cell-08-00654 July 18, 2020 Time: 19:19 # 1
BRIEF RESEARCH REPORT
published: 21 July 2020
doi: 10.3389/ cell.2020.00654
Edi ed by:
Ande Ma heu,
Biodonos ia Heal h Resea ch Ins i u e
(IIS Biodonos ia), Spain
Re iewed by:
Paul J. Lucassen,
Uni e si y o Ams e dam, Ne he lands
Juan Song,
The Uni e si y o No h Ca olina
a Chapel Hill, Uni ed S a es
*Co espondence:
Juan Manuel Encinas
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
S em Cell Resea ch,
a sec ion o he jou nal
F on ie s in Cell and De elopmen al
Biology
Recei ed: 04 May 2020
Accep ed: 01 July 2020
Published: 21 July 2020
Ci a ion:
Ma ín-Suá ez S, Abiega O,
Ricoba aza A, He nandez-Alcoceba R
and Encinas JM (2020) Al e a ions
o he Hippocampal Neu ogenic
Niche in a Mouse Model o D a e
Synd ome.
F on . Cell De . Biol. 8:654.
doi: 10.3389/ cell.2020.00654
Al e a ions o he Hippocampal
Neu ogenic Niche in a Mouse Model
o D a e Synd ome
So aya Ma ín-Suá ez1, Oihane Abiega1, Ana Ricoba aza2, Rubén He nandez-Alcoceba2
and Juan Manuel Encinas1,3,4*
1The Neu al S em Cell and Neu ogenesis Labo a o y, Achuca o Basque Cen e o Neu oscience, Leioa, Spain, 2Gene
The apy P og am CIMA, IdiSNA, Na a a Ins i u e o Heal h Resea ch, Uni e si y o Na a a, Pamplona, Spain,
3Depa men o Neu osciences, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa,
Spain, 4IKERBASQUE, The Basque Founda ion o Science, Bilbao, Spain
Hippocampal neu ogenesis, he p ocess by which neu al s em cells (NSCs) con inuously
gene a e new neu ons in he den a e gy us (DG) o mos mammals including humans,
is chie ly egula ed by neu onal ac i i y. Thus, se e e al e a ions ha e been ound in
samples om epilepsy pa ien s and in he hippocampal neu ogenic niche in mouse
models o epilepsy. Reac i e-like and gliogenic NSCs plus abe an newbo n neu ons
wi h al e ed mig a ion, mo phology, and unc ional p ope ies a e induced by seizu es in
expe imen al models o empo al lobe epilepsy. Hippocampal neu ogenesis pa icipa es
in memo y and lea ning and in he con ol o anxie y and s ess. I has been he e o e
hypo hesized ha pa o he cogni i e symp oms associa ed wi h epilepsy could
be p omo ed by impai ed hippocampal neu ogenesis. We he e analyze o he i s
ime he al e a ions o he neu ogenic niche in a no el mouse model o D a e
synd ome (DS), a gene ic encephalopa hy wi h se e e epilepsy in in ancy and mul iple
neu ological como bidi ies. Scn1aWT/A1783V mice, he ea e e e ed o as DS, ca ying a
he e ozygous and clinically ele an SCN1A mu a ion (A1783V) ecapi ula e he disease
a he gene ic and pheno ypic le els. We demons a e ha in he neu ogenic niche
o young adul DS mice he e a e ewe NSCs, hey ha e impai ed cell di ision and
bea eac i e-like mo phology. In addi ion, he e is signi ican abe an neu ogenesis.
Newbo n imma u e neu ons mig a e abno mally, and se e al mo phological ea u es a e
d as ically changed. Thus, his s udy shows o he i s ime impo an modi ica ions in
hippocampal neu ogenesis in DS and opens enues o u he esea ch on his opic.
