ARTICLE OPEN
The amygdala NT3-T kC pa hway unde lies in e -indi idual
di e ences in ea ex inc ion and ela ed synap ic plas ici y
Gianluca Masella
1,2
, F ancisca Sil a
1,2
, Elisa Co i
1,2
, Ga ikoi z Azkona
3
, Ma ia F ancisca Madei a
1,2
, Ângelo R. Tomé
1,4
,
Sami a G. Fe ei a
1,2
, Rod igo A. Cunha
1,5
, Ca los B. Dua e
1,4
and Mónica San os
1,2,6
✉
© The Au ho (s) 2024
Fea - ela ed pa hologies a e among he mos p e alen psychia ic condi ions, ha ing inapp op ia e lea ned ea and esis ance o
ex inc ion as ca dinal ea u es. Exposu e he apy ep esen s a p omising he apeu ic app oach, he e ficiency o which depends on
in e -indi idual a ia ion in ea ex inc ion lea ning, which neu obiological basis is unknown. We cha ac e ized a model o
ex inc ion lea ning, whe eby ea -condi ioned mice we e ca ego ized as ex inc ion (EXT)-success o EXT- ailu e, acco ding o hei
inhe en abili y o ex inguish ea . In he la e al amygdala, GluN2A-con aining NMDAR a e equi ed o LTP and s abiliza ion o ea
memo ies, while GluN2B-con aining NMDAR a e equi ed o LTD and ea ex inc ion. EXT-success mice showed a enua ed LTP,
s ong LTD and highe le els o synap ic GluN2B, while EXT- ailu e mice showed s ong LTP, no LTD and highe le els o synap ic
GluN2A. Neu o ophin 3 (NT3) in usion in he la e al amygdala was su ficien o escue ex inc ion defici s in EXT- ailu e mice.
Mechanis ically, ac i a ion o opomyosin ecep o kinase C (T kC) wi h NT3 in EXT- ailu e slices a enua ed la e al amygdala LTP, in
a GluN2B-dependen manne . Con e sely, blocking endogenous NT3-T kC signaling wi h T kC-Fc chime a in EXT-success slices
s eng hened la e al amygdala LTP. Ou da a suppo a key ole o he NT3-T kC sys em in in e -indi idual di e ences in ea
ex inc ion in oden s, h ough modula ion o amygdala NMDAR composi ion and synap ic plas ici y.
Molecula Psychia y; h ps://doi.o g/10.1038/s41380-024-02412-z
INTRODUCTION
Fea - ela ed diso de s ga he some o he mos commonly
diagnosed psychia ic condi ions including panic diso de (PAND),
se e al phobias and pos auma ic s ess diso de (PTSD). Wi h a
high p e alence wo ldwide, hese diso de s a e highly dis up i e
o he p o essional and social li e o a flic ed indi iduals and
ep esen conside able go e nmen al and socie al cos s [ e iewed
in [1]. Pa ien s in his g oup o diso de s show impai ed associa i e
lea ning o con ex ual and senso y cues ela ed o hei ‘objec ’o
ea [2,3], esul ing in inapp op ia e and/o excessi e ea , and
inabili y o ex inguish maladap i e ea [4]. These impai men s in
ea ex inc ion nega i ely impac on he e ficiency o exposu e
he apy, he fi s line o ea men o pa ien s wi h anxie y and
ea - ela ed diso de s [5,6]. Indeed, in e -indi idual a ia ion in
ea ex inc ion lea ning is p edic i e o exposu e he apy ou come
[7,8]. We a e s ill lacking mechanism-based he apeu ic
app oaches o hose pa ien s wi h impai ed ex inc ion.
Pa lo ian ea condi ioning and ex inc ion ep esen a simple,
ye obus pa adigm o in es iga e he neu al subs a es and
molecula machine y o acquisi ion and ex inc ion o lea ned
ea . This model has been ins uc i e o iden i y he wide
ne wo k o b ain egions ha a e ec ui ed in he p ocessing o
lea ned ea . The amygdala is he co e b ain egion in he
o ches a ion o ea esponse, which in u n is modula ed by
inpu s om he hippocampus, con eying in o ma ion abou he
con ex , and om he medial p e on al co ex (mPFC), p o id-
ing op-down con ol [9–12]. Wi hin he amygdala, wo dis inc ,
ye in e connec ed, popula ions o glu ama e gic neu ons - ea
neu ons and ex inc ion neu ons – o m opposi e ci cui s ha
egula e ea s a es [13–16]. I is known ha ex inc ion does no
e ase he p e iously acqui ed ea memo ies, o he unde lying
mic oci cui ; ins ead, a e e y condi ioned s imulus (CS) p e-
sen a ion, he wo mic oci cui s compe e o exp ess o supp ess
ea [15].
Fea lea ning and ex inc ion a e associa ed wi h al e a ions in
synap ic s eng h h ough long- e m po en ia ion (LTP) and
dep ession (LTD) o synap ic ac i i y [ e iewed in [17], which a e
conside ed he neu ophysiological basis o lea ning and memo y
[18–20]. Acco dingly, LTP and LTD a amygdala synapses a e
belie ed o unde lie acquisi ion e sus ex inc ion o lea ned ea
by ac ing du ing consolida ion and/o econsolida ion o hese
memo ies [21,22]. Indeed, con ex condi ioning induces a
selec i e inc ease in synap ic s eng h o con ex - esponding
basola e al amygdala (BLA) neu ons [23]. On he o he hand,
ex inc ion lea ning was ound o induce LTD a la e al amygdala
(LA) synapses [22]. In e es ingly, ex inc ion o bo h audi o y and
con ex ual ea memo ies e e ses he condi ioning-induced
po en ia ion o BLA neu ons [23,24].
Recei ed: 13 June 2023 Re ised: 29 Decembe 2023 Accep ed: 4 Janua y 2024
1
CNC –Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, Coimb a, Po ugal.
2
Ins i u e o In e disciplina y Resea ch, Uni e si y o Coimb a (iiiUC), Coimb a,
Po ugal.
3
Depa men o Basic Psychological P ocesses and Thei De elopmen , School o Psychology, Uni e si y o he Basque Coun y (UPV/EHU), San Sebas ian, Spain.
4
Depa men o Li e Sciences, Uni e si y o Coimb a, Coimb a, Po ugal.
