1 in he ipsila e al
co ex inc eased by eigh old compa ed o he con ala -
e al a 7 dpi (Fig.9 E, F, M) Likewise, he a ea occupied
by NRG1 s aining inc eased by h ee old in he SVZ a 7
dpi (Fig.9 G, H, N). In e es ingly, he s udy o he phe-
no ype o NRG1+ cells showed ha a 7 dpi, mo e han
60% o NRG1+ cells we e Iba1+, whe eas only 20% we e
GFAP+ and none o hem we e nes in+ (Fig.9 I-K, O).
Equally, mo e han 80% o NRG1+ cells in he SVZ we e
Iba1+ cells (Fig.9 L, P) and in e es ingly 100% o Iba1
cells exp essed NRG1. Thus, ou esul s sugges ed ha
he inju y acili a ed he appea ance o Iba1+ mic oglial
cells a he SVZ and he pe ilesional a ea ha exp essed
NRG1. We nex elucida ed whe he he ele a ed exp es-
sion o NRG1 led o an inc eased concen a ion o NRG1
in he CSF. We obse ed ha a 7 dpi NRG1 concen a-
ion inc eased by wo old in he inju ed mice compa ed
o con ol mice (sham) (Fig. 9 B). This concen a ion
e u ned o basal le els a 14 dpi (Fig.9 B). In e es ingly,
he ea men o mice wi h EOF2 was able o main ain
(See igu e on nex page.)
Fig. 5 Newly gene a ed neu ons become able o i e an ac ion po en ial s a ing om 28 dpi and a e able o discha ge ac ion po en ials
epe i i ely om 56 dpi. A Memb ane ol age esponses o he minimum cu en equi ed o e oke an ac ion po en ial ( heobase)
in a ep esen a i e neu on om each expe imen al g oup. B Ba cha showing he pe cen age o cell i ing ac ion po en ial in each
expe imen al g oup. I can be seen how he cells a e able o s a i ing om 28 days pos ‑inju y (dpi). Fishe ’s es e ealed signi ican di e ences
in he equency o cells able o i e ac ion po en ials be ween he 15–28 dpi and 29–56 dpi g oups (*p < 0.0001), be ween he 26–56 dpi and 57–90
dpi g oups (*p = 0.0074), and be ween he 57–90 dpi g oup and py amidal neu ons (*p = 0.0033). C‑G Box‑and‑whiske plo s showing he medians
(dashed lines), in e qua ile anges (boxes), minimum/maximum alues (whiske s), and SEM (e o ba s) o heobase (C), ac ion po en ial (AP)
ampli ude (D), AP du a ion (E), ol age depola iza ion (F) and ol age h eshold (G) o each expe imen al g oup. No e ha he heobase inc eased
om he 26–56 dpi g oup o he 57–90 dpi g oup (*p = 0.0033), as did he ac ion po en ial ampli ude (*p = 0.006), while he du a ion dec eased
(*p = 0.036). Addi ionally, he ac ion po en ial ampli ude s ill showed di e ences be ween he 57–90 dpi g oup and he py amidal neu ons g oup.
S a is ical analysis: Repea ed measu es ANOVA wi h Tukey pos ‑hoc. H Ba cha showing he pe cen age o epe i i e esponse occu ence in each
expe imen al g oup. We can see how hey begin o ha e epe i i e i ing p ope ies s a ing a 29 dpi, al hough as we see, e en abo e 56 dpi hese
alues a e a om hose o a pool o py amidal neu ons. Fishe ’s es was used o de e mine di e ences in he obse ed equencies o epe i i e
discha ge, e ealing signi ican di e ences be ween he 15–28 dpi and 29–56 dpi g oups (*p = 0.0045) and be ween he 57–90 dpi g oup
and he py amidal neu on g oup (*p = 0.0039). I Box‑and‑whiske plo s showing he maximum equency alues o each g oup. S a is ical analysis:
Repea ed measu es ANOVA wi h Tukey pos ‑hoc. Fo all g aph, he as e isk (*) deno es s a is ically signi ican di e ences be ween consecu i e
g oups, while he c oss ( +) indica es s a is ically signi ican di e ences be ween ha g oup and he 57–90 dpi g oup. The signi icance le el
was es ablished as p ≤ 0.05

Page 17 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
NRG1 concen a ion ele a ed a 14 dpi (Fig.9 B). Finally,
since EOF2 acili a es he elease o NRG1 h ough ac i-
a ing PKC del a (PKCδ) [43], we es ed o al (pan-PKC)
ac i i y in he ipsila e al and con ala e al co ex and
SVZ, we ound ha o al PKC ac i i y was ele a ed in he
ipsila e al co ex compa ed o he con ala e al a bo h 7
and 14 dpi (Addi onal ile 1: Fig. S5 A). Acco dingly, he
exp ession o PKCδ inc eased in he ipsila e al co ex o
inju ed mice a bo h 7 and 14 dpi (Addi onal ile 1: Fig.
S5 C). No wi hs anding, PKC ac i i y was ele a ed in he
ipsila e al SVZ a 7 dpi bu no a 14 dpi whe eas PKCδ
exp ession was only ele a ed in he ipsila e al SVZ a 14
dpi (Addi onal ile 1: Fig. S5 B).