Keywo ds: neu al s em cells, abe an neu ogenesis, gliosis, D a e synd ome, SCN1A
INTRODUCTION
The hippocampus is one o he mos ulne able s uc u es o he b ain o exci o oxici y and
pa icula ly o seizu es due o i s ecu en ci cui s. The hippocampus o mos mammals, including
humans (E iksson e al., 1998;Mo eno-Jiménez e al., 2019), is able o gene a e new neu ons
h ough adul hood and aging h ough a complex p ocess e med adul hippocampal neu ogenesis
(AHN). Howe e , exci o oxici y and seizu es impai AHN wi h se e al consequences: (a) he
neu ons los by exci o oxici y canno be egene a ed; (b) he no mal unc ions o AHN a e
dis up ed; and (c) he no mally neu ogenic neu al s em cells (NSCs) o he hippocampus u n
o pa icipa e in eac i e gliogenesis. The pe sis ence o AHN in he den a e gy us (DG) o he
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Ma ín-Suá ez e al. Hippocampal Neu ogenesis in D a e Synd ome
hippocampus is consequence o a popula ion o NSCs wi h
neu ogenic (Se i e al., 2001) and gliogenic capaci y (Encinas
e al., 2011). NSCs emain quiescen un il hey ge ac i a ed
o en e he cell cycle and hen gene a e neu onal p ecu so s
du ing a sho pe iod o ime. A e se e al weeks o mig a ion
and di e en ia ion, newbo n neu ons ge inally in eg a ed in o
he hippocampal ci cui y modi ying he p ope ies o he p e-
exis en neu onal ne wo k ( an P aag e al., 2002;Aimone e al.,
2014). Newbo n neu ons pa icipa e in he o ma ion o new
memo ies (Fa ioli-Vecchioli e al., 2008); in lea ning (Zhang
e al., 2008;Deng e al., 2009); in he esponses o s ess, anxie y,
and ea (San a elli e al., 2003;Saxe e al., 2006;Be gami e al.,
2008;Zhang e al., 2008;Aimone e al., 2011;Snyde e al.,
2011), as well as in spa ial and objec ecogni ion memo y
(Jessbe ge e al., 2009) and in pa e n sepa a ion (Sahay e al.,
2011;Nakashiba e al., 2012). Sho ly a e being ac i a ed o
gene a e neu onal p ecu so s, NSCs unde go ul ima e gliogenic
(Bonaguidi e al., 2011;Encinas e al., 2011) o neu ogenic
(Pilz e al., 2018) di e en ia ion. Thus, he popula ion o NSCs
ge s deple ed o e ime in an ac i a ion-dependen manne so
ha he popula ion, and he e o e AHN, is maximal du ing
he pos na al-ju enile pe iod, and is minimal in he aged b ain
(So ells e al., 2018).
P e ious s udies based on epilepsy models in adul mice ha e
shown ha AHN can be a ec ed in wo manne s dependen
on he le el o neu onal hype ac i a ion (Biele eld e al., 2019):
Wi h lowe le els o epilep ic ac i i y and epilep i o m ac i i y,
newbo n neu ons a e he cell ype ha esul s mo e a ec ed. They
mig a e abno mally, hey ex end abno mal basal dend i es, and
hei physiological p ope ies a e modi ied (Pa en e al., 1997).
Toge he , his se o al e a ions has been e e ed o as abe an
neu ogenesis. On he o he hand when using models o mesial
empo al lobe epilepsy (MTLE), in which kainic acid is deli e ed
in o he hippocampus (Bouille e e al., 1999;Sie a e al., 2015)
o he amygdala (Mu o-Ga cía e al., 2019) and he epilep ic
ocus is unila e ally localized in his s uc u e, a mo e d ama ic
al e a ion is induced. NSCs abandon hei neu ogenic p og am
and ans o m in o eac i e-NSCs (Reac -NSCs) which ul ima ely
di e en ia e in o eac i e as ocy es (RAs) ha con ibu e o
hippocampal eac i e gliosis (Sie a e al., 2015;Mu o-Ga cía
e al., 2019). As a esul , a small amoun o abe an neu ogenesis
o no neu ogenesis a all is ound (Mu phy e al., 2012;Sie a
e al., 2015;Mu o-Ga cía e al., 2019).