5
Facul y o Medicine, Uni e si y o Coimb a, Coimb a, Po ugal.
6
Cen e o Inno a i e Biomedicine and
Bio echnology (CIBB), Uni e si y o Coimb a, Coimb a, Po ugal. ✉email: mjpsa[email p o ec ed]
www.na u e.com/mp
Molecula Psychia y
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Amygdala glu ama e ecep o s play a pi o al ole in bo h LTP
and LTD p ocesses ha unde lie ea condi ioning and ex inc ion.
In pa icula , GluN2A- and GluN2B-con aining NMDA ecep o s
(NMDAR) play opposi e oles in ea p ocessing and amygdala
synap ic plas ici y. GluN2A is necessa y o he induc ion o LTP in
he amygdala and is equi ed o he acquisi ion o condi ioned
ea [25]. Fea lea ning induces he ec ui men o GluN2A-
con aining NMDAR o BLA synapses, and hei abundance is
di ec ly co ela ed o he s eng h o he ea memo y [26].
Mo eo e , GluN2A-con aining NMDAR p omo e he es abiliza ion
o ea memo ies in econsolida ion [27]. Con e sely, GluN2B-
con aining NMDAR a e esponsible o amygdala LTD and a e
equi ed o ex inc ion lea ning [25], as well as o he
des abiliza ion o ea memo ies made labile by e ie al [27,28].
Accumula ing e idence sugges s a ole o neu o ophins in he
egula ion o ea [29–32]. In pa icula , he neu o ophin 3 (NT3) -
opomyosin ecep o kinase C (T kC) sys em has been associa ed
o anxie y diso de s in human and non-human p ima es [33–35],
and in mouse models o disease [29,36,37]. In addi ion, ou
ecen s udies unde sco ed he impo ance o T kC signaling in
he o ma ion o ea memo ies in no mal physiological condi ions
[32]. Neu o ophins ha e a c i ical ole in synap ic plas ici y
[38–40] p omp ing hem as candida es o media e he cogni i e-
emo ional egula ion o ea [41].
In his s udy, aking ad an age o in insic in e -indi idual
a ia ion in ea ex inc ion pe o mance, we ha e cha ac e ized a
model o in es iga e he subs a es ha suppo di e ences
be ween mice ha success ully ex inguish ea and hose ha ail.
Using his model, we un eiled a ole o amygdala NT3-T kC
sys em in he egula ion o ea ex inc ion. Ac i a ion o he NT3-
T kC sys em esul s in a enua ion o lea ning-induced LTP, in a
GluN2B-dependen mechanism, which seems pa amoun o
success ul ea ex inc ion.
RESULTS
In e -indi idual a ia ion in con ex ea ex inc ion
Young adul C57BL/6J male mice (n=16) we e ained in he
con ex ual ea condi ioning (CFC) and ex inc ion (EXT) pa adigm
(Fig. 1A). A con ol g oup ha did no ecei e any shock was also
included (CTRL-no shock, n=9). Wi h consecu i e shock admin-
is a ions, condi ioned mice showed a p og essi e inc ease in he
pe cen age o ime spen eezing (Supplemen a y Fig. S1A) and,
when es ed 24 h la e o ea e ie al showed a p ope
condi ioned ea esponse (Supplemen a y Fig. S1B). A e ea
e ie al, mice we e immedia ely ained in ea ex inc ion
acquisi ion. He e, ex inc ion lea ning pe o mance was defined
o each indi idual as he a io (exp essed as pe cen age) be ween
eezing le els in he las ial o ex inc ion acquisi ion (E6) and
hose shown in ea memo y e ie al/fi s ial o ex inc ion
acquisi ion (R/E1). Ex inc ion lea ning pe o mance was used o
e alua e ex inc ion and ca ego ize mice as EXT-success (>30%
educ ion in eezing le els a E6 ela i e o R/E1) o EXT- ailu e
(<30% o no educ ion in eezing).
Th oughou ex inc ion session ials (R/E1 o E6), EXT-success
animals showed a educ ion in eezing o le els compa able o
hose o CTRL-no shock animals, while EXT- ailu e mice inc eased
hei eezing le els (Fig. 1B, ex inc ion ial x g oup in e ac ion
F
(10, 110)
=2.862, p=0.003; R/E1 s. E6, EXT-success =8.675,
p=0.0048; EXT- ailu e =3.215, p=0.0454). In pa icula , in R/E1
ial, bo h EXT-success and EXT- ailu e condi ioned mice oze
significan ly mo e han CTRL-no shock mice (CTRL-no shock s.
EXT-success =4.618, p=0.012, CTRL-no shock s. EXT- ailu e
=6.400, p< 0.001, EXT-success s. EXT- ailu e =0.7485,
p=0.481), showing he p ope o ma ion o a con ex ual ea
memo y. By ial E6, EXT-success mice showed eezing le els
compa able o hose o CTRL-no shock mice, and significan ly
lowe han EXT- ailu e mice (CTRL-no shock s. EXT-success
=1.490, p=0.177, CTRL-no shock s. EXT- ailu e =5.727,
p< 0.001, EXT-success s. EXT- ailu e =2.947, p=0.039). O e all,
EXT-success mice showed an a e age educ ion in eezing le els
o mo e han 40% ela i e o he le els shown in R/E1, as opposed
o a 45% inc ease in eezing le els exhibi ed by EXT- ailu e
animals (Fig. 1C, =4.664, p< 0.001).
In he ex inc ion memo y e ie al phase (EM), EXT-success
animals showed s a is ically significan lowe le els o eezing as
compa ed o R/E1 session (Fig. 1B; ex inc ion session x g oup
in e ac ion, F
(2, 22)
=9.566, p=0.0010; EXT-success, R/E1 s EM
=4.537, p=0.0005), while EXT- ailu e animals did no show a
dec ease in he eezing le els om R/E1 o EM (EXT- ailu e, R/E1
s EM =0.6425, p=0.8943). Ex inc ion memo y pe o mance
(EMP) was defined as he a io (exp essed as pe cen age) be ween
eezing le els in ex inc ion e ie al and hose shown in R/E1.
