scieee Science in your language
[en] (orig)

Valence in the Nucleus Accumbens - identifying neural populations in appetitive and aversive responses

Gaspar, Natacha Sofia Vieitas · 2022

Abstract

O núcleo accumbens (NAc) é reconhecido como um componente essencial do circuito de recompensa, estando associado ao processamento de eventos recompensadores e aversivos e contribuindo para comportamentos motivados. O NAc é uma “interface límbico-motora”, e estudos mostram o seu envolvimento em codificar valência - o valor intrínseco de uma certa experiência e consequentes respostas emocionais e motivacionais. O NAc é constituído maioritariamente por neurónios espinhosos médios GABAérgicos (MSNs), divididos naqueles que expressam o recetor de dopamina D1 (D1-MSNs) e nos que expressam o recetor de dopamina D2 (D2-MSNs). Estas populações foram tradicionalmente segregadas anatomicamente (via direta vs indireta) e funcionalmente (recompensa e valência positiva vs aversão e valência negativa). No entanto, muitos estudos recentes desafiaram esta segregação simplista. Porém, é ainda incerto quais as populações neuronais que codificam valência no NAc, e esta é uma questão crucial na compreensão de distúrbios com défices emocionais. Nesta dissertação, avaliámos os níveis de ativação neuronal associados com estímulos de valência negativa (choque na pata) ou positiva (cocaína) no NAc, usando a amígdala basolateral (BLA) e central (CeA) como regiões controlo, usadas por codificarem valência. Também caracterizámos um vetor viral controlado por c-fos para estabelecer uma estratégia de marcação de ativação neuronal para trabalho futuro. Além disso, efetuámos ativação optogenética de neurónios responsivos a estímulos na BLA, usando o mesmo vetor, para induzir comportamentos de valência e validar a nossa metodologia. Os nossos dados mostram que o NAc core (NAcc) e a BLA contêm populações neuronais que respondem ao choque, medido por uma maior densidade de células c-fos+ em comparação os controlos. Porém, a cocaína não induziu alterações significativas de ativação neuronal. Quanto ao vetor (conduzindo expressão de channelrhodopsin-eYFP), determinámos que 16h pós-exposição a estímulo seria o período mais adequado para observar marcação viral. Ao usar este vetor e expor murganhos a estímulos positivos ou negativos, dados de densitometria de fluorescência mostraram apenas uma tendência para maior ativação neuronal na BLA após o choque na pata, sem efeitos no NAc, em comparação com animais controlo; sem efeitos devido à cocaína. Por último, ativação optogenética de neurónios responsivos a choque na pata da BLA induziu uma tendência de evasão num teste de preferência de lugar em tempo real (RTPP), contudo essa mesma ativação não induziu preferência de lugar condicionada (CPP). Embora os nossos dados mostrem ativação neuronal no NAcc e BLA em resposta a choque, a falta de diferenças na exposição a cocaína, junto com dados de densitometria e optogenética, indicam a necessidade de desenvolver novas ferramentas para marcar neurónios que codifiquem valência no NAc.

Read accessible full text

Valence in the Nucleus Accumbens - identifying neural populations in appetitive and aversive responses

Author: Gaspar, Natacha Sofia Vieitas
Year: 2022
Source: https://repositorium.uminho.pt/bitstreams/76d61bfe-e3bb-4f91-b117-1d7f8009be39/download
ou ub o de 2021
UMinho | 2021
Uni e sidade do Minho
Escola de Medicina
Na acha So ia Viei as Gaspa
Valence in he Nucleus Accumbens -
Iden i ying Neu al Popula ions in Appe i i e
and A e si e Responses
Valence in he Nucleus Accumbens - Iden i ying Neu al
Popula ions in Appe i i e and A e si e Responses
Na acha So ia Viei as Gaspa
ou ub o de 2021
Uni e sidade do Minho
Escola de Medicina
Na acha So ia Viei as Gaspa
Valence in he Nucleus Accumbens -
Iden i ying Neu al Popula ions in Appe i i e
and A e si e Responses
Disse ação de Mes ado
Mes ado em Ciências da Saúde
T abalho e e uado sob a o ien ação de
Dou o a Ana João Gomes Rod igues
e de
Dou o a Ca ina Isabel Soa es da Cunha
ii
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS
Es e é um abalho académico que pode se u ilizado po e cei os desde que espei adas as eg as e
boas p á icas in e nacionalmen e acei es, no que conce ne aos di ei os de au o e di ei os conexos.
Assim, o p esen e abalho pode se u ilizado nos e mos p e is os na licença abaixo indicada.
Caso o u ilizado necessi e de pe missão pa a pode aze um uso do abalho em condições não p e is as
no licenciamen o indicado, de e á con ac a o au o , a a és do Reposi ó iUM da Uni e sidade do Minho.
Licença concedida aos u ilizado es des e abalho
A ibuição-NãoCome cial-SemDe i ações
CC BY-NC-ND
h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/
iii
Ag adecimen os
À Dou o a Ana João Rod igues, minha o ien ado a, po me p opo ciona odo o apoio e meios
necessá ios pa a ealiza es a disse ação. Ob igada po odo o conhecimen o e momen os de
c escimen o, pela disponibilidade con ínua, e po me e dado a opo unidade de abalha nes e p oje o
e na sua equipa.
À Dou o a Ca ina Cunha, minha coo ien ado a, pelos ensinamen os que me ansmi iu, pelo
auxílio, disponibilidade e paciência incessan es, pelas ideias e suges ões que me inci a am a melho a .
Ob igada po oda a ajuda, den o e o a do labo a ó io.
À Bá ba a, Ve ónica, Raquel e ao Rod igo, meus colegas de equipa, po odos os conselhos,
escla ecimen os e pela companhia ao longo do desen ol imen o des a disse ação. Ob igada pelo
cons an e apoio.
À Sónia, à Ca a ina, à Ri a e à Mónica, minhas amigas e colegas de mes ado. Ob igada po
se em inc í eis, po oda a ajuda e po odos os momen os de con í io!
À Adília e à Inês, minhas amigas, po oda a a eição e con í io, mesmo que po ezes i esse de
se à dis ância. Ob igada pelo apoio e pelo en usiamo!
Ao domínio de neu ociências, pelas discussões cien í icas, e, em pa icula , a odos os que
semp e es i e am dispos os a ajuda quando necessá io.
Po úl imo, mas não menos impo an e, mui o ob igada aos meus pais, à minha i mã Ma alda,
aos meus a ós e à Ca la po odo o auxílio e ca inho. Ob igada po me apoia em em odos os momen os,
pelo in e esse no meu abalho, e po odos os enco ajamen os!
O abalho ap esen ado nes a ese oi ealizado no Ins i u o de In es igação em Ciências da Vida
e Saúde (ICVS), da Uni e sidade do Minho. O apoio inancei o oi p es ado a a és de bolsas da Fundação
Bial a a és dos p oje os 30/2016 e 175/20; do Fundo Eu opeu de Desen ol imen o Regional (FEDER),
no âmbi o do p oje o PTDC/MED-NEU/29071/2017 (REWSTRESS), a a és do COMPETE 2020; da
Fundação “La Caixa” (ID100010434; LCF/PR/HR20/52400020); de undos eu opeus
da Eu opean
Resea ch Council
(ERC) – ERC
Consolida o
“
VALENCE
”, No 101003187; da Pla a o ma de Mic oscopia
Cien í ica do ICVS, memb o da
PPBI - Po uguese Pla o m o Bioimaging
(PPBI -POCI-01-0145-FEDER-
022122); e po Fundos Nacionais, po meio da Fundação pa a a Ciência e Tecnologia (FCT) - p oje os
UIDB/50026/2020 e UIDP/50026/2020.

i
STATEMENT OF INTEGRITY
I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used
plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o
i s elabo a ion.
I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o Minho.
Resumo
O núcleo accumbens (NAc) é econhecido como um componen e essencial do ci cui o de
ecompensa, es ando associado ao p ocessamen o de e en os ecompensado es e a e si os e
con ibuindo pa a compo amen os mo i ados. O NAc é uma “in e ace límbico-mo o a”, e es udos
mos am o seu en ol imen o em codi ica alência - o alo in ínseco de uma ce a expe iência e
consequen es espos as emocionais e mo i acionais. O NAc é cons i uído maio i a iamen e po neu ónios
espinhosos médios GABAé gicos (MSNs), di ididos naqueles que exp essam o ece o de dopamina D1
(D1-MSNs) e nos que exp essam o ece o de dopamina D2 (D2-MSNs). Es as populações o am
adicionalmen e seg egadas ana omicamen e (
ia di e a
s
indi e a
) e uncionalmen e (
ecompensa e
alência posi i a
s
a e são e alência nega i a
). No en an o, mui os es udos ecen es desa ia am es a
seg egação simplis a. Po ém, é ainda ince o quais as populações neu onais que codi icam alência no
NAc, e es a é uma ques ão c ucial na comp eensão de dis ú bios com dé ices emocionais.
Nes a disse ação, a aliámos os ní eis de a i ação neu onal associados com es ímulos de
alência nega i a (choque na pa a) ou posi i a (cocaína) no NAc, usando a amígdala basola e al (BLA) e
cen al (CeA) como egiões con olo, usadas po codi ica em alência. Também ca ac e izámos um e o
i al con olado po c- os pa a es abelece uma es a égia de ma cação de a i ação neu onal pa a
abalho u u o. Além disso, e e uámos a i ação op ogené ica de neu ónios esponsi os a es ímulos na
BLA, usando o mesmo e o , pa a induzi compo amen os de alência e alida a nossa me odologia.
Os nossos dados mos am que o NAc co e (NAcc) e a BLA con êm populações neu onais que
espondem ao choque, medido po uma maio densidade de células c- os+ em compa ação os con olos.
Po ém, a cocaína não induziu al e ações signi ica i as de a i ação neu onal. Quan o ao e o (conduzindo
exp essão de
channel hodopsin
-eYFP), de e minámos que 16h pós-exposição a es ímulo se ia o pe íodo
mais adequado pa a obse a ma cação i al. Ao usa es e e o e expo mu ganhos a es ímulos posi i os
ou nega i os, dados de densi ome ia de luo escência mos a am apenas uma endência pa a maio
a i ação neu onal na BLA após o choque na pa a, sem e ei os no NAc, em compa ação com animais
con olo; sem e ei os de ido à cocaína. Po úl imo, a i ação op ogené ica de neu ónios esponsi os a
choque na pa a da BLA induziu uma endência de e asão num es e de p e e ência de luga em empo
eal (RTPP), con udo essa mesma a i ação não induziu p e e ência de luga condicionada (CPP).
Embo a os nossos dados mos em a i ação neu onal no NAcc e BLA em espos a a choque, a
al a de di e enças na exposição a cocaína, jun o com dados de densi ome ia e op ogené ica, indicam a
necessidade de desen ol e no as e amen as pa a ma ca neu ónios que codi iquem alência no NAc.
Pala as-cha e: a i ação neu onal, choque na pa a, cocaína, nucleus accumbens, alência
i
Abs ac
The nucleus accumbens (NAc) is ecognized as an essen ial componen o he ewa d ci cui ,
being associa ed wi h p ocessing o bo h ewa ding and a e si e e en s, and con ibu ing o mo i a ed
beha iou s. The NAc is a “limbic-mo o in e ace”, and e idence shows i s in ol emen in alence
encoding – he in insic alue o a gi en expe ience and consequen emo ional and mo i a ional
esponses. The NAc is mainly cons i u ed by GABAe gic medium spiny neu ons (MSNs), di ided in o hose
exp essing dopamine ecep o D1 (D1-MSNs) and hose exp essing dopamine ecep o D2 (D2-MSNs).
These popula ions ha e been adi ionally seg ega ed ana omically (
di ec
s
indi ec pa hway
) and
unc ionally (
ewa d and posi i e alence
s
a e sion and nega i e alence
). Howe e , many ecen s udies
ha e challenged his simplis ic seg ega ion. Ye , i is s ill unce ain which neu onal popula ions encode
alence in he NAc, and his is a c ucial ques ion in he unde s anding o diso de s wi h emo ional de ici s.
In his hesis wo k we e alua ed he neu onal ac i a ion le els associa ed wi h nega i e-
( oo shock) o posi i e- alence s imuli (cocaine) in he NAc, using he basola e al (BLA) and cen al (CeA)
amygdala as con ol egions, used because hey encode alence. We also cha ac e ized a c- os-d i en
i al ec o in o de o es ablish a neu onal ac i a ion labelling s a egy o u u e wo k. Fu he mo e, we
pe o med op ogene ic ac i a ion o s imulus- esponsi e neu ons in he BLA, using he same ec o , o
induce alence-speci ic beha iou al esponses and alida e ou me hodology.
Ou da a shows ha he NAc co e (NAcc) and he BLA con ain neu onal popula ions ha espond
o shock, measu ed by a highe c- os+ cell densi y in compa ison o con ols. Howe e , cocaine induced
no signi ican changes in neu onal ac i a ion. Rega ding he ec o (d i ing channel hodopsin-eYFP
exp ession), we ound ha 16h-pos s imulus exposu e would be he mo e adequa e ime ame o obse e
neu onal labelling. When using his ec o and exposing mice o posi i e o nega i e s imuli, densi ome y
luo escence da a showed only a endency o highe neu onal ac i a ion in he BLA a e oo shock, wi h
no e ec s in he NAc, in compa ison wi h con ol animals; wi h no e ec s due o cocaine. Las ly,
op ogene ic ac i a ion o oo shock- esponsi e neu ons in he BLA induced a endency o a oidance in a
eal ime place p e e ence es (RTPP), ye his same ac i a ion did no induce condi ioned place
p e e ence (CPP).
While ou da a show neu onal ac i a ion in he NAcc and BLA in esponse o shock, he lack o
di e ences wi h cocaine exposu e oge he wi h densi ome y and op ogene ics da a, indica es he need
o de elop new ools o label alence encoding neu ons in he NAc.
Key wo ds: cocaine, oo shock, neu onal ac i a ion, nucleus accumbens, alence
ii
Table o con en s
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS ........................ ii
Ag adecimen os .................................................................................................................................. iii
STATEMENT OF INTEGRITY ................................................................................................................ i
Resumo...............................................................................................................................................
Abs ac .............................................................................................................................................. i
Lis o abb e ia ions and ac onyms ..................................................................................................... ix
Lis o igu es ...................................................................................................................................... xi
Lis o ables ...................................................................................................................................... xii
CHAPTER 1 – In oduc ion .............................................................................................................. 1
1. In oduc ion ............................................................................................................................. 2
1.1. A ec i e alence ................................................................................................................... 3
1.2. Valence neu oci cui y .......................................................................................................... 5
1.3. Neu oana omy and neu onal popula ions o he nucleus accumbens ..................................... 7
1.4. Func ional e idence on alence encoding in he nucleus accumbens ................................... 11
1.4.1. F om alence o beha iou ......................................................................................... 15
1.5. The ole o D1-MSNs and D2-MSNs in ewa ding and a e si e esponses - Wha is known and
wha is (s ill) unknown .............................................................................................................. 17
1.5.1. Op ogene ic and Chemogene ic s udies……………………………………………..………………18
1.5.2. Pha macological s udies…………………………………………………………………………………19
1.6. Neu oana omy and neu onal popula ions o he amygdala: basola e al and cen al nuclei .... 25
1.7. Func ional e idence on alence encoding in he basola e al and cen al nuclei o he amygdala
................................................................................................................................................. 26
1.7.1. F om alence o beha iou and implica ions o he basola e al and cen al nuclei o he
amygdala ............................................................................................................................. 28
1.8. Open ques ions and u u e o he ield……………………………………………………………………….32
CHAPTER 2 – Objec i es ............................................................................................................... 33
2. Objec i es ............................................................................................................................. 34
CHAPTER 3 – Ma e ials and Me hods ............................................................................................ 35
3. Ma e ials and Me hods .......................................................................................................... 36
3.1. Animals .............................................................................................................................. 36
3.2. Neu al labelling and op ogene ics – cons uc s and i us p epa a ion .................................. 36
2
1. In oduc ion
Daily, indi iduals ha e o il e in o ma ion and ocus on emo ionally- ele an s imuli, and espond
in an adequa e manne . A ec i e alence is a componen ha includes bo h he pleasu e-displeasu e
alue inhe en ly linked wi h a gi en s imulus, and any kind o beha iou al, physiological and emo ional
consequences i induces in an indi idual (Be idge, 2019). Conside ing ha alence aligns expe iences
along a posi i e-neu al-nega i e axis, his concep is hea ily associa ed wi h ewa d and a e sion, such
ha appe i i e/ ewa ding ci cums ances ha e a posi i e alence, while a e si e ci cums ances ha e a
nega i e alence. Some s imuli a e inna ely ewa ding o a e si e o he indi idual, wi hou equi ing any
ype o lea ning such as he uncondi ioned app oach o emale odou by male a s (posi i e alence) o
uncondi ioned a oidance in he p esence o ca (any p eda o ) odou . Howe e , mos s imuli a e ini ially
neu al and equi e lea ning o o m emo ional associa ions be ween hese and he mo i a ionally ele an
ou come (Pa lo , 2010).
The s udy o b ain ci cui s and egions in ol ed in alence is especially pe inen o s udy he
pa hophysiology o a ec i e diso de s wi h de ici s in ewa d and a e sion p ocessing, such as dep ession
and addic ion (Coope e al., 2017; Dich e e al., 2012; Russo & Nes le , 2013).
To s udy alence encoding, esea che s ha e been cha ac e izing neu onal ac i i y pa e ns in
esponse o posi i e o nega i e s imuli o cues p edic ing hose s imuli. In he la es yea s, he
de elopmen o op ogene ics/chemogene ics and new ansgenic models, allowed o causally link he
ac i i y o speci ic neu onal ensembles wi h ewa ding o a e si e esponses. Va ious b ain egions, such
as he nucleus accumbens (NAc), he basola e al amygdala (BLA) and he en al egmen al a ea (VTA),
amongs o he s, ha e been shown o be in ol ed in he ewa d sys em and/o alence encoding,
( e iewed in (Balleine & Killc oss, 2006; Be idge & K ingelbach, 2015; Kelley & Be idge, 2002; Nambu i
e al., 2016; Richa d e al., 2013; Russo & Nes le , 2013; Sesack & G ace, 2010).
The NAc has been implica ed in mo i a ed beha iou s, and in a e si e and appe i i e esponses,
o bo h na u al and d ug ewa ds (Be idge & K ingelbach, 2015; Ca lezon & Thomas, 2009; Roi man
e al., 2005). The majo i y o NAc neu ons a e GABAe gic medium spiny neu ons (MSNs) (Ge en, 1992),
di ided in o hose exp essing dopamine ecep o D1 (DR1; D1-MSNs) o dopamine ecep o D2 (DR2;
D2-MSNs). Fo decades hese neu onal popula ions ha e been conside ed o be seg ega ed bo h
ana omically and unc ionally. Con en ionally, D1- and D2-MSNs we e conside ed as comp ising he di ec
and indi ec s ia al pa hways, espec i ely, which was la e econside ed since D1-MSNs also in eg a e
he indi ec pa hway (Kupchik e al., 2015). On a unc ional le el, s udies ha e associa ed D1-MSNs wi h
ewa d and posi i e ein o cemen (posi i e alence) and D2-MSNs wi h a e sion and punishmen