We hypo hesize ha because he popula ion o NSCs and
AHN a e maximal in pos na al and ju enile s ages, he e ec s
o seizu es in in an epilepsy on he neu ogenic niche could
ha e a g ea e impac on hese ea lie s ages han in la e
ones. D a e synd ome (DS, OMIM 607208), i s desc ibed
in 1978 by Cha lo e D a e (D a e , 1978, 2011) and also
known as se e e myoclonic epilepsy o in ancy (SMEI), is a
a e bu se e e o m o epilepsy a ec ing app oxima ely one
in 15,000 bi hs (D a e e al., 2005;Wu e al., 2015). DS
cha ac e izes by he ea ly onse (4–6 mon hs o age) o ecu en
gene alized seizu es in p e iously heal hy child en, ollowed by
equen p olonged seizu es ha a e esis an o an i-epilep ic
d ugs. As a esul , he symp oms include de elopmen al delays,
speech impai men , dysau onomia, nu i ion issues, au ism-like
beha io , and a high a e o sudden unexpec ed dea h in epilepsy
(SUDEP). App oxima ely 10–20% o he a lic ed pa ien s die
p ema u ely (Hu s , 1990;D a e e al., 2005;Jansen e al., 2006;
Gen on e al., 2011;Sakauchi e al., 2011). Almos he 90% o
cases wi h DS p esen de no o mu a ions in he SCN1A gene,
encoding he alpha subuni o a ol age-ga ed sodium channel
ype 1 (Na 1.1) (Oguni e al., 2005;Ko and No dli, 2006;
Ca e all e al., 2008;Depienne e al., 2009;Ma ini e al., 2009;
Mullen and Sche e , 2009). Newe inducible mouse models ha e
been gene a ed ha o e come he d awbacks o he p e ious
cons i u i e models which we e biased owa d he subse o DS
mice ha su i ed and could b eed (Williams e al., 2019). We
he e analyze o he i s ime he al e a ions o he hippocampal
neu ogenic niche in he p e iously cha ac e ized C57BL/6J
knock-in mouse s ain (Scn1aWT/A1783V, DS). These mice ca y
a clinically ele an he e ozygous SCN1A mu a ion (A1783V)
and p esen he ull spec um o DS mani es a ions (Ricoba aza
e al., 2019). Inc eased suscep ibili y o hype he mia-induced
seizu es (applied o minimize a iabili y in he da a de i ed
om spon aneous seizu es) s a s a pos na al week 3, be o e
he pe iod o maximal mo ali y a e a 8 weeks o li e
(Ricoba aza e al., 2019). One o he mechanisms sugges ed
o igge he onse o seizu es a ha age is he inc ease
o Na 1.1 exp ession compensa ing he no mal decline o
Na 1.3 ea ly a e bi h as obse ed in humans (Cheah
e al., 2013). Al hough he physiopa hology o DS is complex
and no ully elucida ed, he mos accep ed mechanism is
he impai ed exci abili y o pa albumin and soma os a in-
exp essing GABAe gic in e neu ons (Tai e al., 2014) which ips
he neu o ansmission balance in a o o exci a ion.
MATERIALS AND METHODS
Fo an ex ended desc ip ion o me hods, see sec ions “Ma e ials
and Me hods” and “Supplemen a y Ma e ials and Me hods” in
Supplemen a y Ma e ial.
Animals
The condi ional Scn1a-A1783V mice [B6(Cg)-Scn1a m1.1Ds /J,
The Jackson Labo a o y, s ock no. 026133] we e b ed o mice
exp essing C e ecombinase unde he con ol o he CMV
p omo e [B6.CTg(CMV-C e)1Cgn/J, The Jackson Labo a o y,
s ock no. 006054]. B eeding pai s consis ed o he e ozygous
male Scn1a-A1783V and homozygous emale CMV-C e mice.
See h ps://www.jax.o g/s ain/026133 o de ails abou allele
modi ica ion and geno yping. O sp ing ca ying one mu a ed
allele (geno ype he eina e e e ed o as Scn1aWT/A1783V and
he mice e e ed o as DS mice h oughou he ex ) exp ess
he A1783V mu a ion in he Scn1a gene in all body issues,
mimicking wha happens in DS. Animals we e housed ou o
six pe cage wi h ee access o ood and wa e , weighed weekly,
and main ained in a empe a u e and ligh con olled (12/12 h
ligh /da k cycle) en i onmen . The s udies we e pe o med
by compa ing he e ozygous ansgenic Scn1aWT/A1783V o age-
ma ched nega i e li e ma es Scn1aWT/WT ( e e ed o as WT
mice h oughou he ex ). B eeding and expe imen al p o ocols
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Ma ín-Suá ez e al. Hippocampal Neu ogenesis in D a e Synd ome
we e app o ed by he E hical Commi ee o he Uni e si y o
Na a a (in acco d wi h he Spanish Royal Dec ee 53/2013).
These animals ha e been p e iously cha ac e ized (Ricoba aza
e al., 2019). Fi e age-ma ched (7 weeks) animals we e
used o each g oup.
Immunohis ochemis y
Immunohis ochemical, image cap u e, and quan i ica ion
echniques we e pe o med essen ially as desc ibed be o e
ollowing me hods op imized o he use in ansgenic mice
(Encinas and Enikolopo , 2008;Encinas e al., 2011;Sie a e al.,
2015;Mu o-Ga cía e al., 2019).