EXT-success mice showed a be e ex inc ion memo y pe o -
mance as compa ed o EXT- ailu e animals when es ed o
ex inc ion e ie al 24 h la e . He e, EXT-success animals showed
an a e age 45% educ ion in eezing le els ela i e o he le els
shown in R/E1, as opposed an a e age 9% inc ease in eezing
exhibi ed by EXT- ailu e animals (Fig. 1D, U =0, p< 0.001).
Impo an ly, we de ec ed a s a is ically significan co ela ion
be ween eezing le els in E6 and eezing le els in he ex inc ion
e ie al session (Fig. 1E; R
2
=0.5647, p< 0.001), sugges ing ha
ex inc ion lea ning by E6 is p edic i e o ex inc ion memo y
pe o mance.
The p e ious co ela ion sugges s ha indi idual di e ences in
ex inc ion could be a s able ai o he animals, which could be
p edic ed by o he beha io al ai s. To e alua e his hypo hesis,
we in es iga ed how ea ex inc ion and abili y o lea n o
ex inguish ea memo ies could be co ela ed wi h indi idual
di e ences in ai anxie y. Basal anxie y-like beha io and
explo a o y ac i i y we e assessed in he open field (OF) and
ele a ed plus maze (EPM) es s, be o e pe o mance in he CFC
and EXT pa adigm. In he OF, no di e ences we e obse ed
among EXT-success, EXT- ailu e and CTRL-no shock mice in he
o al dis ance a eled no in he pe cen age o ime spen o
dis ance a eled in he cen e o he a ena (Supplemen a y
Fig. S1C–E). Also, no di e ences we e ound among he di e en
g oups in he EPM in he o al dis ance a eled, pe cen age o
open a ms ime o pe cen age o open a ms dis ance (Supple-
men a y Fig. S1F–H). We ound howe e ha basal anxie y le els
a e p edic i e o he s eng h o he ea memo y, bu no o
lea ning pe o mance i sel . Indeed, a s a is ically significan
nega i e co ela ion was ound be ween he pe cen age o ime
spen in he open a ms (OAT), as well as he dis ance a eled in
he open a ms (OAD), and eezing le els in R/E1 and E6 ials o
ex inc ion acquisi ion (Fig. 1F, OAT s. R/E1, R
2
=0.2916, p=0.030;
OAT s. E6, R
2
=0.2809, p=0.035; OAD s. R/E1, R
2
=0.2809,
p=0.035; OAD s. E6, R
2
=0.2025, p=0.077), bu no wi h
ex inc ion lea ning pe o mance and ex inc ion memo y pe o -
mance (Fig. 1F, OAT s. ELP, R
2
=0, 0064, p=0.757; OAT s. EMP,
R
2
=0.0225, p=0.590; OAD s. ELP, R
2
=0, 0009, p=0.913; OAT
s. EMP, R
2
=0.0121, p=0.679).
Success ul ex inc ion lea ning is associa ed wi h weak LTP and s ong
LTD a LA synapses. Synap ic plas ici y in he LA ep esen s a
cellula co ela e o ea condi ioning and ex inc ion [24,42]. In
pa icula , ea condi ioning has been associa ed wi h LTP a LA
synapses [43], while ea ex inc ion is associa ed wi h LTD in he
same egion [22]. He e, we in es iga ed whe he di e ences in
ea ex inc ion lea ning, as hose shown by EXT-success and EXT-
ailu e mice, a e suppo ed by di e ences in LA synap ic plas ici y.
Fea -condi ioned mice we e ained in he EXT pa adigm and
ca ego ized as EXT-success o EXT- ailu e, acco ding o hei
ex inc ion lea ning pe o mance (Supplemen a y Fig. S2A, B).
Ho izon al b ain slices including he amygdala we e ob ained om
EXT-success (n=6 slices om 6 mice) and EXT- ailu e (n=5 slices
G. Masella e al.
2
Molecula Psychia y
om 5 mice) mice a ex inc ion consolida ion window ( wo hou s
a e E6 ial), and elec ical s imula ion and elec ophysiological
eco dings we e pe o med in he LA (Fig. 2A).
No di e ences we e obse ed in he inpu /ou pu (I/O) p ofiles
be ween EXT-success and EXT- ailu e slices (Supplemen a y
Fig. S2C), demons a ing ha he wo g oups showed a simila
LA basal exci abili y. High- equency s imula ion (HFS) p o ocol
( h ee ains o 100 Hz pulses, 1 s du a ion, 5 s in e als)
success ully induced LTP in LA synapses o bo h EXT-success
and EXT- ailu e slices, as shown by an inc ease in popula ion
spikes (PS) ampli ude bo h in he fi s 10 min pos -HFS and in he
las 10 min o eco dings, as compa ed o baseline (Fig. 2C; EXT-
success, baseline s. min 1–10, =2.948 p=0.0146, baseline s.
min 36–45, =2.513 p=0.0307; EXT- ailu e, baseline s. min 1–10,
Fig. 1 In e -indi idual a ia ion in con ex ea ex inc ion. A Schema ic ep esen a ion o he Pa lo ian con ex ual ea condi ioning and
ex inc ion pa adigm o which young adul (8 o 12 weeks old) C56BL/6J male mice we e submi ed h oughou his s udy. BQuan ifica ion o
he pe cen age o ime spen eezing du ing ea ex inc ion acquisi ion and ex inc ion memo y e ie al. Fea -condi ioned mice we e
ca ego ized as EXT-success (n=5) o EXT- ailu e (n=11) acco ding o hei ex inc ion lea ning pe o mance. A CTRL-no shock g oup was
included ha ecei ed no shocks (n=9). Repea ed measu es wo-way ANOVA wi h ei he Sidak o Tukey mul iple compa isons es . * CTRL-no
shock s. EXT- ailu e; £ CTRL-no shock s. EXT-success; # EXT-success s. EXT- ailu e; * EXT-success, R/E1 s. E6 and R/E1 s. EM; * EXT- ailu e, R/
E1 s. E6. CEx inc ion lea ning pe o mance, exp essed as he pe cen age o eezing le els in E6 ela i e o eezing in R/E1. The do ed line
ma ks he h eshold o 30% educ ion om R/E1 used o ca ego ize mice as EXT-success o EXT- ailu e. *** wo- ailed S uden ’s es .