3
(nega i e alence) (Hikida e al., 2010, 2016; K a i z e al., 2012; Lobo e al., 2010; Volman e al., 2013).
Ne e heless, ecen s udies ha e e ealed his model o be o e ly simplis ic. Fo ins ance, op ogene ic
s imula ion o ei he D1- o D2-MSNs suppo s sel -s imula ion (Cole e al., 2018). O he wo ks, including
om ou eam, ha e shown ha bo h popula ions can d i e ewa d and a e sion ((Golden e al., 2019;
Nam a e al., 2019; Na subo i e al., 2017; Soa es-Cunha e al., 2016a, 2018, 2020; S einbe g e al.,
2014) – u he explo a ion o his opic will be shown in a la e sec ion).
In he ollowing sec ions, we ocus on alence encoding in di e en b ain egions, emphasising
he NAc. We will explo e elec ophysiological s udies ega ding alence encoding in he NAc and s udies
ha link he NAc wi h ewa ding and a e si e beha iou s, as well as p esen ing pa allel ele an
in o ma ion o he BLA and he cen al amygdala (CeA) subnuclei.
1.1. A ec i e alence
One o he ea lie wo ks on emo ion was de ised by Da win (1872). He p oposed ha , o some
ex en , emo ions exis ed equally h oughou species, and animal emo ions we e homologues o human
(basic) ones. Emo ions would be linked wi h isible ac ions, which would be selec ed h oughou e olu ion,
and ecognizable ac oss species.
A common heme in emo ion heo y is he associa ion o emo ions wi h in e nal and ex e nal
changes in o ganisms (
e.g.
obse able beha iou s). In humans, a subjec i e componen can be assessed
(Ande son & Adolphs, 2014). Howe e , objec i e and subjec i e le els o a ec i e esponses a e no
necessa ily concu en , con eying he same in o ma ion, no doing so o he same deg ee (Be idge &
K ingelbach, 2015).
Dimensional emo ional heo ies a emp o in eg a e emo ional s a es in o a ious dimensions
(con as ing wi h disc e e heo ies; (Mendl e al., 2010)), and a ec i e alence is a epea edly exhibi ed
c i e ia (
e.g.
(Russell, 1980; Russell & Ba e , 1999; Wa son & Tellegen, 1985)). We highligh he
Ci cumplex Model o A ec (Russell, 1980; Russell & Ba e , 1999) as a con ex ualizing example o he
scope o his disse a ion. This model aligns emo ional neu ophysiological s a es by combining wo axes,
a ousal (engagemen le el) and pleasu e (a ec i e alence) (“co e a ec ”; (Russell, 2003)) (Figu e 1).
Ne e heless, alence mus be dis inguished om o he concep s such as salience and a ousal,
also associa ed wi h s imuli. Salience is conside ed as he le el o pe cep ion o a s imulus (
i.e.
capaci y
o being ecognized and induce beha iou al changes) (Uddin, 2015), while a ousal is linked wi h
inc eased ac i a ion and awa eness o a gi en si ua ion (C i chley e al., 2013), being able o impac
4
salience (Lee e al., 2020). Neu ons may, a imes, appea o p ocess alence, bu ins ead encode he
salience o a ousal o a s imulus, independen ly om he posi i e o nega i e alue.
Figu e 1 - Co e a ec ep esen ed in he Ci cumplex Model o A ec model. a) Alignmen o emo ional s a es in
wo dimensions, a ousal ( om high ac i a ion o low a ousal) and a ec i e alence ( om pleasan o unpleasan ). Posi i e
a ec i e desc ip o s ( igh ) a e opposed o nega i e a ec i e desc ip o s (le ). High a ousal s a es a e usually associa ed wi h
mo i a ion o ei he a oid h ea s ( op le ) o o seek ewa d ( op igh ). Adap ed om (Russell, 1980) and (Russell & Ba e ,
1999).
B ie ly, a ec i e alence has been de ined as no only he in insic alue o a ce ain expe ience,
bu also he esul an hedonic, emo ional, and mo i a ional esponses (Be idge, 2019). On one end o
he spec um, we ha e s imuli wi h a posi i e alence, meaning hey a e in insically pleasan and o igina e
appe i i e eac ions, while nega i e alence s imuli a e in insically unpleasan , ini ia ing a e si e
eac ions (Figu e 1). Speci ically, he s imuli a e conside ed posi i e o nega i e uncondi ioned s imuli
(US), espec i ely, because hey elici such esponses inna ely (Pa lo , 2010). When a p e iously neu al
s imulus (
e.g.
a sound, an odou ) is p esen ed p io o an US, i a ains a pa icula alence, being now
conside ed a condi ioned s imulus (CS) (Pa lo , 2010). F eezing due o a oo shock-pai ed one (nega i e
CS) o licking in esponse o a ligh combined wi h a suc ose ewa d p esen a ion (posi i e CS), o
example, a e such cases, being cases o Pa lo ian condi ioning.
Fo example, ega ding associa ed eac ions, US as ing s imuli such as suc ose o quinine elici
qui e obus and e olu iona ily conse ed esponses. Suc ose, a posi i e alence as ing s imulus, elici s
lip licking, ongue p o usions and, exclusi ely in hominoids, a elaxed, smile-like exp ession. Nega i e
esponses like gaping, headshaking and, in hominoids only, nose-w inkling, and g imacing exp essions
a e seen in esponse o quinine (Be idge, 2000, 2019; G ill & No g en, 1978; S eine e al., 2001).
As s a ed, while om a mo e psychological s andpoin a ec i e alence is o en conside ed and
assessed in a subjec i e manne , he mo e objec i e eac ions ha can de i e om a speci ic alence
5
encoding include physiological and/o beha iou al e ec s which can o he mos pa be measu ed, and
he e o e, s udied.
1.2. Valence neu oci cui y
Valence has been associa ed wi h a ied b ain egions. Fo ins ance, he mesolimbic b ain ewa d
sys em includes b ain a eas implica ed in alence encoding and in ewa ding and a e si e esponses
(Nambu i e al., 2016).
A a ie y o s udies ha e con ibu ed o expand he knowledge on he in ol emen o dis inc b ain
a eas in alence (p esen ed in his sec ion), namely, NAc (Be idge & K ingelbach, 2013; Knowland &
Lim, 2018; Nambu i e al., 2016; Nieh e al., 2013), he VTA (Nambu i e al., 2016; Nieh e al., 2013),
he BLA (Balleine & Killc oss, 2006; Nambu i e al., 2016; O’Neill e al., 2018; Šimić e al., 2021), he
CeA (Balleine & Killc oss, 2006; Šimić e al., 2021) and he hippocampus (Nambu i e al., 2016; Nieh e
al., 2013).
The NAc has been pa icula ly s udied, being a ecognized cen al egion o he mesoco icolimbic
ewa d ci cui , hea ily engaged in ewa ding and mo i a ed beha iou s, p ocessing bo h na u al ewa ds
and d ugs o abuse (such as ood and cocaine), along wi h encoding a e si e condi ions (Be idge &
K ingelbach, 2015; Ca lezon & Thomas, 2009). Ha ing inpu s om an asso men o co ical, sub-co ical
and limbic egions and ou pu s o o he pa s o he basal ganglia, sec ions o he co ex and some
halamic egions (Heime & Alheid, 1991; Sesack & G ace, 2010), i has been p oposed as a majo
in e ace o limbic p ocessing and mo i a ion in o mo o e ec s (Mogenson e al., 1980).
The amygdala nuclei, such as he BLA and CeA, a e also a ocal poin o s udy in emo ional
p ocessing. The BLA is known o eac o s imuli, including alence p ocessing o bo h nega i e and
posi i e con ex s, being also associa ed wi h ea p ocessing, as well as ewa d- ela ed mechanisms (
e.g.
ewa d lea ning and mo i a ion) (Balleine & Killc oss, 2006; Sah e al., 2003; Šimić e al., 2021). The
CeA is implica ed in ea mechanisms (including physiological esponses), along wi h ewa ding and
a e si e lea ning (Balleine & Killc oss, 2006; Sah e al., 2003; Šimić e al., 2021).
O he egions ha we e, a leas o some ex en , linked wi h alence (o ewa d and/o a e sion
p ocessing) include he la e odo sal egmen al nucleus (LDT; (Nieh e al., 2013)), he en al pallidum
(VP; (Be idge & K ingelbach, 2013; Knowland & Lim, 2018)), he la e al hypo halamus (LH; (Nambu i
e al., 2016)), he la e al habenula (LHb; (Knowland & Lim, 2018; Nieh e al., 2013)), he
locus coe uleus
(Nambu i e al., 2016), he pa a en icula nucleus o he halamus (PVT; (Ba son e al., 2020; Ki ouac,
6
2015)), he bed nucleus o he
s ia e minalis
(BNST; (Lebow & Chen, 2016)) and he audi o y co ex
(Concina e al., 2019).
Figu e 2 – Simpli ied alence ci cui y. a) Schema ic sagi al ep esen a ion o he main egions in ol ed in alence and
ewa d/a e sion. Localiza ion ac oss he mediola e al axis, size and shape a e adjus ed o simplici y and ep esen a ion
pu poses. B ain en icles shown in black. NAc – Nucleus Accumbens; VP – Ven al Pallidum; BLA – Basola e al Amygdala;
CeA – Cen al Amygdala; LH – La e al Hypo halamus; VTA – Ven al Tegmen al A ea; LDT - La e odo sal Tegmen al Nucleus;
HPC – Hippocampus. Adap ed om (Paxinos & F anklin, 2001).
I is essen ial o no e ha neu onal popula ions, e en wi hin one pa icula b ain egion, do no
necessa ily encode alence in he same manne . As dis inc ly explained by Nambu i e al. (2015),
neu onal subpopula ions can, o ins ance, be classi ied in a unc ional manne (
i.e.
ac i i y changes in
esponse o s imuli) and by gene ic p o ile and/o ana omical cha ac e is ics (
i.e.
di e ging inpu s o
ou pu s). Following his logic, many s udies ha e de ined alence encoding subpopula ions. Fo example,
Kim e al. (2016), showed wo gene ically dis inc neu onal subpopula ions in he BLA, he RSPO2+
neu ons (shock- esponsi e ha d i e a e sion) and he PPP1R1B+ neu ons (wa e ewa d- esponsi e ha
d i e place p e e ence). Rega ding ana omically dis inc subpopula ions, neu ons p ojec ing om he
p elimbic co ex o he BLA, o om he BLA o he CeA, ha e been shown o p omo e a oidance (Huang
e al., 2020; Nambu i e al., 2015). Occasionally, s udies may combine hese concep s, as seen in S ube
e al. (2011), who examined glu ama e gic NAc- a ge ed BLA neu ons, ound o d i e appe i i e
beha iou s. Las ly, e en he same neu onal popula ions migh be subjec o plas ic changes due o
lea ning a e expe iences wi h a speci ic alence ( o ins ance, inc easing synap ic s eng h o ea -
p ocessing neu ons du ing ea condi ioning), which may in luence beha iou al changes ha occu wi h
said s imuli. Fo example, a s udy has desc ibed plas ici y al e a ions induced by ea and ewa d
condi ioning in BLA-NAc (which can induce posi i e ein o cemen ) and BLA-CeA (which can induce
nega i e ein o cemen ) neu ons (Nambu i e al., 2015). Fea condi ioning lowe ed he AMPAR/NMDAR
a io (a measu e o he s eng h o glu ama e gic synapses) in he in e nal capsule a e en s o BLA-NAc
7
neu ons, and enhanced he AMPAR/NMDAR a io in he in e nal capsule a e en s o BLA-CeA neu ons,
while he e e se occu ed o ewa d condi ioning (Nambu i e al., 2015).
Fu he mo e, alence is no an in lexible quali y by any means, depending on many ac o s
beyond he salience and alue o a s imulus. Fo one, alence can be inna e o lea ned. Inna e alence
can be s udied when p esen ing a s imulus wi hou any p io exposu e, being inhe en ly appe i i e o
a e si e, and induce esponses in indi iduals acco dingly (
e.g.
(G ill & No g en, 1978; S eine e al.,
2001)). Valence can also be assigned o p e iously neu al ci cums ances ia condi ioning – in hese
cases, neu al s imuli u n in o condi ioned s imuli when a lea ning associa ion wi h a alence-s imulus is
c ea ed, as explained in he p e ious sec ion (
e.g.
(Go e e al., 2015a; Roi man e al., 2005)). Simila ly,
e en appe i i e s imuli can be conside ed a e si e when consis en ly p eceding a nega i e- alence
s imulus (Roi man e al., 2010). The same s imulus can also ha e opposing alences depending on he
in e nal s a e o an indi idual – ypically a e si e s imuli can be conside ed ewa ding i he e is a
physiological lack o ha componen (
e.g.
neu ons in sal -dep i ed animals espond o NaCl, usually
a e si e, as a ewa d) (Lo iaux e al., 2011).
Addi ionally, alence is encoded di e en ially depending on ana omic loca ion. Fo ins ance, an
“a ec i e keyboa d” has been desc ibed in he NAc shell, wi h a os ocaudal pa e n om posi i e- o
nega i e- alence esponses, wi h he mo e os al a ea being associa ed wi h appe i i e beha iou s and
he mos caudal NAc shell being associa ed wi h ea - ela ed beha iou s (Be idge & K ingelbach, 2015;
Fau e e al., 2010; Reynolds & Be idge, 2001, 2002; Richa d & Be idge, 2011). E en mo e in e es ingly,
his keyboa d can a y wi h he se ing, wi h he ea -inducing a ea being much mo e caudally es ic ed,
in a home-like en i onmen , and much mo e p ominen in a s ess ul en i onmen , wi h he e e se
occu ing o he posi i ely-associa ed a ea (Be idge & K ingelbach, 2015; Reynolds & Be idge, 2008).
Fu he de ailing o some o he opics men ioned he e will be p esen ed in la e sec ions.
1.3. Neu oana omy and neu onal popula ions o he nucleus accumbens
The NAc is deemed an in eg al pa o he oden en al s ia um (Zahm & B og, 1992), ha ing
been o long di ided in o co e and shell sub egions (Zábo szky e al., 1985). The same occu s o he
human en al s ia um, simila ly spli in o co e and shell (Voo n e al., 1996). Fo he majo i y o he NAc
an e io -pos e io axis p og ession, he an e io commissu e (AC) is a clea s uc u al e e ence poin ,
especially in he co onal plane (Paxinos & F anklin, 2001; Paxinos & Wa son, 2007), being en eloped by
he NAc co e (NAcc), which is in u n en ally, medially, and la e ally en eloped by he NAc shell (NAcs)
(Zábo szky e al., 1985).