S a is ical Analysis
SigmaPlo (San Jose, CA, Uni ed S a es) was used o s a is ical
analysis. A S uden ’s - es was pe o med in all cases o compa e
da a om WT o DS mice o all quan i ica ions excep o da a
om he 3D-Sholl analysis in which a epea ed-measu es wo-
way ANOVA wi h a Bon e oni pos hoc es was employed. When
a iances we e no homogeneous (by Le ene’s es ) a Mann–
Whi ney ank sum es was used ins ead. This case only happened
o he da a o neu oblas s wi h basal dend i es. Only p<0.05 is
epo ed o be signi ican . Da a a e shown as mean ±s anda d
e o o he mean (SEM).
RESULTS
We i s sough o cha ac e ize he p ope ies o NSCs in WT
and DS mice. Fo ha pu pose, we co-s ained b ain slices
om 7-week-old WT o DS mice wi h an ibodies agains GFAP,
which is exp essed by bo h as ocy es and NSCs, and agains
S100β, which is p esen in as ocy es and RAs bu no in
NSCs (Figu e 1A). We cen e ed he analysis in he g anule
cell laye and he subg anula zone (GCL+SGZ) o u he
assu e he iden i y o NSCs. We i s quan i ied he numbe
o NSCs (GFAP+S100β−-cells in he GCL+SGZ) and ound a
s a is ically signi ican dec eased numbe in he DS compa ed o
he WT mice (Figu e 1B). In con as , he numbe o as ocy es
(GFAP+S100β+-cells) in he a ea o in e es was signi ican ly
inc eased (Figu e 1C). We u he analyzed he mo phology
o NSCs by 3D-Sholl analysis as inc eased mo phological
complexi y is a hallma k o eac i e NSCs (Reac -NSCs). Indeed,
NSCs in he DS mice had clea ly al e ed mo phology wi h
signi ican ly highe complexi y (Figu e 1D and Supplemen a y
Figu e S1A). These esul s sugges ha simila ly o wha is
ound in adul mouse models o MTLE, NSCs also de elop
a eac i e mo phology in DS. As we obse ed an inc eased
numbe o as ocy es and inc eased o e all in ensi y o s aining
o GFAP and S100β(Supplemen a y Figu e S1A) we u he
analyzed he p esence o gliosis. The exp ession o GFAP was
signi ican ly inc eased in he SGZ+GCL as measu ed by he
a ea occupied by GFAP+-pixels (Figu e 1B). We he e o e
used S100βas a speci ic as ocy ic ma ke , because GFAP
is also exp essed in NSCs, and measu ed he a ea occupied
by S100β+-pixels in he GCL+SGZ. We ound ha i was
signi ican ly inc eased in he DS mice compa ed o he WT
ones (Supplemen a y Figu e S1B). The same esul was ound
when we measu ed he in eg a ed densi y o he S100β+-pixels
(Supplemen a y Figu e S1C). The mo phological complexi y o
S100β+-cells measu ed by 3D-Sholl analysis was also highe in
he DS animals e sus he WT (Supplemen a y Figu e S1D).
We also obse ed a sligh inc ease in he span o he GCL,
using DAPI nuclea s aining (Supplemen a y Figu e S1E), and
measu ing he dis ance occupied by nuclei om he hilus o he
molecula laye (Supplemen a y Figu e S1F). GCL dispe sion
no mally accompanies gliosis in human and expe imen al
models o MTLE.
We mo ed nex o in es iga e cell p oli e a ion in he
neu ogenic niche and he cell di ision o NSCs in pa icula .
In his case, we co-s ained b ain slices om 7-week-old WT
and DS mice o GFAP, S100β+, and o Ki67, a ma ke o
cells unde going mi osis (Figu e 2A). The o al numbe o
di iding cells (Ki67+-cells), which we e only ound in he SGZ
(Figu e 2B), was signi ican ly lowe in he DS mice han in he
WT mice (Figu e 2C). The numbe o di iding NSCs was also
diminished in he DS mice (Figu es 2D,E), wi h a signi ican d op
in he p opo ion o he al eady no mally low subse o di iding
NSCs (Figu e 2F). Toge he hese esul s show ha hippocampal
NSCs show eac i e mo phology plus impai ed ac i a ion o
en e he cell cycle in DS mice.