In i o elease o neu egulin omSVZ‑isola ed cells
iss imula ed byEOF2
These esul s sugges ed ha EOF2 was esponsible o
inducing NRG1 elease in hese cells. In o de o dem-
ons a e he e ec o EOF2 on NRG1 elease we isola ed
cells om he SVZ ha we e cul u ed as neu osphe es o
-51 mV
-45 mV
V11 mV
dp
Rheobase 120 pA
29-56 dpi
20 ms
20 mV
-60 mV
-39.7 mV
V 20.3 mV
dp
Rheobase 200 pA
100 pA
>56 dpi
20 ms
20 mV
-65 mV
-43.7 mV
V21.3 mV
dp
Rheobase 220 pA
100 pA
Py amidal neu on
A
B
% AP appea ence
100
80
60
40
20
0
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
AP Du a ion (ms)
6
4
2
0
E
Vol age h eshold (mV)
0
-20
-40
-60
-80
GI
-1
Maximum equency (AP·s)
60
40
20
0
C
Rheobase (pA)
400
300
200
100
0
*
D
AP ampli ude (mV)
150
100
50
0
Vol age depola iza ion (mV)
50
40
30
20
10
0
FH
% occu ence o
epe i i e discha ge
100
75
50
25
0
*
*
+
*+
++**+
+
*
+*
*+
*+
*
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu on
s
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu on
s
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
Fig. 5 (See legend on p e ious page.)
Page 18 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
h ee consecu i e passages and cul u ed a ached on o
a subs a e. These cells we e hen ans ec ed wi h an
exp ession ec o ha con ained he sequence o a usion
p o ein in which NRG1 was exp essed lanked by eGFP
and mChe y in he C- e minal and N- e minal domains
espec i ely (Fig.10 A). The exp ession o his cons uc
A
10 ms
1 nA
7-14 dpi
15-28 dpi
29-56 dpi
>56 dpi
Py amidal
neu on
+30 mV
-60 mV
D
7-14 dpi
15-28 dpi
29-56 dpi
>56 dpi
Py amidal
neu on
1 nA
1 ms
-30 mV
-60 mV
G
0 mV
-60 mV
10 ms
1 nA
10 ms
1 nA
10 ms
1 nA
TTX + TEA + 4-AP
TTX
B
300
200
100
0
-1
Cu en densi y (pA·pF)
-60 -40 -20 020 40 60
Vol age (mV)
E
0
-100
-200
-300
-400
-60 -40 -20 0204060
-1
Cu en densi y (pA·pF )
Vol age (mV)
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
**
***
C
50
30
10
0
Ou wa d conduc ance (nS)
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
F
60
40
20
0
Inwa d conduc ance (nS)
*
20
40
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
*
*
*
*
*
*+
+
+
*+
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
Fig. 6 Newly gene a ed neu ons display ol age‑dependen ou wa d and inwa d cu en s. A, D Rep esen a i e cu en esponses o + 30 mV (A)
and o −30 mV (B) s ep depola iza ions in newly neu ons om each expe imen al g oup. Holding po en ial in bo h cases was −60 mV. B, E A e age
cu en densi y – ol age ela ionships o each expe imen al g oup o ou wa d (B) and inwa d cu en s (E). C, F Box‑and‑whiske plo s showing
he medians (dashed lines), in e qua ile anges (boxes), minimum/maximum alues (whiske s) and SEM (e o ba s) o ou wa d (C) and inwa d (F)
cho d conduc ance a + 30 mV and −30 mV, espec i ely. The mos signi ican di e ence in ou wa d cu en s occu s be ween he i s and second
g oups (*p = 0.034), wi h u he inc eases and di e ences be ween he 15–28 days pos ‑inju y (dpi) g oup and 57–90 dpi g oup. No di e ences
a e obse ed be ween he 57–90 dpi g oup and he py amidal neu on g oup. Inwa d cu en s appea la e in he 29–56 dpi g oup, inc easing
in he 57–90 dpi g oup (*p = 0.0048), bu no eaching he alues o he py amidal neu ons g oup (p = 0.018). G. Pha macological dissec ion
o he cu en s in a ep esen a i e 57–90 dpi cell. The cu en was elici ed by a s ep depola iza ion om −60 o 0 mV. TTX (1 μM), TEA (5 mM)
and 4‑AP (2 mM) was applied in he ex e nal solu ion. In all cases, s a is ical di e ences we e analyzed using epea ed measu es ANOVA and Tukey
pos ‑hoc. Fo all g aph, he as e isk (*) deno es s a is ically signi ican di e ences be ween consecu i e g oups, while he c oss ( +) indica es
s a is ically signi ican di e ences be ween ha g oup and he 57–90 dpi g oup. The signi icance le el was es ablished as p ≤ 0.05
Page 19 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
esul s in he in eg a ion in he plasma memb ane o a
NRG1 p o ein in which he C- e minal and N- e minal
domains a luo escen and he a io mChe y/eGFP is
1 (Fig.10 B). Addi ion o EOF2 o he cul u e medium
esul s in a educ ion o he mChe y/eGFP a io due o
he induced elease o NRG1 and he consequen loss o
mChe y luo escence (Fig.10 C, E and Addi onal ile
2: Mo ie S1). No educ ion in mChe y/eGFP a io was
obse ed in con ol cul u es (Fig.10 C, D and Addi onal
ile 3: Mo ie S2). To ensu e ha EOF2 was s imula ing
he elease o NRG1, we used Hek293 cells ans ec ed
wi h he mChe y/eGFP cons uc . These cul u es we e
incuba ed wi h EOF2 o 30 and 180min and mChe y
luo escence was de ec ed in he cul u e medium a e -
wa ds. An inc ease in mChe y luo escence was ound in
he cul u e medium a e 180min o incuba ion (Fig.10
D) suppo ing ha EOF2 s imula es NRG1 elease.