8
The NAc ga he s inpu s om a ied limbic, co ical, and sub-co ical a eas, being a known
con e ging egion o he basal ganglia (Heime & Alheid, 1991; Sesack & G ace, 2010). A e en
p ojec ions a e di e se, including glu ama e gic inpu s om he amygdala (Fulle e al., 1987; Heidb ede
& G oenewegen, 2003; Mcdonald, 1991; Mo gane e al., 2005; Phillipson & G i i hs, 1985; S ube e
al., 2011), halamus (Fulle e al., 1987; Lanciego e al., 2004; Phillipson & G i i hs, 1985), hippocampus
(Heidb ede & G oenewegen, 2003; Kelley & Domesick, 1982; Mo gane e al., 2005), co ical egions
such as he p e on al co ex (PFC) (Fulle e al., 1987; Mcdonald, 1991; Phillipson & G i i hs, 1985; Yin
& Knowl on, 2006) and i s speci ic sub egions such as he p elimbic and in alimbic co ices (Lanciego
e al., 2004; Yin & Knowl on, 2006); choline gic inpu s om he LDT (Dau an e al., 2014) and
dopamine gic inpu s om he VTA (Hnasko e al., 2012; Phillipson & G i i hs, 1985; T i sch e al., 2012).
Rega ding e e en s, he NAc p ima ily p ojec s ia GABAe gic neu ons o egions o he basal
ganglia and some halamic and co ical a eas (Heime & Alheid, 1991; Sesack & G ace, 2010). Majo
ou pu s comp ise p ojec ions o he VP (G oenewegen & Russchen, 1984; Heime e al., 1991; Lu e al.,
1997; Nau a e al., 1978; Zábo sky & Cullinan, 1992; Zhou e al., 2003)
,
he VTA (G oenewegen &
Russchen, 1984; Heime e al., 1991; Lu e al., 1997; Nau a e al., 1978; Zhou e al., 2003) and
diencephalon s uc u es such as he halamus and he la e al hypo halamus (G oenewegen & Russchen,
1984; Heime e al., 1991; Mogenson e al., 1983; Nau a e al., 1978; Williams e al., 1977). The e is
also e idence o e e en s o o he a eas such as he amygdala, globus pallidus,
subs an ia nig a
, LHb,
BNST,
subs an ia innomina a
and sep um (G oenewegen & Russchen, 1984; Heime e al., 1991;
Mogenson e al., 1983; Nau a e al., 1978; Williams e al., 1977; Zhou e al., 2003). A schema ic ou line
o he abo e-men ioned connec ions is p esen ed in Figu e 3.
9
Figu e 3 - Inpu s and ou pu s o he nucleus accumbens. a) Simpli ied and non-comp ehensi e schema ic
ep esen a ion o NAc inpu and ou pu egions. A e en s: glu ama e gic inpu s om he amygdala, halamus, hippocampus,
p e on al, p elimbic and in alimbic co ices (le , g een); choline gic inpu s om he la e odo sal egmen um (le , gold) and
dopamine gic inpu s om he en al egmen al a ea (le , blue). E e en s: GABAe gic p ojec ions o egions as he en al
pallidum, en al egmen al a ea, halamus, la e al hypo halamus, amygdala, globus pallidus,
subs an ia nig a
, la e al
habenula, bed nucleus o he
s ia e minalis, subs an ia innomina a
and sep um ( igh , ed).
A ound 95% o s ia al neu ons a e GABAe gic medium spiny neu ons (MSNs) (Ge en, 1992),
being commonly di ided in o D1-MSNs o D2-MSNs, and he emaining neu ons (≈5%) a e in e neu ons
(G a eland & Di iglia, 1985).
These popula ions ha e di e en ial p ojec ion pa e ns ega ding en al s ia um ou pu s. D1-
MSNs comp ise di ec p ojec ions o ou pu egions o he basal ganglia, such as he VTA (di ec pa hway;
Figu e 4a), while bo h subpopula ions p ojec indi ec ly o hese a eas h ough he VP (indi ec pa hway;
Figu e 4b) (Kupchik e al., 2015).
10
Figu e 4 – Di ec and indi ec en al s ia al pa hways. a) The di ec pa hway in he en al s ia um includes D1-
MSNs p ojec ions om he NAc o he VTA/SN, which p ojec o he mediodo sal halamus. b) The indi ec pa hway in ol es
bo h D1-/D2-MSNs p ojec ions om he NAcc o he dlVP and om he NAcs o he mVP, which in u n p ojec s o he VTA.
Localiza ion ac oss he mediola e al axis, size and shape a e adjus ed o simplici y and ep esen a ion pu poses. B ain
en icles shown in black. NAc – Nucleus Accumbens; VP – Ven al Pallidum; dlVP – do sola e al pallidum; mVP –
en omedial pallidum; VTA – Ven al Tegmen al A ea; SN – subs an ia nig a. Adap ed om (Paxinos & F anklin, 2001; Soa es-
Cunha e al., 2016b).
Conce ning spa ial dis ibu ion, bo h D1-MSNs and D2-MSNs seem andomly dis ibu ed ac oss
he NAc co e, which does no occu o he NAc shell, in which D2-MSNs we e demons a ed o ha e a
nonuni o m dis ibu ion h oughou shell sub egions (Ganga ossa e al., 2013).
Addi ionally, D1-MSNs and D2-MSNs ha e dis inc i e neu ochemical p ope ies, wi h D1-MSNs
shown o exp ess subs ance P and dyno phin, whe eas VP-p ojec ing D2-MSNs exp ess enkephalin
(Ge en, 1992; Ge le e al., 2008; Lu e al., 1997). NAc sub egions also di e ge ega ding
neu ochemical ma ke s. The NAc co e p esen s g ea e le els o p ep oenkephalin (PPE, enkephalin
p ecu so ) (Roga d e al., 1993), calbindin (P ensa e al., 2003), GABAA ecep o s (Chu chill e al., 1992)
and limbic sys em-associa ed memb ane p o ein (LAMP) (P ensa e al., 2003), and lowe le els o
subs ance P (P ensa e al., 2003), cal e inin (P ensa e al., 2003), µ-opioid ecep o s (Chu chill e al.,
1992), se o onin ((Deu ch & Came on, 1992), se o onin ecep o s (Pa el e al., 1995) and dopamine
(Deu ch & Came on, 1992), while he e e se occu s o he NAc shell. Glu ama e deca boxylase has
11
highe mRNA le els in he shell, bu he e ec was asc ibed o highe cell densi y, due o compa able
amoun s o neu ons conside ed posi i e (Roga d e al., 1993).
The s ia al di ision can also be de ined in e ms o compa men s, due o i s pa ch-ma ix mosaic
o ganiza ion. Pa ches a e ypically high in μ-opioid ecep o binding, while he ma ix is cha ac e ized by
ele a ed choline gic ma ke s and calcium-binding p o ein immuno eac i i y (G aybiel & Ragsdale, 1978;
Zahm & B og, 1992). Neu opep ides such as enkephalin, dyno phin and subs ance P a e exp essed in
bo h pa ches and ma ix (50-65% o each compa men s’ neu ons), wi h subs ance P p esen ing lowe
densi y in he en omedial s ia um and dyno phin and enkephalin being e enly sca e ed ac oss he
s ia um (Ge en & Young, 1988).
1.4. Func ional e idence on alence encoding in he nucleus accumbens
Elec ophysiological s udies
The NAc has been demons a ed as selec i ely encoding appe i i e as e s imuli, leading o a
p edominan inhibi o y esponse in a p ima ily (bu no exclusi ely) suc ose- esponsi e popula ion in an
uncondi ioned p esen a ion con ex (inna e esponse) ((Roi man e al., 2005); see Figu e 5a). Simila ly,
his mainly inhibi o y e ec has been obse ed wi h o he s udies ocusing on suc ose and o he ewa ds
(saccha in, (Wheele e al., 2008; Wilson & Bowman, 2004); suc ose, (Nicola e al., 2004b; Roi man e
al., 2010); e iewed in (Wheele & Ca elli, 2009)).
In e es ingly, some NAc popula ions seem o encode no only absolu e hedonic cha ac e is ics,
bu also ela i e ewa d alue (Taha & Fields, 2005; Wheele e al., 2005). A e in ao al adminis a ion
o suc ose solu ions o di e en concen a ions, dis inc neu onal popula ions we e obse ed (Wheele e
al., 2005), wi h no inhibi ion p edominance. This shows ha , while i commonly seems o be he case, a
dec ease in NAc ac i i y is no a ubiqui ous e ec . Some popula ions (“inhibi o y” and “exci a o y”) had,
o he low concen a ion when al e na ing wi h he high concen a ion, educed ac i i y esponses (
i.e.
a
lesse dec ease/inc ease in i ing a e a e ewa d, espec i ely), when compa ed o isola ed
adminis a ion o he low concen a ion. In he e e se scena io, hese neu ons had inc eased ac i i y
esponses (
i.e.
a g ea e dec ease/inc ease in i ing a e a e ewa d, espec i ely).
Rega ding a e sion, in exposu es o unpleasan as e s imuli (speci ically, quinine), deli e ed
wi hou p io exposu e (inna e eac ion), a mainly exci a o y e ec was obse ed in he NAc in a neu onal
popula ion almos solely esponsi e o quinine ((Roi man e al., 2005); see Figu e 5a). In line wi h his,
18
1.5.1. Op ogene ic and Chemogene ic s udies
In his con ex , op ogene ics has shown o be a g ea ool o in es iga e he in luence o D1-MSNs
and D2-MSNs in ewa ding and a e si e con ex s, allowing o selec i e manipula ion o said neu ons o
explo e hei ole in alence- ela ed p ocesses.
A seminal wo k has shown ha op ogene ic ac i a ion o D1-MSNs inc eased cocaine
condi ioning, while D2-MSN ac i a ion educed i (Lobo e al., 2010). Analogous esul s we e achie ed
when using mo phine (Koo e al., 2014). This, oge he wi h s udies showing ha ac i a ing D1- and D2-
MSNs (in he do sal s ia um) gene a es con inual ein o cemen and ansi o y punishmen ,
co espondingly (K a i z e al., 2012), led o he gene al assump ion ha hese wo subpopula ions had
opposing oles in alence associa ed beha iou .
In line wi h a p o- ewa d ole, op ogene ic inhibi ion o D1 neu ons ep esses cocaine sensi i i y
(Chand a e al., 2013), while ac i a ion (using D d1a-iC e mice and designe ecep o s exclusi ely
ac i a ed by designe d ugs; DREADDs) inc eases alcohol consump ion (S ong e al., 2020). Mo eo e ,
modula ion o social beha iou was obse ed by op ogene ically ac i a ing VTA-NAc p ojec ions, and D1-
MSNs we e p o en essen ial o his p osocial modula ion. Also, ac i a ion o NAc D1-MSNs hemsel es
augmen s social in e ac ion (Gunaydin e al., 2014).
Howe e , a ecen s udy showed an inc ease in mo i a ion a e b ie ly op ogene ically s imula ing
ei he D1-MSNs o D2-MSNs (concu en wi h cue- ewa d exposu e) in mice (Soa es-Cunha e al., 2016a).
The same ou come was obse ed wi h D2-MSN ac i a ion in a s, while op ogene ic inhibi ion (wi h
halo hodopsin; NpHR) led o a educ ion in mo i a ion. Ac i a ing D2-MSNs escued mo i a ional de ici s
in a model wi h dis up ion o such mechanisms (Soa es-Cunha e al., 2016a). Op ogene ic ac i a ion o
D2-MSNs was again ound o media e mo i a ion, and D1R and D2R signalling was necessa y o
ewa ding ou comes (Soa es-Cunha e al., 2018). In acco dance, b ie op ogene ic ac i a ion o bo h
subpopula ions p omp ed inc eased p e e ence o cocaine-pai ed con ex and posi i e ein o cemen .
When he op ogene ic app oach was p olonged, he s imula ion became a e si e and, o D2-MSNs,
cocaine-pai ed place p e e ence was educed (Soa es-Cunha e al., 2020). These indings show ha ,
depending on he pa e n o neu onal ac i a ion, bo h subpopula ions can encode ewa d o a e sion.
O he s ha e demons a ed ha , while op ogene ic s imula ion o D2-MSNs does no suppo sel -
s imula ion in some si ua ions (passi e loca ion-based sel -s imula ion), his is no always he case, as
ac i a ing bo h subpopula ions can s ongly suppo his beha iou in a spou sel -s imula ion con ex
(Cole e al., 2018), indica ing ha bo h popula ions can be concu en ly p o- ewa ding (e en i dissimila
in s eng h).