To es whe he hese al e a ions we e also accompanied by
changes in neu ogenesis we s ained b ain slices om 7-week-
old WT and DS mice o doubleco in (DCX) (Figu es 3A,E),
a speci ic ma ke o neu oblas s (imma u e neu ons). We ound
di e en e ec s: The o e all numbe o DCX+neu oblas s
was no signi ican ly changed, al hough a end owa d a
lowe numbe was ound in he DS mice e sus he WT
(Figu e 3B); nume ous neu oblas s we e, howe e , ound in he
hilus o DS mice, which is e y unusual in no mal condi ions
(Figu e 3C). In con as , signi ican ly ewe neu oblas s we e
loca ed o he GCL+SGZ (Figu e 3D). Fu he analysis a
highe magni ica ion (Figu e 3E) allowed he obse a ion o
mo phological al e a ions in neu oblas s o he DS mice. An
inc eased p opo ion o DCX+neu oblas s p esen ed a V-shaped
apical dend i e (de ined as wo dend i es eme ging di ec ly om
he soma o he main one bi u ca ing wi hin he i s 10 µm)
ins ead o he no mal single main dend i e eme ging om he
soma (Figu es 3E,F-le ). In addi ion, a high p opo ion o
hem showed basal dend i es, which neu oblas s a ely ex end in
no mal condi ions (Figu es 3E,F- igh ). Also, DCX+neu oblas s
showed abno mal mig a ion in he DS mice, being dis ibu ed
in he highe laye s o he GCL, owa d he molecula laye
and away om he SGZ whe e hey a e no mally posi ioned
(Figu es 3E,G). Inc eased quan i y o imma u e neu ons wi h
abno mal mig a ion and abno mal mo phology is one o he
hallma ks o abe an neu ogenesis desc ibed in adul models o
MTLE (Pa en e al., 1997). No DCX+neu oblas s we e obse ed
in o he a eas such as he en ho inal co ex o he amygdala in
which cellula plas ici y in he o m o neu ogenesis has been
p oposed o ake place.
Finally, we wan ed o es whe he mic oglia could be also
al e ed in he DG o DS mice as modi ica ions ha e been epo ed
in human and mouse models o MTLE (Abiega e al., 2016).
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FIGURE 1 | Reac i e mo phology o NSCs in DS mice. (A) Rep esen a i e con ocal-mic oscopy images o he DG o WT (le ) and DS ( igh ) mice a e s aining o
GFAP (NSCs and as ocy es) and S100β(as ocy es). DAPI was used o s ain cell nuclei. The numbe o NSCs is signi ican ly dec eased in DS mice (B) bu he
numbe o as ocy es is highe (C). *p<0.05, **p<0.01, by S uden ’s - es (B,C). The mo phology o NSCs was s udied by 3D-Sholl analysis using econs uc ed
cells de i ed om con ocal mic oscopy z-s acks (inse s) and showed an inc ease in he numbe o in e sec ions in DS mice (D) **p<0.01, ***p<0.001 by
epea ed-measu es wo-way ANOVA ollowed by Bon e oni pos hoc es in. Ba s show mean ±SEM. Do s show indi idual da a. n= 5 o each condi ion. Scale
ba in (A) is 20 µm and 10 µmin(B).
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FIGURE 2 | Al e a ions NSC ac i a ion in DS mice. (A) Rep esen a i e con ocal-mic oscopy images o he DG o WT (le ) and DS ( igh ) mice a e s aining o GFAP
(NSCs and as ocy es), S100β(as ocy es), and he p oli e a ion ma ke Ki67. DAPI was used o s ain cell nuclei. Ki67+-cells we e es ic ed o he SGZ in WT and
DS mice (B). Fewe p oli e a ing cells (Ki67+) we e ound in he DS mice (C).(D) High-magni ica ion con ocal-mic oscopy images o di iding NSCs in WT (le ) and
DS ( igh , no e he ami ied mo phology). The o al numbe (E) as well as he p opo ion o di iding (Ki67+) NSCs was lowe in he DS mice (F). *p<0.05 by
S uden ’s - es . n= 5 o each condi ion. Ba s show mean ±SEM. Do s show indi idual da a. Scale ba in (A) is 40 µm, 50 µmin(B) and 5 µmin(D).