Neu egulin‑induced di e en ia ion o SVZ isola ed cells
These esul s indica ed ha EOF2 acili a ed NRG1
elease in SVZ de i ed cells, hus, we analyzed whe he
NRG1 induced di e en ia ion in hese cells. Cells iso-
la ed om he SVZ and cul u ed as neu osphe es we e
g own a ached on o a polyo ni hine subs a e o 72h
in he absence o g ow h ac o s and in he p esence and
absence (con ol) o NRG1. We ound ha he pe cen -
age o be a-III- ubulin+ neu oblas s in con ol cul u es
was a ound 2% (Fig. 10 G, K) whe eas he ea men
wi h inc easing concen a ions o NRG1 inc eased his
pe cen age by wo- old (Fig.10 H–K). On he con a y,
he pe cen age o GFAP+ cells did no inc ease wi h he
ea men (Fig.10 G-J, L). These esul s indica ed ha
NRG1 acili a ed di e en ia ion o SVZ isola ed cells in
ag eemen wi h he in i o indings. No wi hs anding,
he quan i ica ion o he p opo ion o Iba1+ cells ha
exp essed NRG1 in he SVZ e ealed ha all Iba1+ cells
exp ess NRG1, indica ing ha he a i al o mic oglial
cells a he inju ed SVZ will esul in an inc eased exp es-
sion o NRG1 ha could be eleased o he ex acellula
medium.
Discussion
In he p esen wo k, we ha e used a i al labeling ech-
nique ha allows eliable iden i ica ion o newly gen-
e a ed neu ons in co ical inju ies. We ha e ound
neu oblas s ha mig a e om he SVZ owa ds he
mo o co ex in animals ea ed wi h EOF2, which di -
e en ia e in o ully ma u e neu ons in a ime-dependen
manne ha ecapi ula es emb ionic/neona al de elop-
men . These cells ecei e unc ional a e en s, discha ge
ac ion po en ials in esponse o a depola izing exci a-
ion, and de elop an adul pheno ype ha co esponds
o he laye hey a e ins alled on. This comple e se o
unc ional p ope ies endows newly gene a ed neu ons
wi h he capaci y o play a signi ican ole in epai ing
co ical lesions. As an a emp o elucida e he mecha-
nisms in ol ed in he EOF2-induced di e en ia ion we
ha e deepen in o he ole ha mic oglial cells play in he
elease o NRG1 and he capaci y o EOF2 o s imula e
he elease his chemoa ac an .
Inju y‑induced SVZ neu ogenesis isenhanced byEOF2
ea men
A la ge numbe o p e ious e idences show ha co i-
cal inju ies s imula e neu ogenesis in he SVZ [1, 6, 9,
10, 14, 44]. The esul s shown he e, wi h he use o len-
i i al ec o s suppo hese indings. We ha e obse ed
ha in esponse o he co ical inju y pe o med a la ge
abundance o neu oblas s (DCX+) ha inco po a ed
ZsG een was obse ed in he SVZ demons a ing ha
co ical inju ies s imula e neu ogenesis in his egion.
In ag eemen wi h p e ious indings hese neu oblas s
we e ound in he OB, 14 and 28days la e [45] bu no
DCX+ cells we e obse ed in con ol mice by he inju ed
(See igu e on nex page.)
Fig. 7 Mo phological s udy o he newly gene a ed neu ons. A Image o Texas Red an i‑neu obio in s ains showing he mo phological changes
occu ing o e he cou se o ea men in newly gene a ed neu ons o igina ing om he sub en icula zone (SVZ). No e he inc ease in cell size
o e he days o ea men , as well as he inc ease in he numbe , leng h and complexi y o dend i es. B Scholl diag ams showing he mo phology
o he cells in sec ion A. C‑H Box‑and‑whiske plo s showing he medians (dashed lines), in e qua ile anges (boxes), minimum/maximum alues
(whiske s) and SEM (e o ba s) o dend i ic su ace a ea (C), numbe o p ima y dend i es (D), o al dend i ic leng h (E), maximum dend i ic
b anch o de (F) and numbe o e minal endings o each expe imen al g oup. Dend i ic su ace a ea inc eased wi h ea men du a ion, showing
signi ican di e ences be ween he i s wo g oups and he 57–90 days pos ‑inju y (dpi) g oup (+ p = 0.0007; + p = 0.0368). The numbe o dend i es,
hei leng h, and he numbe o e minals inc eased o e ime, wi h he mos signi ican di e ences obse ed be ween he 15–28 dpi and 29–56
dpi g oups (*p < 0.0001, *p = 0.044, and *p = 0.008, espec i ely). The o de inc eased p og essi ely, wi h signi ican di e ences only when compa ing
he i s g oup o he 57–90 dpi g oup (+ p = 0.011). No e how all elec ophysiological pa ame e s a e es ablished wi hin he i s wo mon hs
o ea men . H Do plo showing he numbe o in e sec ions e sus he dis ance o soma. No e ha in he i s and second g oup hese alues a e
e y small, bu om he hi d g oup onwa ds he alues a e p ac ically iden ical o hose o a py amidal neu on. In all cases, s a is ical di e ences
we e analyzed using epea ed measu es ANOVA and Tukey pos ‑hoc. Fo all g aph, he as e isk (*) deno es s a is ically signi ican di e ences
be ween consecu i e g oups, while he c oss ( +) indica es s a is ically signi ican di e ences be ween ha g oup and he 57–90 dpi g oup. The
signi icance le el was es ablished as p ≤ 0.05
Page 20 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
egion a hese imepoin s. In e es ingly, he ea men
wi h EOF2 signi ican ly inc emen ed SVZ neu ogenesis,
pa icula ly in he ipsila e al SVZ gene a ing neu oblas s
ha mig a e no only o he OB bu also o he inju ed
a ea.