19
In ag eemen wi h po en ial p o- ewa ding oles, D1- and D2-MSNs in he en ola e al s ia um
encode goal-di ec ed beha iou o ood a di e en beha iou al s ages, and do no appea o ha e
an agonis ic oles. Op ogene ic inac i a ion o ei he popula ion a ial s a a enua es mo i a ed
pe o mance (Na subo i e al., 2017). Howe e , i inac i a ion is used du ing le e p essing, only D1-MSN
inhibi ion would esul in mo i a ion a enua ion (Na subo i e al., 2017).
In e es ingly, pho os imula ing D2-MSNs does no modi y cocaine beha iou al sensi iza ion,
excep i pe o med du ing wi hd awal, which diminishes cocaine-based sensi iza ion (Song e al., 2014).
Fu he mo e, op ogene ic ac i a ion o dyno phin-exp essing neu ons (dyn; exp essed in D1-
MSNs) in he do sal NAc shell induced place p e e ence, whils in he en al NAc shell induced place
a e sion (Al-Hasani e al., 2015). The e o e, ana omical loca ion o neu onal popula ions is, as has been
shown equen ly, a ac o o conside .
Ano he expe imen no ed ha inhibi ion o D2-MSNs ( ia DREADDs) inc eased cocaine
mo i a ion and op ogene ic s imula ion inhibi ed cocaine sel -adminis a ion (Bock e al., 2013), hin ing
a a ole o hese neu ons in limi ing d ug ein o cemen . Inhibi ion o D2-MSNs (using D d2a-iC e mice
and DREADDs) also inc eases alcohol consump ion (S ong e al., 2020).
D1-MSNs also appea o ha e some in luence in a e si e p ocessing. D1-MSNs a e in ol ed in
agg ession mechanisms, since chemogene ic inhibi ion o D1R leads o lowe le els o agg ession-seeking
and agg ession sel -adminis a ion (Golden e al., 2019).
1.5.2. Pha macological s udies
Pha macological s udies ha e also expanded he oles o D1-MSNs and D2-MSNs, la gely by
manipula ing D1R and D2R ac i i y h ough local adminis a ion o espec i e an agonis s and agonis s.
Fo ins ance, ewa d lea ning acquisi ion is diminished bo h when he di ec pa hway is inhibi ed
using a bila e al blockade ( e anus oxin) and when SCH23390 (D1R an agonis ) is injec ed in he in ac
side o D-aRNB (asymme ic blockade) mice. In iguingly, ac i a ing D2 neu ons in he NAc (quinpi ole,
D2R agonis ) o I-aRNB mice dis up s ewa d lea ning swi ch, as did he bila e al blockade o he indi ec
pa hway, showing ha D2 neu on inac i i y is needed in some aspec s o ewa d lea ning (Yawa a e al.,
2012). Applying simila echniques o a ge ing D2-MSNs (agonism) in he in ac side o I-aRNB mice
dis up ed a e si e a oidance lea ning, showing ha D2R inhibi ion can be essen ial o his p ocess
(Hikida e al., 2013).
In addi ion, ac i i y o D2 neu ons in he NAc co e and shell, as well as D1 neu onal ac i i y in
he co e, a e impo an o a e si e memo y acquisi ion (Managò e al., 2009). In ac , D1R inac i a ion
20
(in a-NAc) educed a oidance eac ions (Wie zikoski e al., 2012), as did using D2R pha macological
inac i a ion (Boschen e al., 2011). D2-MSNs (NAc co e) ha e also been implica ed in eac ing o “less
appe i i e” si ua ions, ollowing deli e y o quinpi ole (D2R agonis ), since he ypical inclina ion owa ds
a ewa d le e (
s
a non- ewa d le e ) was no acqui ed h oughou unexpec ed ewa d omission (Po e -
S ansky e al., 2013). Addi ionally, in a-NAc D1R inac i a ion is associa ed wi h a comp omised
acquisi ion o place p e e ence o cocaine (Bake e al., 1998) and e hanol (Young e al., 2014).
Again, sugges ing pa allel unc ions, coope a ion be ween D1- and D2-MSNs is necessa y o
in ac anial sel -s imula ion ( acili a ed by VTA-NAc dopamine p ojec ions), since an agonism o ei he D1R
(SCH23390) o D2R ( aclop ide) in he NAc mi iga es his beha iou (S einbe g e al., 2014).
An agonism o bo h D1R and D2R lessened pe o mance in a Pa lo ian- o-ins umen al ans e
ask (PIT), showing ha bo h neu on ypes a e p ocessing appe i i e salience (Lex & Haube , 2008).
Inac i a ion o bo h D1 and D2 neu ons educed he ex inc ion s age a e mo phine-based CPP, indica ing
ha bo h popula ions a e a leas pa aking in encoding appe i i e a ibu es o s imuli h oughou
ex inc ion (Nam a e al., 2019).
Likewise, blockade o D1R and D2R ia an agonism in he NAc abolished acquisi ion o
amphe amine-based place p e e ence (Liao, 2008) and inac i a ing bo h ecep o s a ec ed he le el o
cue esponse nega i ely, bu lea es mo i a ion o wai (wi h no ene gy spending) unal e ed (Wakabayashi
e al., 2004). Inac i a ing bo h ecep o s (in a-NAc) educes he quan i y o eeding beha iou and
enhances eeding pe iod, wi hou changing o al ood in ake (Baldo e al., 2002) and inac i a ion in he
co e and shell educes le e p essing wi hou al e ing acquisi ion i sel and e en inc easing ood in ake
as well (Nowend e al., 2001).
In e es ingly, abla ion o s ia opallidal neu ons exp essing D2 ecep o (using C e-media ed
exp ession o diph he ia oxin ecep o , DTR) enhanced amphe amine place p e e ence, sugges ing,
simila ly o p e iously men ioned s udies, a d ug ewa d-limi ing e ec (Du ieux e al., 2009).
Ac i a ion o D1 and D2 neu ons (in a-NAc) is ein o cing in a CPP con ex (Whi e e al., 1991)
and usage o D1R and D2R agonis s (SKF81297 and quinpi ole, espec i ely) in he NAc shell appea s
o e-es ablish cocaine-seeking (Schmid e al., 2006). Compa ably, simul aneous agonism o D1R and
D2R in he NAc shell is ein o cing, causing in ac anial sel -adminis a ion (Ikemo o e al., 1997), and
D1R inac i a ion in he NAc shell educes eins a emen o d ug seeking (Ande son e al., 2003).
D2 neu ons also seem o limi a e si e eac ions in some con ex s, since ac i a ing his
popula ion (in a-NAc, D2 agonism) in addic ed a s inhibi ed physical symp oms o opia e wi hd awal
(Ha is & As on-Jones, 1994).
21
In addi ion (and again showing he impo ance o ana omic loca ion), while only D1-MSNs we e
shown o be c i ical o glu ama e an agonism-induced changes in eeding beha io ( os al NAc shell),
he p oduc ion o a e si e esponses a e glu ama e an agonism equi ed he wo subpopula ions (caudal
NAc shell) (Richa d & Be idge, 2011). Fu he mo e, dopamine an agonism in he co e abolished ca eine-
based place a e sion, while he same s a egy in he shell abolished ca eine-based place p e e ence (Yee
e al., 2020).
22
Table 1 - Summa y o main indings implica ing s ia al neu ons (mainly NAc D1-MSNs and D2-MSNs) in alence encoding.
Neu on Type
(NAc, unless
speci ied)
Me hodology
Beha iou al Task
Valence- ela ed e ec s
Re e ence
Op ogene ic and Chemogene ic s udies
D1-MSNs
D2-MSNs
ChR2
Cocaine-based CPP
Inc eased p e e ence
Reduced p e e ence
(Lobo e al.,
2010)
D1-MSNs
D2-MSNs
ChR2
Mo phine-based CPP
Inc eased p e e ence
Reduced p e e ence
(Koo e al.,
2014)
D1-MSNs
D2-MSNs
ChR2
S imula ion-pai ed
capaci i e igge
Con inued ein o cemen
T ansi o y punishmen
(K a i z e al.,
2012)
(do sal
s ia um)
D1-MSNs
NpHR
Open Field + cocaine
Reduced cocaine
locomo o sensi iza ion
(Chand a e al.,
2013)
D1-MSNs
D2-MSNs
Ac i a ion ia
DREADDs
Inhibi ion ia
DREADDs
In e mi en access o
20% alcohol in a 2-
bo le choice es
Inc eased alcohol
consump ion
Inc eased alcohol
consump ion
(S ong e al.,
2020)
D1-MSNs
ChR2
Social In e ac ion Tes
Inc eased social in e ac ion
(Gunaydin e
al., 2014)
D1-MSNs
D2-MSNs
D2-MSNs
ChR2
NpHR
P og essi e-Ra io ask
(PR) / PIT ask
Inc eased mo i a ion
Inc eased mo i a ion /
Rescued mo i a ion de ici s
Reduced mo i a ion
(Soa es-Cunha
e al., 2016a)
D2-MSNs
D1-MSNs/D2-MSNs
ChR2
D1R, D2R
an agonism
PR ask
Inc eased mo i a ion
Reduced mo i a ion
(Soa es-Cunha
e al., 2018)
D1-MSNs /D2-MSNs
D1-MSNs /D2-MSNs
D1-MSNs /D2-MSNs
D1-MSNs /D2-MSNs
D1-MSNs /D2-MSNs
D2-MSNs
ChR2 (b ie
s imula ion)
NpHR
ChR2 (p olonged
s imula ion)
ChR2 (b ie
s imula ion)
ChR2 (p olonged
s imula ion)
S imula ion-based CPP
RTPP
Cocaine-based CPP
Induced p e e ence
Induced a e sion
Induced a e sion
Induced a e sion
Inc eased p e e ence
Reduced p e e ence
(Soa es-Cunha
e al., 2020)
D1-MSNs
D1-MSNs /D2-MSNs
ChR2
Passi e loca ion-based
sel -s imula ion
Spou sel -s imula ion
Suppo ed sel -s imula ion
Suppo ed sel -s imula ion
(Cole e al.,
2018)
23
D1-MSNs /D2-MSNs
D1-MSNs
A chT (a ial s a )
A chT (du ing le e
p essing)
PR ask
Reduced mo i a ion
Reduced mo i a ion
(Na subo i e
al., 2017)
( en ola e al
s ia um)
D2-MSNs
ChR2 (du ing
wi hd awal)
Open Field + cocaine
Reduced cocaine
locomo o sensi iza ion
(Song e al.,
2014)
Dyn+ (do sal shell)
Dyn+ ( en al shell)
ChR2
CPP (Y-maze) / RTPP /
ICSS
Inc eased p e e ence
Reduced p e e ence
(Al-Hasani e
al., 2015)
D2-MSNs
D2-MSNs
Inhibi ion ia
DREADDs
ChR2
Cocaine sel -
adminis a ion
(PR sessions)
Cocaine sel -
adminis a ion
Inc eased mo i a ion
Reduced mo i a ion
(Bock e al.,
2013)
D1-MSNs
Inhibi ion ia
DREADDs
Agg ession-seeking es
Agg ession sel -
adminis a ion es
Reduced agg ession-
seeking
Reduced agg ession sel -
s imula ion
(Golden e al.,
2019)
Pha macological s udies
S ia onig al D1-
MSNs
S ia opallidal D2-
MSNs
Te anus oxin (TN) +
DOX blockade
S anda d Food
s
Chocola e-based CPP
One- ial inhibi o y
a oidance
Dis up ed ewa d lea ning
Dis up ed a e si e lea ning
(Hikida e al.,
2010)
S ia onig al D1-
MSNs
S ia opallidal D2-
MSNs
TN + DOX blockade
TN + DOX
asymme ic blockade
+ D1R an agonism
(con ala e al)
TN + DOX blockade
TN + DOX
asymme ic blockade
+ D2R agonism
(con ala e al)
Visual Cue Task (VCT),
Response-Di ec ion
Task (RDT)
Dis up ed ewa d lea ning
acquisi ion
Dis up ed ewa d lea ning
acquisi ion
Dis up ed ewa d lea ning
swi ch
Dis up ed ewa d lea ning
swi ch
(Yawa a e al.,
2012)
S ia opallidal D2-
MSNs
TN + DOX
asymme ic blockade
+ D2R agonism
(con ala e al)
One-T ial Inhibi o y
A oidance Task
Dis up ed a e si e lea ning
(Hikida e al.,
2013)
D1-MSNs (co e)
D2-MSNs
D1R an agonism
D2R an agonism
One-T ial Inhibi o y
A oidance Task
Reduced a oidance
Reduced a oidance
(Managò e al.,
2009)
D1-MSNs
D1R an agonism
Two-way ac i e
a oidance
Reduced a oidance
(Wie zikoski e
al., 2012)
D2-MSNs
D2R an agonism
D2R agonism (pos -
aining)
Two-way ac i e
a oidance
Reduced a oidance /
Inc eased escape ailu es
Reduced a oidance
(Boschen e
al., 2011)
D2-MSNs (co e)
D2R agonism
Appe i i e ope an
pa adigm (“ o aging”
p e e ence)
Reduced p e e ence o
ewa d du ing unexpec ed
ewa d omission
(Po e -
S ansky e al.,
2013)
D1-MSNs
D1R an agonism
Cocaine-based CPP
Reduced p e e ence
(Bake e al.,
1998)
D1-MSNs
D1R an agonism
E hanol-based CPP
Reduced p e e ence
(Young e al.,
2014)