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FIGURE 3 | Abe an neu ogenesis in DS mice. (A) Con ocal-mic oscopy images o he DG o WT (le ) and DS ( igh ) immunos ained o DCX (a speci ic ma ke o
neu oblas s) and s ained o DAPI. The e a e no di e ences in he o e all numbe o DCX+-cells (B) bu he e a e mo e neu oblas s in he hilus o DS mice (C) and
co espondingly ewe in he SGZ+GCL (D). In addi ion, neu oblas s in he DS mice p esen ed wi h much highe equency a V-shaped apical dend i e ( wo dend i es
eme ging di ec ly om he soma o he main one bi u ca ing wi hin he i s 10 µm) (E, le ) and basal dend i es (E, igh ), as can be isualized in highe magni ica ion
images (F). The a owheads poin owa d single dend i es eme ging om indi idual neu oblas s whe eas a ows poin owa d wo dend i es eme ging di ec ly om
he soma o bi u ca ing in he mos p oximal segmen . The images in he igh show he abno mal mig a ion o neu oblas s. In he DS mice, DCX+-cells had mig a ed
o uppe laye s o he GCL, close o he molecula laye whe eas in he WT mice hey a e localized in he SGZ (G). *p<0.05, **p<0.01, by S uden ’s - es . n= 5
o each condi ion. Ba s show mean ±SEM. Do s show indi idual da a. Scale ba in (A) is 40 µm and 10 µmin(F) (le ) and 20 µmin(E) ( igh ).
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We s ained b ain slices om 7-week-old WT and DS mice wi h
he speci ic mic oglial ma ke Iba 1 (Figu e 4A) and used he
nuclea s aining DAPI o iden i y apop o ic cells (Figu es 4B,C)
(Sie a e al., 2010). We i s ound ha he numbe o mic oglia,
measu ed in he GCL+SGZ was sligh ly bu signi ican ly educed
in DS mice compa ed o WT (Figu e 4D). Nex , we sough o
es po en ial al e a ions in he unc ional p ope ies o mic oglia
in he neu ogenic niche (Sie a e al., 2010;Abiega e al., 2016)
using phagocy osis as a eadou (Figu e 4C). We i s checked
he numbe o apop o ic cells in he GCL+SGZ (Figu e 4E) as
well as he phagocy ic index (pe cen age o apop o ic cells ha
a e being phagocy osed by mic oglia) (Figu e 4F) and hen he
phagocy osis capaci y dis ibu ion [pe cen age o mic oglia wi h
0, 1, o 2 phagocy osing poaches (Ph)] (Figu e 4G). In e es ingly,
none o hese pa ame e s we e di e en in he DS mice when
compa ed o he WT animals.
DISCUSSION
We he ein desc ibe o he i s ime he al e a ions o he young
adul hippocampal neu ogenic niche in a ecen ly cha ac e ized
mouse model o DS, he Scn1aWT/A1783V mice (Ricoba aza e al.,
2019). This ansgenic line is a condi ional C57BL/6 J knock-in
mouse ha ca ies he he e ozygous and clinically ele an
SCN1A mu a ion (A1783V) and p esen s a ull spec um
o DS mani es a ions. These include cogni i e/beha io al
impai men ; spon aneous in e ic al epilep i o m ac i i y; and
educed h eshold o hea -induced seizu es. Seizu es a e
mul i ocal wi h in a as well as in e hemisphe ic p opaga ion
and gene a e gene alized onic–clonic seizu es. In addi ion, he
Scn1aWT/A1783V mice ha e a 70% mo ali y a e wi hin he i s
8 weeks o age and his is he eason why we chose he 7-week
ime poin as he cen e o ou s udy.
AHN has been shown o pa icipa e in cogni i e asks ela ed
o spa ial memo y and associa i e lea ning (Saxe e al., 2006;
Dup e e al., 2008;Fa ioli-Vecchioli e al., 2008;Imayoshi e al.,
2008;Clelland e al., 2009;Deng e al., 2009), as well as in he
esponses o s ess and dep ession (Snyde e al., 2011). One o
he main egula o s o AHN is neu onal ac i i y and subsequen ly
is g ea ly a ec ed by seizu es. Thus, i has been hypo hesized
ha pa o he cogni i e symp oms ound in epilepsy pa ien s,
such as memo y impai men (Ga ga o e al., 2013), anxie y,
and dep ession (Heuse e al., 2009) could be p o oked o
enhanced by de ec s in AHN. We u he a gue ha he nega i e
e ec o seizu es on AHN can be g ea e i epilep ic seizu es
s a when hippocampal neu ogenesis is maximal, i.e., in ea ly
pos na al pe iods, as i is he case o DS. Ea lie wo ks ocused
mos ly on he al e a ions caused by seizu es on di e en ia ing
newbo n neu ons. Amygdala kindling and piloca pine-induced
epilep ic seizu es induced “abe an neu ogenesis”: neu ons we e
gene a ed in excess, hey mig a ed abno mally (mo ing in o he
hilus and molecula laye ins ead o he GCL) and p esen ed
mo phological anomalies such as basal dend i es (Pa en e al.,
1997, 1998). These pionee ing s udies also ound ec opic GC
neu ons in he hilus and molecula laye in he DG o epilepsy
pa ien s (Pa en e al., 2006). O he s udy, using KA as induc o
o seizu es, sugges ed ha NSCs could also be a ec ed by seizu es,
as hei a e o cell di ision was inc eased, epo ing also ha
gliogenesis was also augmen ed (Hu mann e al., 2003).