The mig a ion pa e n o neu oblas s isal e ed in esponse
o heinju y andEOF2 ea men
The SVZ is an es ablished ich sou ce o new ol ac-
o y neu ons [46]. Physiologically, NSC in he SVZ
p o ides neu oblas s ha mig a e h ough he RMS
7-14 dpi 15-28 dpi29-56 dpi >56 dpi Laye V py amidal neu on
D
L
1
1
0
0
μ
μ
m
m
5
5
0
0
μ
μ
m
m
1
1
0
0
0
0
μ
μ
m
m
1
1
0
0
0
0
μ
μ
m
m
1
1
0
0
0
0
μ
μ
m
m
50 m50 m50 m50 m50 m
2
Dend i ic su ace a ea (m)
40000
30000
20000
10000
0
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
Numbe o dend i es
10
8
6
4
2
0
To al dend i ic leng h (m)
7500
6000
4500
3000
1500
0
O de
15
10
5
0
Numbe o e minal endings
50
40
30
20
10
0-2
0
2
4
6
10
12
14
16
18
8
Nº o In e sec ions
0 200 400 600
Dis ance om soma (μm)
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu ons
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu on
s
Py amidal neu ons
7 - 14 dpi
15 - 28 dpi
29 - 56 dpi
> 56 dpi
A
B
C
DE
FGH
7-14 dpi
15-28 dpi
29-56 dpi
57-90 dpi
Py amidal
neu on
s
+
+
+
+
*
+
+
*
++
*
+
+
+
*
+
*
+
*
*
*
+
+
*+
Neu obio in
Fig. 7 (See legend on p e ious page.)
Page 21 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
owa ds he OB whe e hey di e en ia e in o ma u e
ol ac o y in e neu ons. No wi hs anding, unde pa ho-
logical condi ions, such as ischemic o auma ic inju-
ies, neu oblas s o he SVZ may al e hei mig a ion
pa e n mig a ing owa d he inju ed a ea o p o ide
newly gene a ed neu ons. P e ious e idence show ha
in esponse o local ischemia he SVZ gene a es neu o-
blas ha mig a e o he s ia um whe e hey di e en i-
a e in o ma u e neu ons ollowing he in usion o g ow h
ac o s in he la e al en icles [16]. Mo eo e , he SVZ
has been shown o p o ide neu ons o co ical inju ies
upon he modula ion o signaling cascades ha depend
on ADAM17 [14]. We show he e ha he SVZ esponds
o an inju y by inc easing he numbe o newly gene a ed
neu oblas s and ha his esponse is enla ged when s im-
ula ed by di e pene EOF2. Ou esul s also show ha he
numbe o newly gene a ed SVZ neu oblas s is educed
om 3 dpi compa ed o 14 dpi indica ing ha hese cells
a e mig a ing ei he owa ds he OB o owa ds he pe -
ilesional a ea. In ag eemen , we show in Addi onal ile 1:
Fig. S2 ha hese cells mig a e o he OB and ZsG een
labeled neu oblas s a e ound a 14 and 28 dpi, being
highe in he ipsila e al OB han in he con ala e al and
in EOF2 ea ed mice compa ed o con ol. Thus, an
e ec o he inju y on neu oblas mig a ion owa ds he
OB was obse ed ha was inc eased by EOF2 ea men .
Likewise, we also ound newly gene a ed neu oblas s
wi hin he inju ed a ea, which appea o be mig a ing
om he SVZ owa ds he inju y in a ime dependen
manne p o iding he inju ed mo o co ex wi h newly
gene a ed neu oblas s as soon as 10–14 dpi [9]. Neu o-
blas s seemed o be lea ing he SVZ and c ossing he co -
pus callosum a ound day 5 pos inju y. Many appea ed o
be mig a ing o he damaged mo o co ex on day 7 pos
inju y eaching he lesion by day 14 ollowing he ea -
men wi h EOF2. On day 14 he pe ilesional a ea appea s
eplenished o neu oblas s ha by day 28 pos inju y
s a he di e en ia ion o ma u e neu ons. No mig a ion
was obse ed in he absence o he ea men . Despi e
he numbe o neu oblas s ha we obse ed mig a ing
owa ds he inju y, we canno disca d he possibili y ha
some o he neu oblas s ound wi hin he pe ilesional
a ea ha e gene a ed on si e om NSC o p ogeni o s
ha ha e mig a ed o he inju ed egion and ha e di e -
en ia ed once hey eached he co ex o om ac i a ed
as ocy es ha ha e been ep og ammed as a conse-
quence o he ea men as obse ed ea lie [47, 48] wi h
b ain de i ed neu o ophic ac o (BDNF).