24
VTA-NAc
dopamine gic
ChR2 (+ D1R/D2R
an agonism)
In ac anial sel -
s imula ion (ICSS)
Reduced ICSS
(S einbe g e
al., 2014)
D1-MSNs
D2-MSNs
D1R an agonism
D2R an agonism
PIT ask
Reduced mo i a ion
Reduced mo i a ion
(Lex & Haube ,
2008)
D1-MSNs
D2-MSNs
D1R an agonism
D2R an agonism
Mo phine-based CPP
Reduced ex inc ion s age
Reduced ex inc ion s age
(Nam a e al.,
2019)
D1-MSNs
D2-MSNs
D1R an agonism
D2R an agonism
Amphe amine-based
CPP
Reduced p e e ence
Reduced p e e ence
(Liao, 2008)
D1-MSNs
D2-MSNs
D1R an agonism
D2R an agonism
P og essi e Delay Task
Reduced cue esponding
Reduced cue esponding
(Wakabayashi
e al., 2004)
D1-MSNs
D2-MSNs
D1R an agonism
D2R an agonism
‘‘F ee- eeding’’ Tes
Reduced eeding beha iou
/ Inc eased eeding pe iod
Reduced eeding beha iou
/ Inc eased eeding pe iod
(Baldo e al.,
2002)
D1-MSNs
D2-MSNs
D1R an agonism
D2R an agonism
Fixed-Ra io 5 (FR5)
Schedule o
Rein o cemen /
Choice P ocedu e
Session
Reduced le e p essing /
Inc eased chow in ake
Reduced le e p essing /
Inc eased chow in ake
(Nowend e al.,
2001)
S ia opallidal D2-
MSNs
Diph he ia oxin
ecep o -based
blockade
Amphe amine-based
CPP
Inc eased p e e ence
(Du ieux e al.,
2009)
D1-MSNs
D2-MSNs
D1R agonism
D2R agonism
Agonis -based CPP
Inc eased p e e ence
Inc eased p e e ence
(Whi e e al.,
1991)
D1-MSNs (shell)
D2-MSNs (shell)
D1R agonism
D2R agonism
FR5 Schedule o
Rein o cemen
Reins a emen o cocaine-
seeking
Reins a emen o cocaine-
seeking
(Schmid e al.,
2006)
D1-MSNs + D2-
MSNs (shell)
D1R agonism + D2R
agonism
In ac anial Sel -
Adminis a ion
(ICSA)
Inc eased ICSA
(Ikemo o e al.,
1997)
D1-MSNs (shell)
D1R an agonism
FR5 Schedule o
Rein o cemen
Reduced eins a emen o
cocaine-seeking
(Ande son e
al., 2003)
D2-MSNs
D2R agonism
Mo phine + Sco ing o
Wi hd awal Symp oms
Reduced opia e wi hd awal
physical symp oms
(Ha is &
As on-Jones,
1994)
D1-MSNs ( os al
shell)
D1-MSNs (caudal
shell)
D2-MSNs (caudal
shell)
D1R an agonism
D1R an agonism
D2R an agonism
Tes s o Spon aneous
Mo i a ed Beha iou s
Abolished glu ama e
dis up ion-based enhanced
eeding
Abolished glu ama e
dis up ion-based de ensi e
beha iou s
(Richa d &
Be idge,
2011)
NAc DR+ (co e)
NAc DR+ (shell)
Dopamine
an agonism
Ca eine-based CPP
Reduced a e sion
Reduced p e e ence
(Yee e al.,
2020)
25
1.6. Neu oana omy and neu onal popula ions o he amygdala: basola e al and
cen al nuclei
The amygdala is an almond-shaped b ain egion ha a ises in bo h hemisphe es, ound medial
ela i e o he empo al lobe in humans, and medial and do sal ela i e o ol ac o y a eas in oden s
(Paxinos & F anklin, 2001; Paxinos & Wa son, 2007; Sah e al., 2003). Va ious ana omical
cha ac e iza ions subdi ide he amygdala in 13 nuclei and co ical a eas, wi h conside able simila i ies
be ween oden s, humans and o he p ima es. These nuclei include he basola e al nucleus (BLA), he
la e al nucleus (LA), he cen al nucleus (CeA), he medial nucleus (M) and he co ical nucleus (Co)
(Cha ey on e al., 2011; Sah e al., 2003). The BLA and la e al nucleus a e subdi isions o he basola e al
complex, being su ounded by he CeA, he cauda e pu amen and he ex e nal capsule in oden s. In
u n, he CeA also edges wi h he medial nucleus and wi h he cauda e pu amen, as well as he globus
pallidus (Sah e al., 2003).
Rega ding a e en connec ions, he BLA ecei es p ojec ions om he LA, a connec ion ha is
associa ed wi h senso y in o ma ion inpu and p ocessing (Babae e al., 2018; Du a ci & Pa e, 2014;
Šimić e al., 2021; To o e e al., 2015). O he inpu egions encompass he o bi o on al co ex (OFC),
he basal o eb ain, he do sal aphe nucleus (DRN), he in alimbic co ex and he VTA (Co eia &
Goosens, 2016; Huang e al., 2020; Saddo is e al., 2005).
BLA ou pu s a e a ied, including co ical, subco ical and limbic egions (P ice, 2003; Sah e al.,
2003), such as he OFC and he medial PFC (La iole e & G ace, 2006; Saddo is e al., 2005), he
hippocampus (Felix-O iz & Tye, 2014), he
subs an ia innomina a
(K e ek & P ice, 1978a, 1978b), he
BNST (K e ek & P ice, 1978a, 1978b), he do sal s ia um (Bou geais e al., 2001), he NAc (Bou geais
e al., 2001; K e ek & P ice, 1978a, 1978b), he hypo halamus (K e ek & P ice, 1978a, 1978b), bu
also he la e al amygdala and CeA (Babae e al., 2018; Du a ci & Pa e, 2014; Šimić e al., 2021; To o e
e al., 2015; Tye e al., 2011).
In u n, he CeA p ojec s o a as a ay o a eas (P ice, 2003; Sah e al., 2003; Šimić e al.,
2021; To o e e al., 2015), such as he pe iaqueduc al g ey (PAG) (Babae e al., 2018; LeDoux e al.,
1988), he hypo halamus (Babae e al., 2018; Bou geais e al., 2001; K e ek & P ice, 1978a, 1978b;
LeDoux e al., 1988), he
subs an ia innomina a
(Bou geais e al., 2001), he BNST (Bou geais e al.,
2001; K e ek & P ice, 1978a, 1978b; LeDoux e al., 1988) and e ec o egions, such as, o example,
he b ains em (Veening e al., 1984). I should be no ed ha a conside able numbe o connec ions wi h
mul iple amygdala subnuclei a e bidi ec ional (Mcdonald, 1998; P ice, 2003).
26
Conce ning neu onal popula ions, he BLA is mos ly cons i u ed by la ge spiny py amidal cells,
which a e glu ama e gic in na u e and ha e many s uc u al simila i ies wi h py amidal co ical cells, while
he emaining cells a e GABAe gic in e neu ons (B aak & B aak, 1983; Sah e al., 2003; Spampana o e
al., 2011). The CeA is la gely o med by GABAe gic s ia al-like cells, being o ha conside ed a mo e
“s ia al” segmen o he amygdala (Sah e al., 2003; Swanson & Pe o ich, 1998).
Neu opep ides a e also a poin o di e gence be ween he BLA and CeA. The BLA con ains cells
posi i e o soma os a in, p o ein phospha ase 1 egula o y subuni 1B (PPP1R1B), oo pla e-speci ic
spondin-2 (RSPO2), asoac i e in es inal polypep ide (VIP) and cholecys okinin-oc apep ide (CCK). The
CeA includes cells exp essing me hionine-enkephalin (MET-ENK), soma os a in, p o ein kinase c-δ (PKC-
δ, PRKCD), co ico opin- eleasing ho mone (CRH), neu o ensin, achykinin 2, CCK, dopamine ecep o
D2, subs ance P, calci onin ecep o -like ecep o (CRLR) and se o onin ecep o 5-hyd oxy yp amine 2A
(HTR2A), wi h some pep ides o e lapping in exp ession wi hin he egion (Kim e al., 2016, 2017;
McCullough e al., 2018; Robe s e al., 1982). Neu ochemical ma ke s in he cen al nucleus a e
unequally dis ibu ed ac oss di e en subnuclei (Kim e al., 2017; McCullough e al., 2018).
1.7. Func ional e idence on alence encoding in he basola e al and cen al nuclei
o he amygdala
The amygdala, as a whole, p ocesses bo h ewa d- and a e sion- ela ed mechanisms, some hing
which has been explo ed in elec ophysiological s udies. Fo example, neu ons in his b ain a ea a e
implica ed in encoding appe i i e alue, wi h a equen ly exci a o y p o ile ha appea s o ack ewa ds
wi h di e en magni udes (Be mudez & Schul z, 2010). A p ima e s udy depic ed di e se amygdala
popula ions, including neu ons encoding unexpec ed wa e ewa d (and expec ed ewa d, wi h lowe i ing
a es); esponding p e e en ially o unexpec ed a e sion (
i.e.
ai -pu s); and p ocessing bo h unexpec ed
ewa d and a e sion (“non- alenced” speci ici y) (Belo a e al., 2007). Amygdala also encodes ol ac o y,
audi o y, isual, and soma osenso y s imuli, wi h mul imodal- esponsi e cells occu ing specially in he
BLA and CeA (Uwano e al., 1995). This b ain egion encodes ewa d acquisi ion and ex inc ion, wi h
subpopula ions p ocessing opposing alences (Li neh & Paz, 2012), and p ocessing posi i e and nega i e
alue o isual s imuli h oughou lea ning (Pa on e al., 2006).
BLA cells eac o p ima y ewa ds. F om glucose- esponding neu ons, a sizable numbe o cells
exhibi ed exci a ions, simila ly o he popula ion encoding he glucose-p edic ing cue (Mu amo o e al.,
1993). Addi ionally, i was equen (bu no uni e sal) ha ac i i y changes (
i.e.
exci a ions o inhibi ions)
o ewa d ma ched he ones o igina ed by he p edic ing cue (Mu amo o e al., 1993). In a p ima e s udy
27
using di e en ewa d schedules wi h isual cues, a conside able numbe o BLA cells esponded o cues
(o en exci a o y ac i i y), while some eac ed o ewa d deli e y (o en an ac i i y inc ease) (Sugase-
Miyamo o & Richmond, 2005). Fu he mo e, cue- eac i e cells (exci a o y e ec ) we e also no ed in an
ope an ask in which animals ecei ed a cue and suc ose i hey execu ed a nosepoke. Some BLA
neu ons esponded o cues only i a po en y o suc ose occu ed, while ano he subse sus ained cue-
esponse du ing eins a emen , e en i no po en y occu ed (Tye & Janak, 2007). Using a ewa d
ex inc ion pa adigm wi h suc ose, Tye e al. (2010) ound a ied BLA popula ions wi h phasic ac i i y
changes. One o hese popula ions ini ially esponded o po en ies only i suc ose was deli e ed, bu
du ing ex inc ion esponded o un ewa ded en ies (mainly inhibi o y in bo h ins ances, bu no only) (Tye
e al., 2010). Some esponded o emp y po en ies only in ex inc ion, o he s esponded o po en ies
wi h suc ose deli e ed only and o he s esponded o all po en ies wi h a p edominan dec ease in i ing
(Tye e al., 2010). This links his b ain a ea wi h p ocessing ewa d ou come and a ailabili y,
ein o cemen and mo i a ional cues in gene al. Las ly, o he s udy ound ha BLA p ojec ing neu ons
espond wi h inc eases in ac i i y p ac ically exclusi ely o ewa d-p edic i e cues (Lee e al., 2016).
Cu iously, he subpopula ion wi h signi ican exci a ions h oughou cue- ewa d p esen a ions appea s o
depend on he lea ned esponding beha iou (Lee e al., 2016). The CeA also esponds o a leas some
ewa d- ela ed mechanisms, such as eeding, seeing ha high- a die has an e ec in exci a o y synap ic
unc ion in
Pnoc
(p ep onocicep in) neu ons o his subnucleus (Ha daway e al., 2019).
The BLA also encodes p ima y a e si e s imuli. A e oo shock exposu e, esponsi e neu ons in
he BLA p esen ed simila le els o exci a ions and inhibi ions, as did he shock-p edic ing cue (Mu amo o
e al., 1993). Mo eo e , BLA subpopula ions ha e been shown o p ocess alence using a ea pa adigm
and compa ing neu onal ac i i y du ing cue exposu e wi h deli e y o dis inc luids (one a e si e and one
ewa ding) (Shabel e al., 2011). In his s udy, neu onal ac i i y a ia ions in a subse o neu ons we e
ound o be mo e compa able in wo a e si e si ua ions o di e en modali ies ( ea
s
unpala able as e)
han when compa ing ea wi h a pala able as e (Shabel e al., 2011).
Bo h egions seem o encode cue exposu es in a e si e lea ning. A e sion-p edic i e cues
induced exci a ions in some BLA p ojec ion neu ons, a subse wi h almos no o e lap wi h he cue- ewa d-
esponsi e popula ion (Lee e al., 2016). In ano he condi ioned app oach, when saccha in was used by
Yasoshima e al. (1995) as a cue o deli e y o li hium chlo ide, bo h BLA and CeA neu ons esponded
s ongly o he cue, once as e a e sion has been es ablished. In he BLA, a conside able popula ion
inc eased spiking in esponse o saccha in (Yasoshima e al., 1995). In con as , he main e ec de ec ed
in he CeA was inhibi o y, being also obse ed due o exposu e o o he unpala able s imuli e en be o e
34
2. Objec i es
The aim o he p esen disse a ion was o s udy he con ibu ion o NAc neu onal popula ions in
alence encoding, o be e comp ehend ewa ding and a e si e mechanisms. The majo ocus o his
disse a ion was he NAc, while he BLA and he CeA we e used as expe imen al con ols, conside ing
hei p e iously es ablished oles in alence encoding and in ewa d and a e sion.
This hesis was subdi ided in h ee main objec i es:
1. Iden i y neu onal popula ions encoding posi i e- and nega i e- alence s imuli by e alua ing c- os+
cell densi y in he NAc, BLA and CeA;
2. Cha ac e ize he exp ession and unc ion o a neu onal ac i i y (c- os)-dependen i al ec o a e
cocaine adminis a ion (posi i e alence) o exposu e o elec ic oo shock (nega i e alence);
3. Induce posi i e- o nega i e- alence associa ed beha iou al esponses in a neu al con ex , by
op ogene ic ac i a ion o posi i e o nega i e s imuli- esponsi e neu onal ensembles (in a eal
ime place p e e ence (RTPP) and a condi ioned place p e e ence (CPP) pa adigm).