We ha e ecen ly shown, using an in ahippocampal and an
in amygdala injec ion o KA as models o MTLE, ha NSCs
a e d ama ically a ec ed by seizu es and ha he neu ogenic
niche is se e ely dis up ed oo (Sie a e al., 2015;Mu o-Ga cía
e al., 2019). We ha e p e iously discussed ha he di e en
le els o neu onal hype exci a ion will a ec he neu ogenic
niche di e en ially (Biele eld e al., 2019): Milde e en s would
ansla e in o a empo a y inc ease o p ogeni o p oli e a ion
wi hou o he changes bu mo e ex eme ones, such as he
case o expe imen al MTLE, can induce NSCs o con ibu e
o eac i e gliosis by con e ing in o RAs a he expense o
abandoning hei neu ogenic p og am. In he DS mice, we ound
ha NSCs p esen ed eac i e mo phology and ha he e was
inc eased as ogliosis. In con as wi h MTLE, NSCs di ided
wi h lowe equency. This inding could be a ibu ed as hem
being in he second phase o educed ac i a ion, due o as oglial
di e en ia ion and neu oin lamma o y ac o s, a e he ea ly
massi e ac i a ion igge ed by he ini ial episodes o seizu es
(Hu mann e al., 2003;Sie a e al., 2015;Mu o-Ga cía e al.,
2019). Fu u e ex ensi e ime-cou se expe imen s and lineage-
acing analysis would help cla i y whe he indeed an ini ial phase
o NSC o e ac i a ion and inc eased as oglial di e en ia ion
akes place. Long- e m s udies will show whe he neu ogenesis
d ops below basal le els and whe he i emains abe an .
A any a e, he le el o eac i e gliosis in he DS model is
lowe han ha ound in MTLE, and i is ac ually pe missi e o
AHN as shown by he p esence o DCX-exp essing imma u e
neu ons (neu oblas s). The o e all numbe o neu oblas s was
no ac ually changed in compa ison o con ols, bu ypical
ea u es o abe an neu ogenesis (Pa en e al., 1997, 1998;
Jessbe ge e al., 2007) we e ound. We specula e ha due o
he con e sion o NSCs in o eac i e-like NSCs wi h educed
a e o cell di ision, neu ogenesis would be deple ed in he long
e m i he animals su i ed mo e han 8 weeks. Rega dless
o he o al numbe s o neu oblas s, i is clea ha hei
mo phology and loca ion a e abno mal. These changes ha e been
shown o co ela e wi h changes in hei synap ic unc ional
p ope ies (Pie ce e al., 2011) and ep esen a majo inding
in his s udy. The ea u es o abe an neu ogenesis desc ibed
he ein a e simila o hose induced by s ess and knockdown
o glucoco icoid ecep o s (Fi zsimons e al., 2013). A his
s age, howe e , and due o lack o s udies on his issue, we
canno make any claim abou his mechanism d i ing abe an
neu ogenesis in his DS model. In spi e o he con o e sy
ega ding he exis ence o neu ogenesis in adul and aged humans
wi h s ong a gumen s in a o and agains —see Lucassen e al.
(2019) and Pe ik and Encinas (2019) o c i ical e iews— he
da a a ailable coincide in ha neu ogenesis is maximal du ing
he ea ly pos na al pe iods, i s weeks in mice, and i s yea s
in humans, exac ly he pe iod in which seizu es s a and a e
mo e equen in DS.
Seizu es in MTLE models also inc ease apop osis in he
neu ogenic niche and neighbo ing egions (Bengzon e al., 1997;
Mu o-Ga cía e al., 2019), and mic oglia has been shown
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 7July 2020 | Volume 8 | A icle 654
cell-08-00654 July 18, 2020 Time: 19:19 # 8
Ma ín-Suá ez e al. Hippocampal Neu ogenesis in D a e Synd ome
FIGURE 4 | Al e a ion o mic oglia in DS mice. (A) Con ocal-mic oscopy images o he DG o WT (le ) and DS ( igh ) immunos ained o Iba 1 (a speci ic ma ke o
mac oglia in he b ain) and s ained o DAPI. DAPI DNA s aining was used o iden i y apop o ic (condensed) nuclei a highe magni ica ion (B). Phagocy osis was
iden i ied as mic oglial pouches ex ended o m mic oglia cells (Iba 1+) wholly engul ing apop o ic nuclei (C). The e we e ewe mic oglia in he SGZ+GCL o DS mice
(D). The numbe o apop o ic nuclei did no change om WT o DS mice (E). The numbe o apop o ic cells being engul ed by mic oglia was no al e ed ei he (F).