Neu egulin asachemoa ac an o SVZ neu oblas s
In ligh o he abo e-men ioned indings he ques-
ion a ises as o whe he he mig a ion o neu oblas s is
d i en by cell-au onomous mechanisms o media ed by
a chemoa ac an . Knowledge on he mechanisms ha
lead neu oblas mig a ion has been ob ained by s udy-
ing he mig a ion o hese cells owa d he OB. Neu o-
blas s mig a e in chains a e acqui ing an elonga ed
mo phology wi h a long leading p ocess [49, 50]. Acco d-
ing ea lie epo s SVZ neu oblas s a e sessile and need
o ini ia e di e en ia ion in o de o mig a e long dis-
ances [51]. The mechanism o di e en ia ion may be
media ed by he ecep o o he E bB amily E bB4, and
i s ligands neu egulins [42, 52]. NRG2 leads neu oblas s
owa d he OB whe eas NRG1 ac s as a chemoa ac -
an leading neu oblas s owa d he NRG1 sou ce [42].
We ha e ound he e ha in esponse o an inju y, mic o-
glial cells a i ed a he pe ilesional a ea and he SVZ.
As shown in Fig.9, mic oglial cells exp essed NRG1. As
a consequence, NRG1 exp ession inc eased in he SVZ
and co ex as soon as 7 dpi, which was accompanied
by ele a ed concen a ion o soluble NRG1 in he CSF.
Bo h NRG1 exp ession and he soluble ligand e u ned
o no mal le els a 14 dpi. In e es ingly, he exp es-
sion o NRG1 ecep o E bB4 did no inc ease un il 14
dpi, when NRG1 concen a ion had e u ned o no mal
le els. Howe e , he ea men wi h EOF2 main ained
NRG1 concen a ion ele a ed in he CSF a 14 dpi. P e-
ious e idence shows ha EOF2 induced ac i a ion o
Fig. 8 Mo phological and elec ophysiological dis inc ions be ween newly gene a ed neu ons in he uppe and deepe laye s. A The igu e
illus a es he mo phological and elec ophysiological di e ences based on neu on loca ion. Neu ons in he uppe laye s (le ) a e smalle
han hose in he lowe laye s ( igh ). Lowe laye neu ons exhibi mo e, longe , and mo e complex dend i es. Addi ionally, hei i ing pa e ns di e ,
wi h uppe laye neu ons showing highe equency i ing, as indica ed by he equency eco dings and equency‑cu en g aph a he op. The
cen al panel shows he dis ibu ion o eco ded neu ons om he 29–56 days pos inju y (dpi) and 57–90 dpi g oups. Dashed lines illus a e lesion
size changes o e ime: ini ially la ge a 7–14 dpi (black), and p og essi ely smalle wi h ea men [15–28 dpi in ed, 29–56 dpi in blue, 57–90 dpi
in g een]. B‑H Box‑and‑whiske plo s displaying he medians (dashed lines), in e qua ile anges (boxes), minimum/maximum alues (whiske s)
and SEM (e o ba s) o o al su ace a ea (B), dend i e leng h (C), numbe o o al segmen s (D), numbe o e minal endings (E), heobase (F), inpu
esis ance (G), and i ing gain (H) o each g oup. I is ema kable ha neu ons in deepe laye s we e la ge (*p = 0.036), longe (*p = 0.013), and mo e
complex (segmen s, *p = 0.004; endings, *p = 0.034). They had highe heobases (*p = 0.043) and lowe i ing equencies (*p = 0.0034). S uden ’s
‑ es assessed di e ences be ween g oups. As e isks (*) indica e s a is ically signi ican di e ences be ween g oups, wi h a signi icance le el
o p ≤ 0.05
(See igu e on nex page.)

Page 22 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
no el PKCδ p omo es NRG1 elease ia he phospho-
yla ion o he NRG1 p o-ligand [43]. We show in he e
ha p o ein kinase C ac i i y is inc eased in he inju ed
SVZ and co ex up o 14 dpi. This is concomi an wi h
an inc eased exp ession o PKCδ. These esul s suppo
he hypo hesis ha NRG1 elease is s imula ed by EOF2
h ough he ac i a ion o PKCδ. All hese esul s ag ee
wi h hese p e ious epo s [43]. As he abo e e idence
sugges ed, he e is an inc ease in he le els o NRG1 in
ea ed mice, no only because o he ele a ed numbe o
mic oglial cells ha exp ess NRG1 bu also by he no el
PKC-induced NRG1 shedding d i en by EOF2 ea men .
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
300
200
100
0
Inpu Resis ance (MΩ)
Rheobase (pA)
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
400
300
200
100
0
*
VI
V
IV
140 pA
-50 mV -70 mV
340 pA
40000
30000
20000
10000
0
To al su ace a ea (m²)
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
50 ms
20 mV
50 ms
20 mV
To al dend i ic leng h (m)
8000
6000
4000
2000
0
Numbe o Segmen s
80
60
40
20
0
Numbe o e minal endings
50
40
30
20
10
0
A
B
100 m
100 m
CDE
Cells w/ mo phology econs uc ion Cells w/ econs uc ion & Repe i i e Fi ing p ope ies
29 - 56 Inju y
7 - 14 Inju y 15 - 28 Inju y 57-90 Inju y
I
II - III
IV
II - III
IV
V
VI
0
20
40
60
80
100
-1
F equency (AP·s )
80 120 160 200 240 280 320 360
Cu en (pA)
I
II-III
500 μm
*
**
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
*
Uppe
Laye s
I-IV
Bo om
Laye s
V-VI
1000
750
500
250
0
-1 -1
Fi ing Gain (PA·s·nA )
FGH
*
Fig. 8 (See legend on p e ious page.)