DEx inc ion memo y pe o mance, exp essed as he pe cen age o eezing le els in ex inc ion e ie al ela i e o eezing in R/E1. ***
Mann–Whi ney U es . ECo ela ion o he eezing le els du ing he las ial o ex inc ion acquisi ion (E6) wi h he eezing le els in
ex inc ion e ie al. Pea son . FCo ela ion ma ix o he o al dis ance a eled, dis ance in open a ms and ime spen in he open a ms in he
EPM es wi h he pe cen age o ime spen eezing du ing ex inc ion acquisi ion ials and wi h ELP and EMP; s a is ics using Pea son . CS
condi ioned s imulus, E1 o E6 ex inc ion ials, EM ex inc ion memo y e ie al, ELP ex inc ion lea ning pe o mance, EMP ex inc ion memo y
pe o mance, EPM ele a ed plus maze, R ea e ie al, US uncondi ioned s imulus.; *
,£,#
p≤0.05, **p≤0.01, ***p≤0.001.
G. Masella e al.
3
Molecula Psychia y
=3.141 p=0.0138, baseline s. min 36–45, =2.313 p=0.0495).
Howe e , slices om EXT-success mice showed a s a is ically
significan weake po en ia ion han hose om EXT- ailu e mice
in he fi s 10 min a e HFS (Fig. 2D, =2.383, p=0.0410).
Di e ences in LTP we e los in he las 10 min o eco dings
(Fig. 2E, =1.532, p=0.1592), sugges ing ha al e a ions in he
LTP induc ion phase unde lie di e ences in EXT-success and EXT-
ailu e beha io al pe o mance.
Low- equency s imula ion (LFS, 900 s imuli a 1 Hz) success ully
induced LTD in EXT-success slices, causing a dec ease in PS
ampli ude bo h in he fi s 10 min pos -LFS and in he las 10 min
o eco dings, as compa ed o baseline (Fig. 2G; EXT-success, base-
line s. min 1–10, =7.391 p< 0.001, baseline s. min 36–45
=4.611 p=0.001). The same p o ocol did no induce LTD in EXT-
ailu e slices (Fig. 2G; EXT- ailu e, baseline s. min 1–10,
=2.764 p=0.0506, baseline s. min 36–45 =0.6347
p=0.6347). B ain slices om EXT-success mice showed lowe PS
ampli ude han hose om EXT- ailu e mice in he fi s 10 min
pos -LFS (Fig. 2H, =2.614, p=0.0347; EXT-success n=6 slices
om 5 mice, EXT- ailu e n=3 slices om 3 mice), which was
main ained in he las 10 min o eco dings (Fig. 2I, U =0,
p=0.0238).
Fig. 2 EXT-success mice show a enua ed LTP and inc eased LTD in LA synapses. A Schema ic ep esen a ion o expe imen al condi ions
and localiza ion o s imula ing and eco ding elec odes in ho izon al slices con aining he LA. B–ELTP was induced ex- i o in he LA o EXT-
success (n=6) and EXT- ailu e (n=5) b ain slices wi h a HFS p o ocol ( h ee 1 s du a ion ains o 100 Hz pulses, wi h 5 s in e - ain in e al;
e ical dashed line). BRep esen a i e aces o EXT-success and EXT- ailu e slices a baseline (
__
), upon 10 min (
…
) and upon 45 min (---) a e
HFS. CTime cou se o LTP eco ded o 45 min ollowing LTP induc ion. PS ampli ude was a e aged o he (D)fi s 10 min and (E) las 10 min
o eco dings ollowing LTP induc ion. F–ILTD was induced ex- i o in he LA o EXT-success (n=6) and EXT- ailu e (n=3) b ain slices wi h a
low- equency s imula ion p o ocol (900 s imuli a 1 Hz; be ween he wo e ical lines). FRep esen a i e aces o EXT-success and EXT-
ailu e slices a baseline (
__
), upon 10 min (
…
) and 45 min (---) o LTD induc ion wi h he LFS ain. GTime cou se o LTD eco ded o 45 min
ollowing induc ion. PS ampli ude was a e aged o he (H)fi s 10 min and (I) las 10 min o eco dings. D,E,H,ITwo- ailed S uden ’s es
and Mann–Whi ney U es . BLA basola e al amygdala, Ce cen al amygdala, E6 ex inc ion ial 6, HFS high- equency s imula ion, LA la e al
amygdala, LFS low- equency s imula ion, PS popula ion spikes. *p≤0.05.
G. Masella e al.
4
Molecula Psychia y
Taken oge he wi h he beha io al da a, hese esul s show
ha indi iduals wi h na u ally occu ing di e ences in ea
ex inc ion ha e di e en synap ic plas ici y p ope ies in he LA.
Amygdala GluN2A- and GluN2B-con aining NMDA ecep o s unde -
lie g oup di e ences in ea ex inc ion. A he molecula le el,
amygdala GluN2A- and GluN2B-con aining NMDAR play sepa a e
oles in ea condi ioning and ex inc ion, and in he unde lying
synap ic plas ici y e en s. In pa icula , GluN2A has been
associa ed o ea lea ning and LTP, while GluN2B has been
associa ed o ea ex inc ion and LTD [25,44]. Mo eo e , while
GluN2A/B a e ela ed wi h he induc ion phase o LTP and LTD,
AMPA ecep o s a e linked o he main enance o basal and
po en ia ed synap ic ansmission [45,46]. We in es iga ed
whe he changes in he su ace densi y o glu ama e ecep o s
s and on he basis o beha io al and cellula di e ences be ween
EXT-success and EXT- ailu e animals. To add ess his ques ion,
ea -condi ioned mice we e ained in he EXT pa adigm and
ca ego ized as EXT-success o EXT- ailu e, acco ding o hei
ex inc ion lea ning pe o mance (Supplemen a y Fig. S3A, B). Two
hou s a e E6 ial, a ex inc ion consolida ion window, amygdalae
synap oneu osomes we e isola ed om EXT-success and EXT-
ailu e mice (Fig. 3A) and s ained o su ace GluN2A- and GluN2B-
con aining NMDAR (Fig. 3B, E) and GluA1- and GluA2-con aining
AMPA ecep o s (AMPAR) (Supplemen a y Fig. S3C, F).