35
CHAPTER 3 – Ma e ials and Me hods
36
3. Ma e ials and Me hods
3.1. Animals
Wild- ype C57BL/6J mice (males and emales) (Cha les Ri e Labo a o ies, Ba celona, Spain)
we e kep in s anda d housing condi ions (ligh /da k cycle o 12/12h wi h ligh s u ned on a 08:00h;
22°C±2°C ambien empe a u e) wi h ood and wa e
ad libi um
. Animals we e housed up o a maximum
o 6 mice pe homecage ( ype 2L). All beha iou al expe imen s we e execu ed du ing he ligh pe iod o
he ligh /da k cycle.
All expe imen s we e conduc ed wi h 2–4-mon h-old subjec s, which we e kep di ided acco ding
o gende om pos na al day 21, and handled egula ly o 5-10min in he week be o e beha iou al es ing
o educe handling-associa ed s ess. Animals we e also habi ua ed o all beha iou al appa a uses in a
leas h ee occasions o 10min p io o beha iou al es s.
All p o ocols we e app o ed by he E hics Commi ee o he Li e and Heal h Sciences Resea ch
Ins i u e and by he na ional compe en en i y, he Di eção-Ge al de Alimen ação e Ve e iná ia (DGAV)
(#19074). All p ac ices and echniques we e pe o med ollowing es ablished e hical and legal s anda ds,
including he Eu opean Union Di ec i e 2010/63/EU. Heal h moni o ing was ca ied ou acco ding o
FELASA guidelines and all expe imen e s and animal acili ies we e acc edi ed by DGAV.
3.2. Neu al labelling and op ogene ics – cons uc s and i us p epa a ion
We used he pAAV-c- os-hChR2(H134R)-eYFP-PEST-noWPRE plasmid, unde he con ol o he c-
os minimal p omo e segmen (767bp), and including he 500bp in on 1 coding egion comp ising key
egula o y elemen s, as well as pAAV-c- os-eYFP-PEST-noWPRE (di e ing only by no including he
hChR2(H134R) exon sequence). Bo h cons uc s included he ampicillin esis ance (
AmpR
) gene. The
plasmids we e kindly p o ided by D . Ka l Deisse o h, om S an o d Uni e si y ( ec o desc ip ion
a ailable a (Ye e al., 2016)).
Plasmid DNA was used o ans o m DH5α compe en
E. coli
cells, ollowed by DNA isola ion and
simul aneous gene a ion o glyce ol socks. B ie ly, 5ul o plasmid DNA ecei ed om he collabo a o was
added o an aliquo o DH5α compe en cells, kep on ice o 30min and hen apidly exposed o hea
shock (42°C; 1min) and pu on ice o 3min. 500ul o SOC medium (supe op imal b o h wi h ca aboli e
ep ession) was added o he mix u e and sample was incuba ed o 1h wi h agi a ion a 37°C. This
medium was hen pla ed in LB (lysogeny b o h) aga pla es wi h ampicillin a 100µg ml-1, so ha only
cells ha had in eg a ed he plasmid would su i e, and le o e nigh a 37°C. Single colonies we e hen
37
inocula ed in o LB liquid medium wi h added ampicillin a 100µg ml-1, and bac e ia we e g own a 37°C
(o e nigh a 180 pm).
To ex ac and pu i y he eplica ed plasmid DNA, ob aining a high-yield p oduc while ha ing low
le els o con aminan s such as endo oxins, he NucleoBond® X a EF plasmid pu i ica ion ki was used
(Mache ey-Nagel, Dü en, Ge many). P o ocol was ollowed exac ly as desc ibed in he ki -p o ided
ins uc ions o a midip epa a ion. DNA quan i ica ion was assessed ia UV-Vis spec opho ome y
(NanoD op™ 1000; The mo Fishe Scien i ic, MA, USA).
The i al cons uc s we e packaged in o an adeno-associa ed i us ( AAV) 5 se o ype by he UNC
Gene The apy Cen e Vec o Co e (Uni e si y o No h Ca olina, NC, USA). AAV5 ec o i e s we e 1.2-
9.9X1012 i us molecules/ml. Schema ic ep esen a ion o he sequenced ec o s was pe o med using
SnapGene®.
AAV5-c- os-ChR2-eYFP leads o AAV c- os-d i en exp ession o channel hodopsin (ChR2(H134R))
used wi h enhanced yellow luo escen p o ein (eYFP) o op ogene ic ac i a ion o c- os-ac i a ed
neu ons, while AAV5-c- os-eYFP simply exp esses eYFP in c- os-ac i a ed neu ons.
3.3. T acing su ge ies and cannula implan a ion
Wild- ype C57BL/6J mice (>2 mon hs old) we e anaes he ized wi h 75mg kg-1 ke amine
(Imalgene, Me ial, Lyon, F ance), oge he wi h 1mg kg-1 mede omidine (Do bene, Cymedica, Ho o ice,
Czech Republic), o s e eo axic su ge ies.
The animals used o ini ial ec o cha ac e iza ion we e injec ed wi h he ChR2 cons uc (AAV5-
c- os-ChR2-eYFP; 500nl pe injec ion) bila e ally in he NAc (s e eo axic coo dina es om b egma (Paxinos
& F anklin, 2001): +1.3mm an e opos e io (AP), +0.9/-0.9mm mediola e al (ML), and -4.0mm
do so en al (DV)); and in he BLA (s e eo axic coo dina es om b egma: -1.6mm an e opos e io (AP),
+3.3/-3.3mm mediola e al (ML), and -4.6mm do so en al (DV)).
The mice used o assessing he ec ui men o neu onal popula ions we e unila e ally injec ed
wi h he YFP cons uc (AAV5-c- os-eYFP; 1000nl o al pe coo dina e) in h ee NAc s e eo axic coo dina es
(Paxinos & F anklin, 2001) in wo-s ep injec ions (500nl in he i s coo dina e ollowed by 500nl injec ion
in a di e en DV coo dina e); Injec ion 1: +1.6mm AP, +1.2mm ML, and -4.5mm DV, hen up o -3.9mm
DV; Injec ion 2: +1.3mm AP, +1.2mm ML, and -4.7mm DV, hen up o -4.0mm DV; Injec ion 3: +1.0mm
AP, +1.2mm ML, and -4.7mm DV, hen up o -3.9mm DV. Simila sequen ial injec ions (500nl each s ep)
we e pe o med in he BLA, wi h wo s e eo axic coo dina e se s (Paxinos & F anklin, 2001); Injec ion 1:
38
-1.4mm AP, +3.2mm ML, and -4.7mm DV, hen up o -4.2mm DV; Injec ion 2: -1.6mm AP, +3.3mm ML,
and -4.7mm DV, hen up o -4.2mm DV.
Fo op ical s imula ion in he BLA, animals we e unila e ally injec ed wi h he ChR2 cons uc
(AAV5-c- os-ChR2-eYFP; 500nl pe indi idual injec ion) in he BLA (s e eo axic coo dina es om b egma:
-1.6mm AP, +3.3mm ML, and -4.6mm DV). To allow op ogene ic s imula ion, animals we e also
implan ed wi h an op ic ibe cannula (200µm co e ibe op ic; Tho labs, Ge many), wi h 2.5mm s ainless
s eel e ule (Tho labs, Ge many), in he same coo dina es wi h he excep ion o DV (-4.0mm DV), being
ixed o he skull using den al cemen (C&B ki , Sun Medical, Shiga, Japan).
P esen ed alues a e s e eo axic coo dina es om b egma acco ding o Paxinos & F anklin
(2001) and all injec ions we e pe o med using a 30-gauge needle 2 Hamil on sy inge (Hamil on
Company, Reno, NV, USA), a a a e o 100nl min-1. A e injec ion, he sy inge was le in place o 5min
o allow i al di usion.
A he end o he su gical p ocedu es, mice we e emo ed om he s e eo axic ame, su u ed
and injec ed wi h anaes hesia e e sal (1mg kg-1 a ipamezole; An isedan). Pos ope a i e ca e was ca ied
ou by adminis e ing analgesia (0.05mg kg-1 bup eno phine; Bupaq, Rich e Pha ma, Aus ia) 6h pos -
p ocedu e, as well as once e e y 24h du ing h ee successi e days. A mul i i amin supplemen and saline
we e also adminis e ed pos -p ocedu e when necessa y.
3.4. Beha iou al Assessmen
3.4.1. Appa a us
Th ee iden ical ope an chambe s (21.59cm leng h x 18.08cm wid h x 12.7cm heigh - wo king
a ea; Med Associa es, S . Albans, VT, USA) accommoda ed in ligh - and sound-a enua ing boxes, we e
used in he es . All chambe s con ained a cen al magazine and we e equipped wi h a g id loo and a
shocke . The sou ce o illumina ion was a 100mA, 2.8W house-ligh ins alled on he op-cen e o he wall
opposi e o he magazine wall. A compu e equipped wi h Med-PC so wa e (Med Associa es, S . Albans,
VT, USA) was used o con ol he equipmen and eco d he da a, and webcams (Mic oso Li eCam HD-
3000) we e used o acqui e ideo oo age o each indi idual s imulus exposu e.
3.4.2. Rec ui men o NAc and amygdala ensembles upon exposu e o posi i e
s
nega i e alence s imuli
Wild- ype C57BL/6J mice, 2-4 mon hs o age we e andomly dis ibu ed in o ou g oups: Shock
(n=9), No Shock (n=5), Cocaine (n=8) and Saline g oup (n=5). In he week p io o beha iou al es ing,
39
handling was egula ly pe o med in 5-10min sessions in all mice, aside om which hey we e
expe imen ally naï e o p e en condi ioning. The animals we e habi ua ed o he appa a us in h ee
10min habi ua ion sessions a ew days p eceding he beha iou al p ocedu e. Animals we e also
acclima ized o he expe imen oom 30min p io o es ing. The animals we e exposed o one o wo
s imuli, ei he cocaine (appe i i e s imulus) o elec ic oo shock (a e si e s imulus).
Mice we e adminis e ed wi h cocaine ia in ape i oneal (I.P.) injec ion (20mg/kg) (Cocaine
g oup) o exposed o 20 1.5mA oo shocks o e 10min (Shock g oup) be o e being e u ned o he
homecage. Fo his and he emaining o his disse a ion, shock p o ocol was de ined in ag eemen wi h
Go e e al. (2015a). Con ols we e injec ed wi h saline ia I.P. (Saline g oup) o le o explo e in he same
chambe s o 10min (No Shock g oup). The house-ligh emained ON independen ly o he g oup. All
animals we e sac i iced 90min a e s imulus exposu e.
3.4.3. Tempo al cha ac e iza ion o a c- os-d i en i al ec o
Wild- ype C57BL/6J mice, 2-4 mon hs o age, we e used. All mice we e p e iously subjec ed o
su ge y o injec he AAV5-c- os-ChR2-eYFP cons uc , bila e ally in he NAc and in he BLA. These animals
we e andomly dis ibu ed in o h ee g oups, designa ed he ea e as Shock 8h (n=4), Shock 16h (n=4)
and he Con ol g oup (n=3). Mice we e handled egula ly in 5-10min sessions in he week p io o es ing.
All animals we e accus omed o he appa a us a ew days p eceding he beha iou al es ing in h ee
10min habi ua ion sessions. The subjec s we e mo ed o he expe imen oom 30min p io o he
beha iou al p ocedu e o acclima iza ion. Only one s imulus session was execu ed wi h all subjec s, and
he house-ligh was kep on independen o he g oup.
The s imulus used was an elec ic oo shock (a e si e s imulus), in which he animals we e
exposed o 20 1.5mA oo shocks e e y 30s, o e 10min. Bo h he Shock 8h and Shock 16h g oups we e
subjec ed o elec ic oo shock, being sac i iced 8h and 16h a e s imuli exposu e, espec i ely. Con ols
we e simply le o explo e he same appa a us o 10min and sac i iced wi h he Shock 16h g oup. All
animals e u ned o he homecage a e he p ocedu e.
3.4.4. T ansien labelling and iden i ica ion o neu onal popula ions esponsi e o
posi i e
s
nega i e alence s imuli
Wild- ype C57BL/6J mice, 2-4 mon hs o age, we e o me ly subjec ed o su ge y o injec he
AAV5-c- os-eYFP cons uc in he NAc and in he BLA. Mice we e andomly assigned in ou g oups: Shock
(n=9), No Shock (n=5), Cocaine (n=8) and Saline g oup (n=5). In he week p eceding beha iou al es ing,