Page 23 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
Rega ding he cellula mechanisms in ol ed in he mig a-
ion o neu oblas s. Since we obse e ha hey mig a e in
chains owa ds he inju y, i is possible ha hey mig a e
assis ed by blood essels and eac i e as ocy es as p e-
iously epo ed in inju ies like he ones es ed by us [5,
53].
Newly gene a ed neu ons acqui e physiological
cha ac e is ics o ma u es neu ons ina ime dependen
manne ha obeyed emb yonic/neona al de elopmen
Ou ol age-clamp eco dings e ealed ha newly gen-
e a ed neu ons ecei e exci a o y synap ic inpu s when
hey had jus a i ed o he co ical lesion, indica ing
ha he ne wo k o ma ion was es ablished e y ea ly
and be o e eaching ma u a ion o he ac i e memb ane
p ope ies, as also seen in newly gene a ed neu ons in
den a e g anule cells o he hippocampus [54–59] and
ischemia in he s ia um [16]. The highes equency
alues o sEPSCs we e ound in ou s udy in ZsG een+
newly gene a ed neu ons a 28 dpi and dec eased p o-
g essi ely o each alues a 57–90 dpi simila o hose
ound in py amidal neu ons. These da a seem o indica e
ha mig a ing ZsG een+ cells ecei e inpu om nume -
ous neu ons when hey a i e a he mo o co ex and a
compe i i e selec ion may be occu ing o de ine which
ci cui hey will inally in eg a e in o. An al e na i e
explana ion could be ha he equency o sEPSCs is he
sum o glu ama e gic and GABAe gic e en s a 15–28
dpi. I is known ha GABA ini ially depola izes newbo n
den a e g anule cells in he adul b ain, a depola iza-
ion ha is essen ial o he es ablishmen o unc ional
synapses [60]. The e o e, we can p opose ins ead ha
he equency o sEPSCs would dec ease wi h ime
as GABAe gic cu en s become inhibi o y du ing he
ma u a ion.
Elec ophysiological p ope ies o he newly bo n neu-
ons unde wen changes wi h a simila ime cou se o
he ma u a ion o he unc ional p ope ies as desc ibed
in de eloping py amidal neu ons o he mo o co ex
[61]. The newly gene a ed neu ons ha we ound in he
inju ed mo o co ex exhibi ed imma u e cha ac e is ics,
such as a depola ized es ing memb ane po en ial, highe
inpu esis ance, and a e no able o i e ac ion po en ials
up o 29 dpi. A a la e s age, hey g adually exhibi ed sig-
ni ican ly mo e hype pola ized es ing po en ial, lowe
alues o inpu esis ance and mul iple ac ion po en ials
wi h depola izing pulses. This d i in he es ing mem-
b ane po en ial could be explained by a highe exp es-
sion o he K+/Cl− co anspo e in ma u e neu ons ha
would u n GABAe gic synap ic inpu s om depola iz-
ing o hype pola izing [62], [63].
The educ ion in inpu esis ance could be due o
an enla ged exp ession o he po assium leak channels
and a ise in neu onal size [61, 64]. Ou wa d cu en s
we e p esen in he ea ly s ages o ma u a ion in newly
gene a ed neu ons as epo ed p e iously in cul u es
[65], p eceding he appea ance o inwa d cu en s.
The e inemen in ac ion po en ial shape obse ed
in newly gene a ed neu ons could be mainly gi en by
an inc ease in densi y o he ol age-ga ed po assium
and sodium. This inc ease in cu en densi ies o e
ime esembles he desc ibed changes in a neoco ex
(See igu e on nex page.)
Fig. 9 Ele a ed NRG1 exp ession in he SVZ and co ex o inju ed mice, localized in mic oglial cells and de ec ed in he CSF. A Scheme
o expe imen al p ocedu es. Mechanical co ical lesions we e unila e ally pe o med in he adul mouse p ima y mo o co ex (CTX). Mice we e
sac i iced 7‑ o 14‑days pos inju y (dpi). Only he g oup “14 dpi + EOF2” was in anasally ea ed wi h EOF2 5 µM om he day o he inju y
un il sac i ice. B Mouse ce eb ospinal luid (CSF) we e ob ained om non‑inju e mice (sham) and om inju ed mice a day 7 and day 14 pos ‑inju y
(dpi). The g aph shows neu egulin 1 (NRG1) de ec ion (ng/mL) in CSF. *p = 0.04 Sham s. 7dpi; p = 0.042 Sham s. 14dpi + EOF2 in one‑way ANOVA.
6 animals we e used pe g oup. C Rela i e mRNA exp ession o NRG1, and E bB4 in he inju ed co ex (ipsila e al, ipsi) and in non‑inju ed co ex
(con ala e al, con a) a 7 dpi and a 14 dpi. mRNA exp ession was measu ed using eal ime qPCR. *p = 0.042 7dpi CTX NRG1 (con a s. ipsi).