We obse ed ha , as compa ed o EXT-success mice, synap o-
neu osomes isola ed om he amygdalae o EXT- ailu e animals
showed a highe in ensi y o GluN2A signal (Fig. 3C, U =180587,
p=0.0482; synap oneu osomes: EXT-success n=631, EXT- ailu e
n=612) and a highe pe cen age o GluN2A-posi i e synap o-
neu osomes (Fig. 3D, =3.312, p=0.0162; synap oneu osomal
p epa a ions: EXT-success n=4, EXT- ailu e n=4). In u n, he
in ensi y o GluN2B signal was highe in EXT-success han in EXT-
ailu e amygdalae synap oneu osomes (Fig. 3F, U =113552,
Fig. 3 Di e en ial su ace densi y o GluN2A and GluN2B subuni s o NMDA ecep o s in amygdala synap oneu osomes om EXT-
success and EXT- ailu e mice. A Schema ic ep esen a ion o expe imen al condi ions and biologic ma e ial used. B,ERep esen a i e images
o synap oneu osomes isola ed om he amygdalae o EXT-success and EXT- ailu e mice, li e s ained o GluN2A and GluN2B subuni s o
NMDA ecep o s. Synap oneu osomes we e iden ified wi h co-s aining agains he pos synap ic ma ke PSD95 and he p esynap ic ma ke
VGlu 1 and inspec ion o in ac memb anes by phase con as . C,FIn eg a ed densi y o GluN2A and GluN2B signal was quan ified in
synap oneu osomes isola ed om he amygdalae o EXT success (GluN2A n=631, GluN2B n=529) and EXT- ailu e (GluN2A n=612; GluN2B
n=518) mice. D,GThe pe cen age o GluN2A- and GluN2B-posi i e amygdala synap oneu osomes was calcula ed o EXT-success and EXT-
ailu e animals (n=4 independen expe imen s). C,FMann–Whi ney U es , (D,G) wo- ailed S uden ’s es . E6 ex inc ion ial 6, GluN2A
subuni 2A o NMDA ecep o , GluN2B subuni 2B o NMDA ecep o , PSD95 pos synap ic densi y 95, VGluT1 esicula glu ama e anspo e 1.
Scale ba 0.5 µm. *p≤0.05, ***p≤0.001.
G. Masella e al.
5
Molecula Psychia y
p< 0.001; synap oneu osomes: EXT-success n=529, EXT- ailu e
n=518). No di e ences we e obse ed in he pe cen age o
GluN2B-posi i e synap oneu osomes isola ed om he amygdalae
o EXT-success and EXT- ailu e animals (Fig. 3G, =1.268,
p=0.2607; synap oneu osomal p epa a ions: EXT-success n=3,
EXT- ailu e n=4).
Fo AMPA ecep o s, we did no obse e any di e ence in he
pe cen age o GluA1- and GluA2-posi i e synap oneu osomes, no
in he in ensi y o GluA1 and GluA2 signals, be ween EXT-success
and EXT- ailu e mice (Supplemen a y Fig. S3C–H).
O e all, in e indi idual di e ences in ea ex inc ion lea ning
e idenced using a wi hin-session con ex ual ea ex inc ion
model a e co obo a ed by cellula and molecula subs a es
o lea ning, and add o he pool o ools al eady
a ailable o in es iga e he neu al and molecula mechanisms o
ex inc ion.
Amygdala T kC ac i a ion co ela es wi h success ul ea ex inc ion.
In p e ious s udies, we ha e shown he in ol emen o NT3-T kC
pa hway in he egula ion o ea memo y o ma ion and
ex inc ion in bo h pa hological and no mal physiological condi-
ions [29,32,37]. Taking ad an age o his p e ious knowledge,
we in es iga ed he ole o he NT3-T kC pa hway in ou ea
ex inc ion model.
Fea -condi ioned mice we e ained in he EXT pa adigm and
ca ego ized as EXT-success o EXT- ailu e, acco ding o hei ea
ex inc ion lea ning pe o mance (Supplemen a y Fig. S4A, B). Two
hou s a e E6 ial, wi hin he ex inc ion consolida ion window,
mice we e killed and he amygdalae, hippocampi, and PFC b ain
egions we e dissec ed (CTRL-no shock n=7; EXT-success n=7;
EXT- ailu e n=14). Wes e n blo s o o al p o ein ex ac s we e
pe o med o measu e T kC ac i a ion and densi y (Fig. 4A, B,
Supplemen a y Fig. S4).
In he amygdala, we obse ed an inc ease in ela i e T kC
ac i a ion, as measu ed by pT kC/ ull-leng h T kC a io, in EXT-
success mice as compa ed o CTRL-no shock and EXT- ailu e mice
(Fig. 4C; F
(2, 25)
=5.121, p=0.0137; CTRL-no shock s. EXT-success
q=4.101, p=0.0202, CTRL-no shock s. EXT- ailu e q=0.8053,
p=0.8374, EXT-success s. EXT- ailu e q=3.931, p=0.0266).
Al hough no s a is ically significan , he same end was obse ed
in he o al le els o phospho yla ed T kC (Fig. 4D; F
(2, 25)
=3.685,
p=0.040; CTRL-no shock s. EXT-success q=3.350, p=0.065,
CTRL-no shock s. EXT- ailu e q=0.4027, p=0.956, EXT-success
s. EXT- ailu e q=3.466, p=0.054). No di e ences we e obse ed
in he o al le els o ull-leng h T kC (Fig. 4E; F
(2, 25)
=1.917,
p=0.1680).
In he PFC, we obse ed a dec ease in pT kC/ ull-leng h T kC
a io in EXT-success mice as compa ed wi h EXT- ailu e
Fig. 4 Amygdala T kC ac i a ion is associa ed wi h a success ul ea ex inc ion pe o mance. A Schema ic ep esen a ion o expe imen al
condi ions and dissec ed b ain egion. BRep esen a i e images o wes e n blo s o phospho yla ed T kC and o al T kC pe o med in b ain
ex ac s om he amygdala. The panel shows non-con iguous lanes om he same memb ane. Quan ifica ion o (C) ela i e T kC ac i a ion as
measu ed by pT kC/ ull-leng h T kC a io, (D) o al pT kC le els, (E) o al ull-leng h T kC le els, (F) ull-leng h T kC/ unca ed T kC a io and (G)
unca ed T kC le els. CTRL-no shock (n=7), EXT-success (n=7) and EXT- ailu e (n=14) mice. β-ac in was used as a loading con ol. C–GOne-
way ANOVA wi h Tukey’s mul iple compa isons es . CTRL con ol g oup, E6 ex inc ion ial 6, pT kC phospho yla ed T kC, T kC opomyosin
ecep o kinase C. *p≤0.05.