40
handling was conduc ed equen ly in 5-10min sessions in all mice. Animals we e o he wise
expe imen ally naï e o p e en condi ioning. Mice we e amilia ized wi h he appa a us in h ee 10min
habi ua ion sessions a ew days p io o he beha iou al p ocedu e. Animals we e also acclima ized o he
expe imen oom 30min be o e es ing. The animals we e exposed o one o wo s imuli, ei he cocaine
(appe i i e s imulus) o elec ic oo shock (a e si e s imulus).
These animals we e adminis e ed wi h cocaine ia in ape i oneal (I.P.) injec ion (20mg/kg)
(Cocaine g oup) o exposed o 20 1.5mA oo shocks o e 10min (Shock g oup) be o e being e u ned o
he homecage. Con ols we e injec ed wi h saline ia I.P. (Saline g oup) o le o explo e in he same
chambe s o 10min (No Shock g oup). The house-ligh emained ON independen ly o he g oup. All
animals we e sac i iced 16h a e s imulus exposu e.
3.4.5. Op ogene ic manipula ion o neu ons esponsi e o nega i e alence s imuli
Wild- ype C57BL/6J mice, 2-4 mon hs o age, we e used in his expe imen . Animals we e
o me ly subjec ed o su ge y o injec he ChR2 cons uc (AAV5-c- os-ChR2-eYFP) in he BLA and we e
hen implan ed wi h an op ical ibe in he same b ain egion.
Fo condi ioned place p e e ence (CPP), animals we e di ided in o Shock (n=5) and No Shock
g oups (con ols; n=4). Fo eal ime place p e e ence (RTPP), all animals we e exposed o shock (n=9).
In he week p eceding beha iou al es ing, handling was conduc ed equen ly in 5-10min sessions. Th ee
10min habi ua ion sessions in he appa a us we e pe o med in he week p io o shock exposu e. Animals
we e also acclima ized o he beha iou al oom 30min p io o es ing.
3.4.5.1. Real Time Place P e e ence (RTPP)
Animals we e exposed o elec ic oo shock 15h30min p io o he beha iou al es (20 1.5mA
oo shocks (1 shock e e y 30s) o e he cou se o 10min) o induce AAV5-c- os-ChR2-eYFP exp ession
imed wi h he beha iou al es ing.
RTPP appa a us co esponded o a cus omized ac ylic a ena (60cm x 60cm x 40cm), comp ised
by wo indis inguishable chambe s ailo ed wi h a con inual s iped pa e n and connec ed by a cen al
opening.
RTPP was pe o med in ag eemen wi h he p ocedu e used by Soa es-Cunha e al. (2020), and
mice could explo e he appa a us eely o a pe iod o 15min. One chambe (ON/S imula ion chambe )
was pai ed wi h ligh s imula ion, while he o he was pai ed wi h no ligh s imula ion (OFF/No S imula ion
chambe ). Mice s a ed he es in he No S imula ion chambe and op ical s imula ion would be ac i a ed
41
immedia ely upon c ossing in o he S imula ion chambe . The ime spen in each chambe was manually
e alua ed. The S imula ion chambe was coun e balanced ac oss subjec s. Da a a e p esen ed as o al
ime spen (s) in each chambe .
3.4.5.2. Condi ioned Place P e e ence (CPP)
Mice we e subjec ed o elec ic oo shock 15h30min p io o he condi ioning sessions (20 oo
shocks a 1.5mA (1 shock e e y 30s) ac oss 10min), and con ols we e le o explo e he appa a us o
10min. This was done o ime he AAV5-c- os-ChR2-eYFP exp ession induc ion.
CPP was execu ed in a h ee compa men appa a us, wi h wo main chambe s (“whi e” and
“black”) di ided by a neu al a ea (Med Associa es Inc., S . Albans, VT, USA). The wo side chambe s had
dis inc designs on bo h he loo ing and walls.
CPP was execu ed in ag eemen wi h (Coimb a e al., 2017), comp ising h ee sepa a e s ages
ac oss h ee days. On day 1, a p e- es was ca ied ou , in which he animals we e ee o mo e in he
appa a us o 15min, wi h no s imula ion. Shock exposu e was pe o med la e on he same day. On day
2, wo 30min condi ioning sessions we e pe o med, in which animals we e con ined o a speci ic
chambe (session 1 was in he ON/S imula ion chambe , while session 2 was in he OFF/No S imula ion).
The chambe designa ed as s imula ion-pai ed was andomly assigned and coun e balanced h oughou
animals. On day 3 (pos - es ), mice we e again allowed o eely explo e he appa a us o 15min and he
ime spen on each chambe was e alua ed by an au oma ed pho o-beam sys em (Med Associa es Inc.,
S . Albans, VT, USA). Subjec s we e placed in he neu al a ea, wi h he doo s o he main chambe s
being opened a he s a o he wo es sessions. Resul s we e p esen ed in wo a ios: a io 1 – di e ence
be ween he ime spen in he S imula ion chambe in pos - es day and he ime in he same chambe
on p e- es day; a io 2 – di e ence be ween he ime spen in he S imula ion chambe in pos - es day
and he ime spen in he No S imula ion chambe in pos - es day.
3.4.5.3. Op ical s imula ion
Op ogene ic s imula ion was pe o med using blue ligh , a 20Hz, wi h 60 5ms pulses o ligh
being deli e ed e e y 5s o he condi ioned place p e e ence, and o all he pe iod emaining in he
S imula ion side in he RTPP.
Blue ligh was p oduced by a 473nm DPSS lase (CNI Lase , Changchun, China) and supplied
o he b ain ia a ibe op ic pa ch co d (0.22 NA, 200μm diame e ; Tho labs, New on, NJ, USA), which
was connec ed o he implan ed e ule du ing he es . Lase ou pu was egula ed by a Mas e -8 pulse
gene a o (A.M.P.I., MN, USA).
42
3.5. His ological p ocedu es
3.5.1. Sac i ice and b ain sec ioning
Mice we e deeply anes he ized by a mix u e o ke amine/mede omidine 90min/8h/16h a e
s imulus exposu e. Animals we e hen ansca dially pe used wi h 0.9% saline, ollowed by 4%
pa a o maldehyde (PFA) solu ion. B ains we e ca e ully emo ed and imme sed o 48h in 4% PFA o
ixa ion and hen insed and s o ed in 30% o suc ose a 4°C un il sec ioning. Sec ioning was pe o med
co onally, in 40µm slices, on a ib a ing mic o ome (VT1000S, Leica, Ge many) and slices we e s o ed
a 4ºC on 12-well pla es (o long- e m s o age in c yop o ec an solu ion a -20°C) un il use. Slices om
he a eas o in e es (NAc and Amygdala) we e selec ed using he Mouse B ain A las (Paxinos & F anklin,
2001).
3.5.2. Immuno luo escence (IF) o c- os and GFP de ec ion
Below ollows he gene al immuno luo escence p ocedu e, ollowed by he speci ic an ibodies
and incuba ion condi ions u ilized o each pa icula case. Speci ic an ibodies and incuba ion condi ions
a e de ailed in Table 2.
B ain slices we e washed i s wi h phospha e bu e ed saline (PBS; 1X), and hen wi h
PBS/T i on-X100 (0.3%) (PBS-T). An an igen e ie al s ep was pe o med using hea ed ci a e bu e (1X),
o unco e epi opes and dis up po en ial p o ein c oss-links. Blocking, o a oid unspeci ic binding, was
execu ed o 30min using 10% e al bo ine se um (FBS; In i ogen, MA, USA) in PBS-T a oom
empe a u e (RT). P ima y an ibody incuba ion was pe o med o e nigh , ollowed by PBS-T washes and
subsequen incuba ion wi h he app op ia e seconda y luo escen an ibody. All an ibodies we e dilu ed
in PBS-T wi h 2% FBS. Slices we e washed wi h PBS-T, incuba ed wi h DAPI (4’,6-diamidino-2-
phenylindole; 1:1000), washed wi h PBS (1X) and moun ed using Pe ma luo (moun ing media;
In i ogen, MA, USA). Slides we e s o ed a 4°C and kep p o ec ed om ligh .
43
Table 2 – An ibody in o ma ion o all immuno luo escence (IF) p o ocols used.
IF
Species
Binding
Concen a ion
Incuba ion
Condi ions
No e
c- os
abbi
an i-c- os
1:1000
o e nigh ,
RT
AB_2314042 *
c- os
seconda y
goa
an i- abbi
1:1000
2h, RT
Alexa Fluo ® 488
**
GFP
goa
an i-GFP
1:500
o e nigh ,
4°C
ab6673 ***
GFP
seconda y
donkey
an i-goa
1:500
2h, RT
Alexa Fluo ® 488
* Me ck Millipo e, Bu ling on, MA, USA ** In i ogen, Ca lsbad, CA, USA *** Abcam, Camb idge, UK
3.6. Image acquisi ion and analysis
Images we e collec ed and analysed by in e ed luo escence mic oscopy (Olympus Wide ield
In e ed Mic oscope IX81). Abou 5-10 slices o each animal we e used o each analysis. Slices we e
classi ied in e ms o s e eo axic coo dina es using he Mouse B ain A las (Paxinos & F anklin, 2001) and
he e alua ed egions (
i.e.
he BLA, CeA and he main NAc sub egions, co e and shell) we e d awn and
measu ed in e ms o a ea (mm2) using Fiji (ImageJ) so wa e (Schindelin e al., 2012).
Quan i ica ion o neu onal ac i a ion (c- os+ cell densi y) was pe o med using he Cell Coun e
plugin in he Fiji (ImageJ) so wa e (Schindelin e al., 2012). Cell densi ies we e p esen ed as cells pe
mm2.
In densi ome y analysis, images (g een luo escen p o ein – GFP - s aining) we e con e ed o
g eyscale. Fluo escence le els, se ing as a ec o exp ession measu e, we e ob ained as he mean g ey
alue (a e age g ey alue o pixels wi hin he selec ion). Fluo escence a ea (pe cen age o labelled a ea
wi hin a gi en selec ion, ob ained by de ining a pixel in ensi y h eshold o he backg ound) was also
e alua ed. This assessmen was ully execu ed in he Fiji (ImageJ) so wa e (Schindelin e al., 2012).
Op ic ibe placemen was assessed in he animals subjec ed o op ogene ic manipula ion o
con i m i ligh s imula ion was accu a ely conduc ed in he in ended BLA egion. Fo ha , slices whe e
he op ic ibe was de ec ed we e classi ied acco ding o Paxinos & F anklin (2001) o es ima e he
s e eo axic coo dina es.
50
Figu e 9 – Iden i ica ion o neu onal popula ions esponsi e o nega i e alence s imuli – endogenous c- os+
cell densi y in he Amygdala. Rep esen a i e GFP immuno luo escence images showing endogenous c- os exp ession in
he BLA and CeA o a a) Shock- and a b) No Shock-exposed mouse (20x - le ; 40x - igh ). A ows poin o a ew examples o
c- os+ cells. Scale ba 400µm (le ), 40µm ( igh ). c) c- os+ cell densi y (cells mm-2) in he BLA 90min pos - oo shock exposu e
(Shock, n=9; No Shock, n=5). d) c- os+ cell densi y (cells mm-2) in he CeA 90min pos - oo shock exposu e (Shock, n=9; No
Shock, n=5). **
p
≤ 0.001. Da a a e ep esen ed as mean ± SEM.

51
Figu e 10 – Iden i ica ion o neu onal popula ions esponsi e o posi i e alence s imuli – endogenous c-
os+ cell densi y in he Amygdala. Rep esen a i e GFP immuno luo escence images showing endogenous c- os
exp ession in he BLA and CeA o a a) Cocaine- and a b) Saline-exposed mouse (20x - le ; 40x - igh ). A ows poin o a ew
examples o c- os+ cells. Scale ba 400µm (le ), 40µm ( igh ). c) c- os+ cell densi y (cells mm-2) in he BLA 90min pos -
cocaine injec ion (Cocaine, n=8; Saline, n=5). d) c- os+ cell densi y (cells mm-2) in he CeA 90min pos -cocaine injec ion
(Cocaine, n=8; Saline, n=5). Da a a e ep esen ed as mean ± SEM.
52
4.2. Tempo al cha ac e iza ion o a c- os-d i en i al ec o
In o de o label neu ons p e iously esponsi e o posi i e o nega i e alence s imuli (c- os-
exp essing neu ons) we used a i al ec o ha con ains channel hodopsin (ChR2) unde he con ol o
he c- os p omo e egion (AAV5-c- os-ChR2-eYFP ec o ) (Figu e 11a), which has been p e iously
epo ed in (Ye e al., 2016). This cons uc allows exp ession o ChR2 used wi h a yellow luo escen
p o ein (eYFP), in neu ons ha a e ac i a ed. I has a window o exp ession (
i.e.
labels neu onal ac i i y)
o ≈6h a e c- os ac i a ion and pe du ance (
i.e.
allows obse a ion and manipula ion o labelled neu ons)
o up o one day (DeNa do & Luo, 2017). The i us AAV5-c- os-eYFP (Figu e 11a) which does no con ain
he ChR2 sequence was used as a con ol.
Wild- ype C57BL/6J mice we e injec ed wi h AAV5-c- os-ChR2-eYFP bila e ally in he NAc and in
he BLA (Figu e 11b). Th ee weeks a e su ge y, animals we e subjec ed o elec ic oo shocks o allow
c- os-induced ChR2-eYFP exp ession in ac i a ed neu ons (Figu e 11c). To selec an op imal imepoin
o i al labelling and op ical s imula ion o be used in he emaining wo k, eYFP exp ession was assessed
8h and 16h a e oo shock exposu e (Figu e 11).
Fo bo h NAc and BLA, animals ecei ing oo shock 8h p io o sac i ice (Shock 8h g oup) had
e y low le els o i al exp ession (Figu e 11d and 11 , espec i ely), while animals ecei ing oo shock
16h p io o sac i ice (Shock 16h) had s onge i al exp ession (Figu e 11e and 11g, espec i ely; No
Shock was used as he con ol g oups - Figu e 11h and 11i). Thus, 16h imepoin was chosen o
u u e expe imen s.
53
54
Figu e 11 – Expe imen al design o i al ec o empo al cha ac e iza ion. a) Schema ic ep esen a ion o he
plasmid ec o s used in he expe imen ; AAV5-c- os--eYFP (le ); AAV5-c- os-ChR2-eYFP ( igh ). b) S e eo axic su ge ies o
injec ion o he i ally encapsula ed cons uc pAAV5-c- os-ChR2-eYFP we e pe o med bila e ally in he NAc and in he BLA.
c) Mice we e exposed o 20 1.5mA oo shocks o e 10min. Con ols we e le o explo e he chambe s o 10mim (No Shock
g oup). Sac i ice was pe o med ei he 8h (Shock 8h g oup) o 16h (Shock 16h g oup); US – uncondi ioned s imulus.
Rep esen a i e GFP immuno luo escence images showing i al exp ession in he NAc in he d) Shock 8h and in he e) Shock
16h g oup (20x). Rep esen a i e GFP immuno luo escence images showing i al exp ession in he BLA in he ) Shock 8h
g oup and in he g) Shock 16h (20x). Rep esen a i e GFP immuno luo escence images showing i al exp ession in he h) NAc
in he No Shock g oup and in he i) BLA in he No Shock g oup (20x). Scale ba s 400µm.
4.3. T ansien labelling and iden i ica ion o neu onal popula ions esponsi e o
posi i e
s
nega i e alence s imuli
Wild- ype C57BL/6J mice we e injec ed wi h he AAV5-c- os-eYFP o allow eYFP exp ession in he
NAc and BLA in esponse o ei he a posi i e o nega i e alence s imulus.
Fou weeks a e in ec ion, animals we e subjec ed o a oo shock o cocaine injec ion as
desc ibed be o e (16h imepoin ). As an indica o o neu onal ac i a ion, we calcula ed luo escence
in ensi y o each b ain egion gi en in he o m o he mean g ey alue (MGV), and he pe cen age (%) o
he a ea p esen ing YFP exp ession (see me hods o de ails abou he calcula ion). Foo shock exposu e
did no inc ease eYFP exp ession gi en by he MGV nei he in he NAc co e no shell (NAc co e – Figu e
12c;
(12) = 0.9221;
p
= 0.3746; Shock: 31.31±2.296; No Shock: 27.68±3.314; NAc shell – Figu e
12d;
(12) = 0.8334;
p
= 0.4209; Shock: 30.41±2.295, No Shock 27.19±3.137). In e ms o a ea o
i al exp ession, analysis o luo escen a ea e ealed no di e ences be ween Shock and No Shock
animals in bo h NAc sub egions (NAc co e – Figu e 12e;
(11) = 1.463,
p
= 0.1715; Shock:
65.15±3.769%; No Shock: 73.98±2.673%; NAc shell – Figu e 12 ;
(11) = 0.6649,
p
= 0.5198; Shock:
68.78±3.338%; No Shock: 63.74±8.134%).
Simila ly, conce ning cocaine adminis a ion, no di e ences we e ound in e ms o luo escence
in ensi y o bo h NAc sub egions (NAc co e – Figu e 13c;
(11) = 0.3018
p
= 0.7684; Cocaine: 28.09±-
2.976; Saline: 26.84±2.086; NAc shell – Figu e 13d;
(11) = 0.3744;
p
= 0.7153; Cocaine:
25.14±2.818, Saline: 23.72±1.487. Rega ding luo escence a ea, no di e ences we e obse ed due o
he cocaine injec ion (NAc co e – Figu e 13e;
(11) = 1.064,
p
= 0.3102; Cocaine: 62.87±5.734%;
Saline: 71.68±4.901%; NAc shell – Figu e 13 ;
(11) = 0.5023,
p
= 0.6254; Cocaine: 52.40±9.244%;
Saline: 59.27±8.786%).
55
Rega ding he BLA analysis, his b ain egion showed a endency o inc eased ac i a ion by
oo shock, as e lec ed in he MGV da a (Figu e 14c;
(11) = 1.913,
p
= 0.0821; Shock: 46.15±1.884;
No Shock: 36.31±5.901). No changes we e ob ained in e ms o i al exp ession a ea (Figu e 14d;
U
= 18,
p
= 0.8329; Shock: 75.45±6.465%; No Shock: 74.63±15.04%). Fu he mo e, no e ec s we e ound
in luo escence in ensi y in ol ing cocaine exposu e in his egion (Figu e 15c;
(10) = 0.04114;
p
=
0.9680; Cocaine: 35.61±4.929; Saline: 35.24±8.250). Simila ly, i al exp ession a ea e lec ed no
al e a ions due o cocaine adminis a ion (Figu e 15d;
(10) = 0.2899,
p
= 0.7778; Cocaine:
63.05±11.45%; Saline: 69.00±17.97%). I should be no ed ha wo animals we e emo ed om BLA
analysis due o he lack o i al exp ession in he sec ions, which likely indica es a p oblem wi h i al
injec ion, as we expec o always see some deg ee o YFP exp ession.
In sum, endogenous c- os (c- os+ cell densi y) and densi ome y luo escence da a sha ed simila
indings, namely a simila endency o highe neu onal ac i a ion in he BLA in esponse o oo shock,
bu no signi ican changes in esponse o cocaine.