6 animals we e used pe g oup D Rela i e mRNA exp ession o NRG1 and E bB4 in he sub en icula zone (SVZ) o he inju ed ipsila e al side
and non‑inju ed side 7 dpi and a 14 dpi. mRNA exp ession was measu ed using eal ime qPCR. The da a show he means ± SEM o 6 animals
pe g oup. *p = 0.0472 7dpi SVZ NRG1 con a s. ipsi; *p = 0.0313 14dpi SVZ E bB4 con a s. ipsi. E–H Rep esen a i e con ocal images showing
he CTX (E, F) and SVZ (G, H) o 7 dpi mice. Images we e p ocessed o he immunode ec ion o NRG1. The do ed line indica es he limi
o he lesion (L) o la e al en icle (LV), and he scale ba ep esen s 25 μm. I‑L Rep esen a i e con ocal images showing he colocaliza ion
o NRG1 wi h GFAP (I), Iba1 (J,L) and Nes in (K) in he Co ex CTX (I‑K) and SVZ (L) o 7 dpi mice. Scale ba s ep esen 25 µm. M G aph ep esen s
he numbe o NRG1 cells in he con ala e al co ex compa ed o he pe ilesional a ea o 7 dpi mice. The da a show means ± SEM o 6 animals
pe g oup. S a is ical analysis: *p < 0.0001 compa ed he ipsila e al si e wi h he con ala e al side in a S uden ’s es o equal‑ a iance unpai ed
samples. N G aph shows NRG1 bu den as a pe cen age o he o al SVZ a ea in con ala e al and ipsila e al SVZ o 7 dpi mice. Da a show
Means ± SEM o 6 animals pe g oup. S a is ical analysis: *p < 0.0001 compa ed he ipsila e al si e wi h he con ala e al side in a S uden ’s es
o equal‑ a iance unpai ed samples. O Quan i ica ion o he colocaliza ion o NRG1 wi h GFAP, Iba1 o Nes in in he inju ed co ex o 7 dpi mice.
The da a show he means ± SEM o 6 animals pe g oup; *p < 0.0001 ipsi NRG1+GFAP+ s NRG1+Iba1+; *p = 0.0431 NRG1+GFAP+ s NRG1+Nes in+;
*p < 0.0001 NRG1+Iba1+ s NRG1+Nes in+ in one‑way ANOVA, ollowed by pos hoc Tukey es . P Quan i ica ion o he colocaliza ion o NRG1
wi h Iba1 in he ipsila e al SVZ o 7 dpi mice. Da a a e he mean ± SEM; n = 6 animals pe g oup. Q Pe cen age o he numbe o Iba1+ cells pe mm3
ha also exp ess NRG1 (Iba1+NRG1+ cells pe mm3) in con ala e al and ipsila e al SVZ o 7 dpi mice. n = 6 animals pe g oup
Page 24 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
B
C
Ipsila e al CTX 7dpiCon ala e al CTX 7dpi Ipsila e al SVZ 7dpiCon ala e al SVZ 7dpi
NRG1 in CSF (ng/mL
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Sham
CTX mRNA exp ession
14 dpi CTX7 dpi CTX
NRG1 E bB4 NRG1 E bB4 NRG1 E bB4 NRG1 E bB4
L
D
NRG1
LV
LV
day 0 7 Days
Pos -Inju y
Lesion
A
7 dpi
Con a Ipsi
day 0 14 Days
Pos -Inju y
Lesion 14 dpi
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Sham
SVZ mRNA exp ession
*
*
*
ipsila e al
con ala e al
Ipsila e al SVZ 7dpi
Ipsila e al CTX 7dpi
NRG1
Nes in
L
NRG1
GFAP
L
25 m
NRG1
Iba1
L
LV
14 dpi SVZ7 dpi SVZ
0.0
2.5
5.0
7.5
10.0
12.5
15.0
Sham 7dpi 14dpi 14dpi
+
EOF2
*
E
G
JK
I
L
F
H
+/+ cells (%NRG1+ cells)
in ipsi SVZ
0
20
40
60
80
100
NRG1-Iba1
P
+/+ cells (%NRG1+ cells)
in ipsi CTX
0
20
40
60
80
NRG1-GFAP
NRG1-Iba1
NRG1-Nes in
**
*
O
0
5
10
15
20
25
30
NRG1 bu den (% a ea SVZ)
*
N
Con a Ipsi
*
Con a Ipsi
NRG1
+
cells/mm
3
(CTX)
0
2000
4000
6000
8000
10000
M
Con a Ipsi
0
20
40
60
80
100
Q
Iba1+/NRG1+ cells
(%Iba1+ cells) in ipsi SVZ
25 m
Fig. 9 (See legend on p e ious page.)
Page 25 o 30
Pa dillo‑Díaze al. S em Cell Resea ch & The apy (2025) 16:1
de elopmen [66, 67]. To be e unde s and he na u e
o he cu en s, we pe o med a classical pha maco-
logical cha ac e iza ion. The applica ion o TTX 1μM
abolished comple ely he inwa d componen , indi-
ca ing ha a sodium cu en was esponsible o he
in lec ion o he cu en . Al hough we canno disca d
he idea ha he e we e an unde lying calcium com-
ponen , due o he essen ial ole o calcium cu en s
in ea ly s ages o he de elopmen [68] and he p es-
ence in laye V py amidal neu ons [69]. The candida e
channels esponsible o hese inwa d cu en s could
be NaV1.2 [70] o NaV1.6 [71, 72], which has been
de ec ed in co ical neu ons. Following his, he appli-
ca ion o non-speci ic po assium inhibi o s, such as
TEA (5mM) and 4-AP (2mM), signi ican ly educed
he ampli ude o he cu en s. The non-blocked cu -
en is p obably pa o he leakage cu en s, which a e
insensi i e o hese d ugs, al hough i was p oposed
ha he e a e componen s o po assium cu en s pa -
ially insensi i e o TEA o 4-AP in co ical neu ons
[73, 74]. While in he adul neu ogenic egions (sub-
g anula zone o he hippocampus and SVZ-OB) new
neu ons acqui e ma u e elec ophysiological p ope -
ies wi hin 4weeks [54, 75, 76], he newly gene a ed
neu ons ha we e ound in he do sola e al s ia um
a e ischemia [16] and in ou s udy in he inju ed
mo o co ex displayed epe i i e discha ge o ac ion
po en ials wi hin 12–20 weeks. The e o e, he ime
scale o unc ional ma u a ion in inju ed b ains was
longe han in he adul neu ogenic egions. This di -
e ence migh imply ha he local en i onmen a e
he b ain insul was less a o able o new neu ons due
o he diminu ion o ophic suppo . The e o e, his
hos ile en i onmen o he auma ic b ain inju y i sel
can cause e ec s in he exp ession [77] and cha ac e -
is ics o he biophysical p ope ies o ol age-ga ed ion
channels [78].