G. Masella e al.
6
Molecula Psychia y
(Supplemen a y Fig. S4D). Di e ences we e no due o dec eased
le els o pT kC, as no changes we e obse ed in any o he g oups
(Supplemen a y Fig. S4E), bu o di e ences in ull-leng h T kC
densi y, inc eased in EXT-success as compa ed wi h EXT- ailu e
mice (Supplemen a y Fig. S4F).
In he hippocampus, we did no obse e any di e ence among
g oups in T kC ac i a ion as measu ed by pT kC/ ull-leng h T kC
a io (Supplemen a y Fig. S4J), o al pT kC le els (Supplemen a y
Fig. S4K) o o al T kC le els (Supplemen a y Fig. S4L).
The unca ed iso o m o T kC lacking he phospho yla ion
domain can ac as a dominan nega i e, dime izing wi h ull-
leng h T kC and p e en ing i s ac i a ion [47]. O e all, no
di e ences we e obse ed in he ull-leng h T kC/ unca ed T kC
a io, no in he densi y o unca ed T kC in he amygdala
(Supplemen a y Fig. S4F, G), PFC (Supplemen a y Fig. S4G, H) o
hippocampus (Supplemen a y Fig. S4M, N).
Amygdala NT3-T kC signaling escues ea ex inc ion defici s. Nex ,
we es ed whe he he e is a causal link be ween T kC ac i a ion in
he amygdala and ea ex inc ion pe o mance. To his end, mice
we e bila e ally implan ed wi h cannulas posi ioned abo e he
BLA and ained in he CFC and EXT pa adigm. A he ex inc ion
consolida ion window, EXT- ailu e mice we e in used wi h NT3
and es ed o ex inc ion memo y (Fig. 5E).
To confi m ha NT3 in usion a a dose o 1 µg/µL selec i ely
ac i a es T kC, NT3 was in used unila e ally (con ala e al side was
sham-manipula ed) in a fi s ba ch o uncondi ioned animals
(n=4) and amygdalae we e collec ed 15 min la e . Wes e n blo
analysis showed an inc ease in he le els o pT kC in he NT3-
in used amygdalae as compa ed o he con ala e al no -in used
amygdalae (Fig. 5A, B, =6.486, p< 0.001). Conside ing ha NT3
can also bind o T kB ecep o s, e en hough wi h low a fini y [48],
we measu ed T kB ac i a ion le els. We did no obse e
Fig. 5 NT3 in usion in he BLA escues ea ex inc ion defici s in EXT- ailu e mice. Rep esen a i e images o wes e n blo s o (A) pT kC and
(C) pT kB pe o med using p o ein ex ac s om NT3-in used amygdalae e sus con ala e al no -in used amygdalae (n=4 pe condi ion).
Quan ifica ion o o al (B) pT kC and (D) pT kB le els. β-ac in was used as a loading con ol. Two- ailed S uden ’s es . ESchema ic
ep esen a ion o expe imen al condi ions and ea men s. FSchema ic ep esen a ion o cannulas placemen in he BLA. Ci cles ep esen he
ip o he in e nal cannula. Mice wi h misplaced cannulas we e excluded om he analysis (EXT-success n=2, EXT- ailu e n=1).
GQuan ifica ion o he pe cen age o ime spen eezing du ing ex inc ion acquisi ion session. Fea -condi ioned mice we e ca ego ized as
EXT-success (n=6) o EXT- ailu e (n=12), acco ding o hei ex inc ion lea ning pe o mance. Repea ed measu es wo-way ANOVA wi h Tukey
mul iple compa isons es . * EXT-success, R/E1 s. E6. HEx inc ion lea ning pe o mance, exp essed as he pe cen age o eezing le els in E6
ela i e o eezing in R/E1. Mann–Whi ney U es . IEx inc ion memo y pe o mance, exp essed as he pe cen age o eezing le els in
ex inc ion memo y e ie al ela i e o eezing in R/E1. The do ed line ma ks he h eshold o 30% educ ion om R/E1 used o ca ego ize
mice as EXT-success o EXT- ailu e. One-way ANOVA wi h Tukey mul iple compa isons es . E1-E6, ex inc ion ial 1 o 6; ELP, ex inc ion
lea ning pe o mance; EMP, ex inc ion memo y pe o mance; NT3, neu o ophin 3; pT kC, phospho yla ed opomyosin ecep o kinase C;
pT kB, phospho yla ed opomyosin ecep o kinase B; R/E1, ea e ie al/ex inc ion ial 1. *p≤0.05, **p≤0.01, ***p≤0.001.
G. Masella e al.
7
Molecula Psychia y
al e a ions in he le els o pT kB in he NT3-in used e sus
con ala e al no -in used amygdalae (Fig. 5C, D, =0. 07351,
p=0.9887). Taken oge he , da a confi m ha a a dose o 1 µg/µL
NT3 in usion in he BLA selec i ely ac i a es T kC ecep o .
Nex , a second ba ch o cannula-implan ed mice was ea
condi ioned and ained in he EXT pa adigm, and ca ego ized as
EXT-success o EXT- ailu e, acco ding o hei ex inc ion lea ning
pe o mance. Th oughou ex inc ion acquisi ion ials, EXT-suc-
cess, bu no EXT- ailu e mice, showed a educ ion in hei eezing
le els (Fig. 5G, ex inc ion ial x g oup in e ac ion F
(5, 80)
=7021,
p< 0.001, R/E1 s. E6, EXT-success =4.185, p< 0.001; EXT- ailu e
=2.052, p=0.1989). In pa icula , in R/E1 ial bo h EXT-success
and EXT- ailu e mice showed simila obus le els o eezing (EXT-
success s. EXT- ailu e, =0.7927, p=0.9696), showing he p ope
o ma ion o a con ex ual ea memo y. By E6, EXT-success mice
showed an a e age 44% educ ion in eezing le els ela i e o he
le els shown in R/E1, as opposed o a e age 35% inc ease in
eezing le els by EXT- ailu e mice (Fig. 5H, U =0, p< 0.001).