The e we e no changes in he p opo ion o mic oglia wi h ei he none, 1, o 2 phagocy osing poaches (Ph) (G). *p<0.05 by S uden ’s - es in (D).n= 5 o each
condi ion. Ba s show mean ±SEM. Do s show indi idual da a. Scale ba in (A) is 40 µm, 10 µmin(B), and 20 µmin(C).
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 8July 2020 | Volume 8 | A icle 654
cell-08-00654 July 18, 2020 Time: 19:19 # 9
Ma ín-Suá ez e al. Hippocampal Neu ogenesis in D a e Synd ome
o become se e ely impai ed (Abiega e al., 2016). In he
Scn1aA1783V mice, we ha e only encoun e ed a sligh dec ease
in he numbe o mic oglia in he neu ogenic niche, bu none o
he unc ional pa ame e s ela ed o phagocy osis we e al e ed.
These esul s a e somehow expec ed, as neu onal loss is no a
p ominen cha ac e is ic in DS (Le Gal e al., 2010;Ha a e al.,
2020), which in u n can be due o he absence o massi e
eac i e gliosis and neu oin lamma ion in he DS hippocampus.
We hypo hesize ha his is a e lec ion o he lowe le el o
neu onal exci o oxici y and neu onal dea h localized in he DG
due o he a iable o igin o seizu es in compa ison wi h he
maximal le el o hype exci a ion and exci o oxici y igge ed by
on-si e hippocampal seizu es ypical o MTLE. Indeed, cell dea h
was no changed in DS mice, p o iding a i s explana ion as o
why mic oglia is mos ly unchanged. In his scena io, as ocy es
and NSCs would be mo e sensi i e o changes in neu onal ac i i y
han mic oglia, an aspec ha emains o be explo ed. The s udy
o he p e ious mouse models o DS was mainly ocused in he
al e a ions o neu onal elec ophysiological p ope ies and he
cha ac e iza ion o he newe inducible models o DS ha e been
ocused on also neu onal ac i i y and beha io asks (Ricoba aza
e al., 2019;Williams e al., 2019). Thus, he mechanisms o
eac i e gliosis and o he induc ion o eac i e NSCs and
abe an neu ogenesis emain unexplo ed as hey ha e only been
ecen ly desc ibed.
DATA AVAILABILITY STATEMENT
The aw da a suppo ing he conclusions o his a icle will be
made a ailable by he au ho s, wi hou undue ese a ion.
ETHICS STATEMENT
The animal s udy was e iewed and app o ed by E hical
Commi ee o he Uni e si y o Na a a.
AUTHOR CONTRIBUTIONS
SM-S pa icipa ed in he expe imen al design, pe o med he
expe imen s and da a analysis, p epa ed he igu es, and co-
w o e he manusc ip . OA pa icipa ed in he expe imen al
design, pe o med he expe imen s and da a analysis, and
p epa ed he igu es. AR pa icipa ed in he expe imen al design,
gene a ed he mice, p o ided he samples, and helped w i ing
he manusc ip . RH-A supe ised sample collec ion and helped
w i ing he manusc ip . JE designed he p ojec , pa icipa ed in
he expe imen al design, pe o med he expe imen s and da a
analysis, p epa ed he igu es, p o ided unding, and w o e he
manusc ip . All au ho s con ibu ed o he a icle and app o ed
he submi ed e sion.
FUNDING
This wo k was suppo ed by Spanish Minis y o Economy and
Compe i i eness (MINECO) G an /Awa d Numbe s SAF-2015-
70866-R (wi h FEDER Funds) and RyC-212-11137 o JE and
RTI2018-097730-B-I00/MCI/AEI/FEDER, UE, and AC17/00029
(ISCIII)/FEDER o RH-A. SM-S ecei ed a Fundación Ta iana
p edoc o al ellowship. OA is he ecipien o a Basque
Go e nmen pos doc o al ellowship.
ACKNOWLEDGMENTS
We hank he es o he Neu al S em Cells and Neu ogenesis
Labo a o y o aluable echnical inpu and discussion.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound online
a : h ps://www. on ie sin.o g/a icles/10.3389/ cell.2020.00654/
ull#supplemen a y-ma e ial
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