Newly gene a ed neu ons de elop mo phological
cha ac e is ics simila o hose o ma u e neu ons
ina ime‑dependen manne , esembling hep ocesses
obse ed du ingemb yonic andneona al de elopmen
The a ailable da a on he de elopmen o dend i ic
ees in newly o med neu ons in adul b ains a e
limi ed, mainly coming om he adul den a e gy us
neu ogenic niche [79, 80]. Neu onal di e en ia ion
in he den a e gy us ollows a pa e n simila o ha
desc ibed in emb yonic s ages. New neu ons acqui e
hei inal mo phological cha ac e is ics o e se e al
mon hs, going h ough s ages o mig a ion, dend i e
and axon o ma ion, dend i ic and axonal g ow h, syn-
apse es ablishmen , and synap ic modi ica ions [59, 76].
In he pi i o m co ex, imma u e (do man ) neu ons
ma u e wi h an inc ease in leng h and complexi y o
he dend i ic ee las ing o 6mon hs [81, 82]. Du -
ing pos na al de elopmen , neu ons in newbo n ani-
mals expe imen mo phological changes as hey ma u e
h oughou his pe iod [29, 34, 83]. Pos na ally, he den-
d i ic a bo o bo h hypoglossal and oculomo o mo o-
neu ons g ows in size and leng h owa ds a eas om
which new synap ic inpu s o igina e, bu he complex-
i y o hei dend i ic a bo is al eady es ablished a bi h
[29, 34] as also desc ibed in hippocampal in e neu ons
[84] o co ical neu ons in he isual o p e on al co -
ex [85, 86]. By con as , in ou s udy mig a ing new
gene a ed neu ons expe ience an inc ease in dend i ic
complexi y. Cells esemble he ypical mo phology o
neu oblas s wi h ounded soma and e y sho den-
d i ic p ocesses when a i ed o he mo o co ex, and
hen became bipola and inally each a dend i ic a bo-
iza ion wi h a dend i ic complexi y ha esembled
ma u e co ical neu ons, a a ime when hey do no
ye display ully inwa d cu en s. We hypo hesize ha
he augmen a ion in dend i ic complexi y may mi -
o a phenomenon ha akes place du ing emb yonic
Fig. 10 Molecula mechanisms unde lying he e ec s o EOF2: ole o NRG1. A Scheme o mChe y‑NRG1‑eGFP cons uc . B Mechanisms
o NRG1‑bound luo escence elease. C Quan i a i e analysis o he mic oscopic images ob ained om he ime‑lapse assays o neu al p ogeni o
cells (NPC) exp essing mChe y‑NRG1‑eGFP and s imula ed wi h EOF2 (5 μM). mChe y/eGFP a ios we e no malized o he a e age mChe y/
eGFP a io measu ed be o e s imula ion. The mean no malized mChe y/eGFP a ios a e shown, n = 3. A leas 10 cells we e analyzed pe condi ion
in each expe imen . D. mChe y luo escence in he cul u e medium o cells ans ec ed wi h he cons uc mChe y/eGFP ea ed wi h EOF2
o 30 and 180 min. E–F mChe y/eGFP a io images o SVZ‑de i ed cul u ed NPC ea ed wi h diluen (con ol) o EOF2 (5 µM) a e shown
in he in ensi y‑modula ed display mode a he indica ed ime poin s. The colo ange goes om ed o blue o ep esen mChe y/eGFP a io.
The uppe and lowe limi s o he a io ange a e shown. Scale ba ep esen s 25 μm. G‑J Rep esen a i e luo escence images o SVZ‑de i ed
cul u ed NPC ea ed wi h diluen (con ol) o he soluble NRG1 ligand (1, 5 o 10 ng/mL). Cells we e g own wi hou g ow h ac o s and allowed
o di e en ia e o 72 h a e ea men . β‑III‑ ubulin ma ke was used o neu onal immunode ec ion ( ed), and glial cells we e iden i ied
by he immunode ec ion o GFAP (g een). To al nuclei we e coun e s ained wi h DAPI (blue). K G aph ep esen s he pe cen age o o al cells
(de ec ed by DAPI nuclea s aining) ha we e posi i e o β‑III‑ ubulin. L G aph ep esen s he pe cen age o o al cells (de ec ed by DAPI nuclea
s aining) ha we e posi i e o GFAP. Da a a e he mean ± SEM o six independen cul u es (n = 6). Di e ences we e de ec ed by one‑way ANOVA
ollowed by he Tukey b es s con ol (*p = 0.0063). All in i o expe imen s we e done in iplica e. A iplica e was conside ed a uni and six
iplica es we e used o he s a is ical analysis
(See igu e on nex page.)