Two hou s a e E6, coinciding wi h he ex inc ion consolida ion
window, a subse o EXT- ailu e animals was bila e ally in used
wi h NT3 in o he BLA. EXT- ailu e no -in used and EXT-success
animals we e sham manipula ed. Impo an ly, when es ed o
ex inc ion e ie al 24 h la e , EXT- ailu e NT3-in used animals
showed an imp o ed ex inc ion memo y pe o mance (60%
educ ion in eezing le els) as compa ed o EXT- ailu e sham
animals (1% educ ion in eezing le els), and compa able o ha
o EXT-success animals (67% educ ion in eezing le els) (Fig. 5I,
F
(2, 15)
=12.47, p< 0.001; EXT-success s. EXT- ailu e q=6.404,
p=0.001, EXT-success s. EXT- ailu e-NT3 q=0.6208, p=0.900,
EXT- ailu e s. EXT- ailu e-NT3 q=5.783, p=0.003; n=6 pe
g oup).
This se o expe imen s shows ha ac i a ion o he NT3-T kC
pa hway in he BLA du ing ex inc ion consolida ion is su ficien o
escue ea ex inc ion defici s.
NT3-T kC signaling modula es LTP s eng h in a GluN2B-dependen
mechanism. Finally, we sough o in es iga e he cellula and
molecula mechanisms unde lying amygdala NT3-induced escue
o ea ex inc ion defici s. He e, ea condi ioned mice ained in
he EXT pa adigm we e ca ego ized as EXT-success o EXT- ailu e,
acco ding o ex inc ion lea ning pe o mance (Supplemen a y
Fig. S5A, B). A e E6, a ex inc ion consolida ion window, mice
we e killed o p epa e ho izon al b ain slices including he
amygdala and ex i o elec ophysiological eco dings we e
pe o med (Fig. 6A).
Fi s , ecapi ula ing in i o expe imen s, we ea ed EXT- ailu e
slices wi h NT3 o es whe he neu o ophin could also dec ease
LA LTP (Fig. 6B–E). In he fi s 10 min ollowing HFS, EXT- ailu e
slices ea ed wi h NT3 showed lowe po en ia ion han un ea ed
EXT- ailu e slices (Fig. 6D; EXT- ailu e s. EXT- ailu e-NT3 q=4.101,
p=0.0447; EXT- ailu e n=6 slices om 6 mice; EXT- ailu e-NT3
n=6 slices om 3 mice), an e ec ha was main ained in he las
10 min o eco dings (Fig. 6E; NT3 e ec F
(1, 17)
=18.59,
p=0.0005).
We hypo hesized ha he e ec s o NT3 on LA LTP could be
media ed by GluN2B-con aining NMDAR. LTP eco dings we e
pe o med in he p esence o NT3 and i enp odil, an an agonis o
GluN2B-con aining NMDAR. We obse ed ha in he fi s 10 min
ollowing HFS, i enp odil adminis a ion p e en ed he e ec s o
NT3 on LTP (Fig. 6D; NT3 x i enp odil in e ac ion F
(1, 17)
=5.171,
p=0.0362; EXT- ailu e s. EXT- ailu e-NT3+i enp odil, q=0.1917,
p=0.9991, EXT- ailu e-NT3 s. EXT- ailu e-NT3+i enp odil,
q=4.101, p=0.0447; EXT- ailu e-NT3+i enp odil n=5 slices om
4 mice). Howe e , in he las 10 min o eco dings i enp odil was
no longe able o p e en he e ec s o NT3 (Fig. 6E; NT3 x
i enp odil in e ac ion F
(1, 17)
=0.04139, p=0.8412), fi ing wi h he
a ibu ed ole o NMDAR in he induc ion phase o LTP [46].
Despi e blocking he e ec s o NT3 on LTP induc ion, when
adminis e ed alone i enp odil did no show an e ec nei he in he
fi s 10 min a e HFS (Fig. 6D; EXT- ailu e s. EXT- ailu e-i enp odil,
q=0.5621, p=0.9780; EXT- ailu e-i enp odil n=4 slices om 3
mice) no in he las 10 min o eco dings (Fig. 6E; i enp odil e ec
F
(1, 17)
=0.4615, p=0.5061), demons a ing ha GluN2B-
con aining NMDAR inhibi ion selec i ely a ec s NT3 e ec s on
LA LTP.
Finally, we ea ed EXT-success slices wi h T kC-Fc chime a o
sca enge endogenous NT3 and in his way block NT3 signaling
(Fig. 6F–I). In he fi s 10 min ollowing HFS, EXT-success slices
ea ed wi h T kC-Fc showed a s onge po en ia ion han EXT-
success un ea ed slices (Fig. 6H; =3.084, p=0.0150; EXT-
success n=6 slices om 5 mice, EXT-success-T kC-Fc n=4 slices
om 2 mice). Howe e , he di e ence in LTP was los in he las
10 min o eco dings (Fig. 6I; =1.884, p=0.0964).
The obse ed e ec s we e specific o LTP since none o he
d ugs a ec ed he basal synap ic ansmission, as no di e ences
we e obse ed in he I/O cu es be o e and a e ea men s wi h
NT3, NT3+i enp odil o T kC-Fc (Supplemen a y Fig. S5A–C).
Taken oge he , ou esul s show ha NT3 leads o an
a enua ion o LA LTP induc ion in EXT- ailu e slices, h ough a
GluN2B-dependen mechanism. Con e sely, blocking endogenous
NT3 signaling wi h T kC-Fc in EXT-success slices po en ia ed LTP,
demons a ing ha endogenous NT3 is necessa y o LA LTP
weakening and he eby ea ex inc ion.
In e -indi idual a ia ion in ea ex inc ion elies on a win o lose
compe i ion be ween he ea and ex inc ion mic oci cui s [49].
Ou cu en findings suppo a heo e ical model whe e NT3-T kC
sys em is a key playe in media ing his balance (Fig. 7).
DISCUSSION
In e -indi idual di e ences in he abili y o ex inguish ea ha e a
dual ou come: fi s on se ing he ulne abili y o de elop anxie y
and ea - ela ed diso de s, and second on de e mining he
e ec i eness o exposu e he apy owa ds pa ien s in his g oup
o diso de s. Indeed, ea ex inc ion mechanisms ha suppo
exposu e he apy p inciples [8] a e o en impai ed in pa ien s wi h
ea - ela ed diso de s [50]. He e, aking ad an age o he
indi idual a ia ion in ea ex inc ion pe o mance, we cha ac e -
ized (a he beha io al, cellula , and molecula le el) a model in
oden s o s udy in insic g oup di e ences in con ex ual ea
ex inc ion. Mo eo e , we ound ha NT3-T kC signaling in he
amygdala is su ficien o escue ea ex inc ion defici s and he
unde lying synap ic plas ici y, in a GluN2B-dependen mechanism.
The beha io al model he ein p oposed ep esen s a powe ul
ool o s udy g oup di e ences in ea ex inc ion. Using his
model, 40% o ea -condi ioned mice we e able o ex inguish ea
(EXT-success) when ained in a con ex ual ex inc ion pa adigm, as
opposed o EXT- ailu e who ailed o show a educ ion, and in
some ins ances e en showed an inc ease, in eezing le els. Bo h
EXT-success and EXT- ailu e mice acqui e and o m equally s ong
ea memo ies, sugges ing ha he obse ed beha io al di e -
ences a e specific o he ex inc ion p ocess. Impo an ly,
beha io al di e ences in EXT-success and EXT- ailu e mice we e
main ained 24 h a e ex inc ion lea ning, when mice we e es ed
o ex inc ion e ie al, highligh ing he p edic i e alue o
lea ning pe o mance o ex inc ion ou come.
G oup di e ences in ea ex inc ion ha e been s udied be o e,
pa icula ly using models o s ess-enhanced ea lea ning [51–53].
In hese models, a p io ‘ auma ic’e en esul s in he
enhancemen o ea lea ning and defici s in ea mi iga ion upon
ex inc ion [54]. A modified e sion o such models o s udy ea
ex inc ion di e ences in s ess- esis an and s ess-suscep ible
mice was p e iously epo ed, wi h he la e showing ea
ex inc ion defici s [53]. In hese s ess-enhanced ea lea ning
models, he di e en esponse o he animals o a auma ic e en
se s he basis o he di e en ex inc ion pe o mances. Ins ead, in
G. Masella e al.
8
Molecula Psychia y
ou model, animals wi h equally s ong ea memo ies show
indi idual di e ences in ex inc ion ha a e inhe en o he
animals and obse ed in he absence o any gene ic o
en i onmen al manipula ion.
A model o app oach/a oidance conflic ask, mo e closely
ela ed o ou model, was es ablished o assess indi idual
di e ences in esponse o conflic ing s imuli [55]. The au ho s
ound ha animals ca ego ized a p io i as ‘a oiding’,in
compa ison o ‘balancing’, did no ex inguish ea [55,56].
Impo an ly, beha io al di e ences we e suppo ed by mo pho-
logical, unc ional and ansc ip omic signa u es in he mPFC and
amygdala py amidal neu ons, ha p edispose o maladap i e ea
ex inc ion [56].
The o ma ion o ea memo ies and hei ex inc ion is
dependen on synap ic plas ici y e en s occu ing a amygdala
ea and ex inc ion mic oci cui s [42]. In pa icula , he o ma ion
Fig. 6 NT3 a enua es LTP a LA synapses in a GluN2B-dependen mechanism. A Schema ic ep esen a ion o expe imen al condi ions and
localiza ion o s imula ing and eco ding elec ode in ho izon al b ain slices con aining he LA. A HFS p o ocol ( h ee 1 s du a ion ains o
100 Hz pulses, wi h 5 s in e - ain in e al; dashed e ical line) was used o induce ex- i o LTP in he LA o (B–E) EXT- ailu e slices un ea ed
(n=6), ea ed wi h NT3 (n=6, 50 ng/mL), ea ed wi h i enp odil (n=4, 3.25 µg/mL), ea ed wi h NT3+i enp odil (n=5, NT3 50 ng/mL and
i enp odil 3.25 µg/mL) and o (F–I) EXT-success slices un ea ed (n=6) and ea ed wi h T kC-Fc chime a (n=4, 0.5 µg/mL). Tes ed d ugs we e
added 45 min be o e HFS and we e main ained in he medium un il he end o he expe imen . BRep esen a i e aces o EXT- ailu e slices
un ea ed and ea ed wi h NT3, i enp odil and NT3+i enp odil a baseline (
__
), upon 10 min (
…
) and 45 min (---) o HFS. CLTP ime cou se
eco ded o 45 min ollowing induc ion. Ampli ude o PS was a e aged o he (D)fi s 10 min and (E) las 10 min o eco dings ollowing LTP
induc ion. Repea ed measu es wo-way ANOVA wi h Tukey mul iple compa isons es . FRep esen a i e aces o EXT-success slices un ea ed
and ea ed wi h T kC-Fc a baseline (
__
), upon 10 min (
…
) and 45 min (---) o HFS. GLTP ime cou se eco ded o 45 min ollowing induc ion.
PS ampli ude was a e aged o he (H)fi s 10 min and (I) las 10 min o eco dings ollowing LTP induc ion. Two- ailed S uden ’s es . BLA
basola e al amygdala, Ce cen al amygdala, E6 ex inc ion ial 6, HFS high equency s imula ion, LA la e al amygdala, NT3 neu o ophin 3, PS
popula ion spikes. T kC-Fc opomyosin ecep o kinase C–Fc chime a. *p≤0.05.
G. Masella e al.
9
Molecula Psychia y
esul s. EC pe o med he elec ophysiology expe imen s. GA helped wi h he
s e eo axic su ge ies and in i o d ug in usions. MFM pe o med wes e n blo s and
his ology, and p epa ed he g aphical abs ac and he figu e o he p oposed model.
AT and SF assis ed wi h he elec ophysiology eco dings and discussed he da a.
RAC and CBD p o ided esou ces and con ibu ed o he discussion o he esul s. MS
pe o med and analyzed beha io al es s and assis ed in s e eo axic su ge ies and
in i o d ug in usions, designed he s udy, supe ised esea ch and da a analysis, and
was a majo con ibu o in w i ing he manusc ip and unding acquisi ion.
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