56
57
Figu e 12 – Quan i ica ion o c- os d i en eYFP exp ession in he NAc in esponse o nega i e alence s imuli.
Rep esen a i e GFP immuno luo escence images showing i al exp ession in he NAc o a a) Shock- and a b) No Shock-
exposed mouse (4x – op igh ; 20x – bo om le ; 40x – bo om igh ) and espec i e b ain a las scheme ( op le ) (AP:
0.98mm; 0.86mm). Scale ba 400µm (bo om le ), 40µm (bo om igh ) 1mm ( op igh ). c) Immuno luo escence in ensi y
(16h pos - oo shock exposu e) measu ed by he mean g ey alue (MGV) wi hin he de ined NAc co e (Shock, n=9; No Shock,
n=5). d) Immuno luo escence in ensi y (16h pos - oo shock exposu e) measu ed by he MGV wi hin he de ined NAc shell
(Shock, n=9; No Shock, n=5). e) A ea o immuno luo escence (16h pos - oo shock exposu e) labelled wi hin he de ined NAc
co e (Shock, n=9; No Shock, n=4). ) A ea o immuno luo escence (16h pos - oo shock exposu e) labelled wi hin he de ined
NAc shell (Shock, n=8; No Shock, n=5). Da a a e ep esen ed as mean ± SEM.
58
59
Figu e 13 – Quan i ica ion o c- os d i en eYFP exp ession in he NAc in esponse o posi i e alence s imuli.
Rep esen a i e GFP immuno luo escence images showing i al exp ession in he NAc o a a) Cocaine- and a b) Saline-exposed
mouse (4x – op igh ; 20x – bo om le ; 40x – bo om igh ) and espec i e b ain a las scheme ( op le ) (AP: 0.86mm;
1.70mm). Scale ba 400µm (bo om le ), 40µm (bo om igh ) 1mm ( op igh ). c) Immuno luo escence in ensi y (16h pos -
cocaine injec ion) measu ed by he MGV wi hin he de ined NAc co e (Cocaine, n=8; Saline, n=5). d) Immuno luo escence
in ensi y (16h pos -cocaine injec ion) measu ed by he MGV wi hin he de ined NAc shell (Cocaine, n=8; Saline, n=5). e) A ea
o immuno luo escence (16h pos -cocaine injec ion) labelled wi hin he de ined NAc co e (Cocaine, n=8; Saline, n=4). ) A ea
o immuno luo escence (16h pos -cocaine injec ion) labelled wi hin he de ined NAc shell (Cocaine, n=8; Saline, n=5). Da a
a e ep esen ed as mean ± SEM.
66
CHAPTER 5 – Discussion, Conclusion
and Fu u e Pe spec i es

67
5.1. Discussion
Comp ehending he neu onal mechanisms unde lying ewa d and a e sion can ad ance he
knowledge on he pa hophysiology o emo ional diso de s wi h de ici s in hese p ocesses such as
dep ession and addic ion (Coope e al., 2017; Dich e e al., 2012; Russo & Nes le , 2013). To
accomplish ha i is essen ial o de ine how posi i e and nega i e alence is encoded and p ocessed in
e ms o neu onal popula ions, which may occu in a comple ely seg ega ed o , o some ex en ,
o e lapped manne . How a speci ic alence is encoded may be ela ed wi h, o example, inpu s and
ou pu s o dis inc gene ic p o iles o he neu ons in ol ed, o e en a combina ion o ana omical and
gene ic cha ac e is ics. Though se e al b ain egions o he limbic sys em, including he nucleus
accumbens (NAc) (Be idge & K ingelbach, 2013; Knowland & Lim, 2018; Nambu i e al., 2016; Nieh e
al., 2013; O’Neill e al., 2018), a e known o encode alence, he ype o cha ac e is ics ha seg ega es
posi i e om nega i e alence neu ons emains a om being disclosed.
Rec ui men o NAc and amygdala ensembles upon exposu e o posi i e
s
nega i e alence
s imuli
NAc neu ons a e known o espond o bo h uncondi ioned and condi ioned s imuli o posi i e and
nega i e alence (Ca lezon & Thomas, 2009). NAc neu ons change hei ac i i y in esponse o p ima y
a e si e s imuli (
e.g.
quinine; ai pu s) (Roi man e al., 2005; Yanagimo o & Maeda, 2003) and p ima y
na u al ewa ds, such as suc ose and saccha in (Roi man e al., 2005; Wheele e al., 2005, 2008;
Wilson & Bowman, 2004). They a e also essen ial o p ocessing appe i i e s imuli deli e ed ia sel -
adminis a ion pa adigms, such as suc ose (Nicola e al., 2004a), ood (Ca elli, 2002; Ca elli e al., 2000),
wa e (Ca elli e al., 2000), cocaine (Chang e al., 1998; Peoples e al., 1998; Peoples & Wes , 1996),
he oin (Chang e al., 1997, 1998) and e hanol (Janak e al., 1999). Gi ing c edi o compa a i e
expe imen s wi h a ied s imuli, neu onal p ocessing is no equal ac oss ewa d ypes (“na u al” and
“d ugs o abuse”) o e en be ween speci ic ewa ds o simila na u e (wa e
s
cocaine, (Ca elli &
Deadwyle , 1994; Ca elli & Wondolowski, 2003); wa e
s
ood
s
cocaine, (Ca elli, 2002; Ca elli e al.,
2000); cocaine
s
he oin, (Chang e al., 1998); wa e
s
suc ose, (Roop e al., 2002)).
Simila ly, amygdala neu ons encode a a ie y o appe i i e and a e si e s imuli, such as wa e
and ai -pu s, espec i ely (Belo a e al., 2007). The BLA in pa icula , has popula ions esponding o
ewa ds like suc ose (Mu amo o e al., 1993), wa e (Kim e al., 2016) and nico ine (Go e e al., 2015a)
and nega i e s imuli like oo shock (Go e e al., 2015a; Kim e al., 2016; Mu amo o e al., 1993) and
unpala able as es (Shabel e al., 2011). Likewise, he CeA has neu onal popula ions eac ing o oo shock
68
(nega i e) and suc ose (posi i e) (S einbe g e al., 2020). Due o he ac ha di e en s udies ha e shown
ha amygdala neu ons espond o bo h posi i e and nega i e alence s imuli, we chose his egion o be
used as a
posi i e con ol
o he expe imen s p esen ed in his hesis.
Tools o iden i y alence esponsi e neu ons
Endogenous c- os o quan i y neu onal popula ions esponsi e o posi i e
s
nega i e alence
s imuli
A common basis in s a egies used o unde s and i /how neu onal popula ions a e in ol ed in
ce ain beha iou s, o esponsi e o pa icula s imuli, is he e alua ion o he exp ession o immedia e
ea ly genes (IEG) (
i.e.
c- os
), which a e exp essed apidly upon neu onal ac i i y (Deubne e al., 2019).
Labelling endogenous c- os is ypically used o assess neu onal ac i a ion on sho - e m analysis, as
mRNA le els peak a ound 30-45min, wi h p o ein le els peaking a ound 1-2h (depending on speci ic b ain
egion and condi ions) (Bisle e al., 2002; Mülle e al., 1984; Zangenehpou & Chaudhu i, 2002).
Reac ion o s imuli wi h a speci ic alence has hus been obse ed ia c- os exp ession le els in a ied
egions - ei he ia mRNA le els, o p o ein, usually by c- os+ cell coun s.
To iden i y neu onal popula ions encoding posi i e- and nega i e- alence s imuli we s a ed by
injec ing mice wi h cocaine o exposing hem o oo shock, espec i ely, and hen labelled endogenous c-
os o quan i y neu onal ac i a ion in he NAc co e and shell, as well as in he BLA and in CeA. Elec ic
oo shock was deli e ed a an in ensi y known o induce neu onal ac i a ion in he BLA, as well as
gene a ing nega i e- alence esponses (Go e e al., 2015a). Cocaine was injec ed a a concen a ion
p e iously shown o enhance mo o ac i i y and induce CPP, hus ha ing a obus appe i i e alue (Ca low
& Ki s ein, 2005; I zhak & Ma in, 2002).
Ou da a showed ha elec ic oo shock appea s o be encoded by neu ons in he NAc co e, since
neu onal ac i a ion was highe in Shock animals in compa ison o con ol animals, while he NAc shell
did no p esen changes in he c- os+ posi i e cell densi y. A summa y o ou main esul s is p esen ed in
Table 3. This da a is in ag eemen wi h p e ious li e a u e, showing an o e all inc ease in NAc c- os-
labelled ac i a ion o many a e si e e en s, such as he case o o malin injec ion (Senba & Ueyama,
1997), acu e o ced swim s ess (Cullinan e al., 1995), audiogenic s ess (Campeau & Wa son, 1997)
and es ain s ess (Cullinan e al., 1995; Senba & Ueyama, 1997). On he con a y, we saw no s a is ical
di e ence in he neu onal ac i a ion in cocaine g oup in compa ison o saline, o nei he NAc sub egion.
This esul was unexpec ed, conside ing ha he NAc is known o be a cen al ewa d sys em egion
(Be idge & K ingelbach, 2015; Ca lezon & Thomas, 2009; Russo & Nes le , 2013), and in pa icula
69
since i has been shown o exhibi an inc ease in NAc c- os le els a e exposu e o bo h na u al ewa ds
(suc ose; (Koekkoek e al., 2021)) and d ugs o abuse such as mo phine (Liu e al., 1994), cocaine
(Johansson e al., 1994) and amphe amine (Dalia & Wallace, 1995; Johansson e al., 1994), along wi h
social no el y (Gómez-Gómez e al., 2019).
We pe o med simila analysis o he amygdala subnuclei - BLA and CeA, as a con ol s a egy,
expec ing neu onal ac i a ion o bo h alence s imuli. c- os+ cell densi y was highe in he BLA ollowing
shock exposu e. These esul s we e an icipa ed, pa icula ly conside ing he wo k o Go e e al. (2015a),
who ha e epo ed an inc ease in BLA c- os+ neu ons a e oo shock. No di e ences we e ound in he
CeA a e oo shock exposu e (al hough a endency was p esen ), which was somewha su p ising,
conside ing his egion’s gene al in ol emen in a e si e lea ning (Gio anniello e al., 2020) and p e ious
desc ip ions o shock- esponsi e neu ons p ojec ing om he CeA o he
subs an ia nig a
(S einbe g e
al., 2020). Rega ding he cocaine g oup, no e ec was ound in nei he he BLA no he CeA, wi h no
di e ences in neu onal ac i a ion be ween d ug and ehicle (saline) injec ion. Bo h subnuclei ha e been
obse ed o include neu onal popula ions esponding o appe i i e s imuli: Go e e al. (2015a), ha ing
demons a ed an inc ease in BLA c- os-labelled neu ons a e nico ine exposu e; and suc ose- esponsi e
cells p esen ed in he CeA (S einbe g e al., 2020).
While da a in he NAc co e and he BLA egions seemed in line wi h p e ious s udies using
a e si e condi ions, he da a wi h cocaine was unan icipa ed. In his con ex , we can pinpoin some
echnical issues as po en ial explana ion o he indings. Fi s , i is impo an o conside ha he numbe
o animals is ela i ely low, which may hampe he iden i ica ion o sub le neu onal ac i a ion di e ences
be ween g oups. Second, i is impo an o e e ha animals we e expe imen ally naï e p io o
beha iou al exposu es, wi h handling in he week p e ious o es ing and wi h only h ee 10min
habi ua ion sessions o he appa a us ( o Shock and No Shock animals). I is plausible ha inc easing
he ime and equency o he habi ua ion o he appa a us and expe imen al ooms could educe
“unspeci ic” neu onal ac i a ion due o no el y ( a he han o shock). Rega ding posi i e alence s imulus
expe imen , we should ha e habi ua ed animals o I.P. injec ions p io o he beha iou al p ocedu e
(cocaine and saline g oups) o minimize neu onal ac i a ion due o he injec ion ( ha is a e si e) a he
han cocaine
pe se,
as his p ocedu e is pain ul and can cause acu e dis ess in he animals. We belie e
ha hese condi ions would minimize unspeci ic neu onal ac i a ion le els, and lead o clea e esul s.
Apa om expe imen al condi ions ha could be imp o ed, one should also conside some o he
explana ions o he esul s. Fi s , maybe NAc neu ons do no encode cocaine posi i e alence as we
assumed i would, hough his is unlikely conside ing he elec ophysiological da a suppo ing changes in
70
neu onal i ing wi hin he NAc in esponse o ewa d consump ion, and in pa icula cocaine (Ca lezon &
Thomas, 2009; Chang e al., 1997; Peoples & Wes , 1996; Roi man e al., 2005; Wheele e al., 2005,
2008; Wilson & Bowman, 2004), as well calcium imaging da a suppo ing an inc ease o NAc ac i i y in
esponse o cocaine-associa ed cues (Calipa i e al., 2016). Second, one could hypo hesize ha c- os
ac i a ion is no he i es ma ke o e alua e alence ec ui men in he NAc, and ha o he IEGs could
be mo e sensi i e – such as
A c
o
Zi 268
(Choi e al., 2020; Go e e al., 2015b; Ko ács, 2008).
Tempo al cha ac e iza ion o a c- os-d i en i al ec o o label neu onal popula ions esponsi e
o posi i e
s
nega i e alence s imuli
Addi ional con en ional c- os-dependen echniques inco po a e he exp ession o speci ic genes
associa ed wi h IEG gene p omo e s (Deubne e al., 2019). A cons uc can be encapsula ed in a i al
ec o and injec ed in b ain egions o in e es o locally exp ess (in an ac i i y- elian manne ), o
ins ance, luo escen labels o neu onal acing (
e.g.
eYFP) (Deubne e al., 2019), o ex insic ion
channels o induce neu onal ac i a ion (
e.g.
ChR2) (Ye e al., 2016). Fo ou app oach, we selec ed a
simila s a egy, using a c- os-dependen i al ec o o assess neu onal ac i a ion a e exposu e o
elec ic oo shock (nega i e alence) and cocaine adminis a ion (posi i e alence). The i al app oach
was p e iously used o analyse he molecula p ope ies o neu ons in he PFC ha a e associa ed wi h
dis inc beha iou al expe iences (Ye e al., 2016).
We i s s a ed by alida ing his cons uc , ega ding op imal ime ame and he abili y o
ansien ly label NAc and amygdala neu ons ac i a ed by speci ic- alence s imuli (simila ly o endogenous
c- os). By using bo h endogenous c- os and c- os-d i en i al exp ession o a cons uc , wo s imuli o
appe i i e and a e si e alence could be combined, and esponsi e popula ions could be hus compa ed
in e ms o o e lap and seg ega ion and ana omical dis ibu ion, amongs o he ac o s. A compa able
app oach (wi h len i i us) has been used success ully in he BLA by Go e e al. (2015a), demons a ing
ana omically dis inc ye ma ginally o e lapped popula ions esponding o nega i e- and posi i e- alence
s imuli.
We i s cha ac e ized he ec o in e ms o empo al exp ession le els, and selec ed 16h pos -
s imulus imepoin due o s onge exp ession le els in compa ison o 8h imepoin . Conce ning ou
s imuli-induced luo escence da a, he cons uc showed e y limi ed success in labelling salien s imuli
o nega i e and posi i e alence. The BLA did exhibi a end ega ding pAAV5-c- os-eYFP luo escence
in ensi y a e oo shock exposu e (nega i e alence), hough no signi ican . No di e ences we e ound
ega ding cocaine g oup. I is, howe e , no de ini e whe he he lack o signi ican c- os-dependen i al
71
exp ession in ou da a is due o he ine iciency o he i al app oach i sel o , o ins ance, he
a o emen ioned possibili y o p ocedu al con ounding e ec s in he beha iou al p o ocol.
Rega ding he i al app oach i sel , i is ele an o conside ha ansduc ion a es luc ua e
ac oss s a egies. When u ilizing ecombinan adeno-associa ed i uses ( AAVs) – equen ly used due o
hei e icacy –, ansduc ion a es o he many se o ypes change no only ac oss issues, bu h oughou
di e en b ain egions and neu on ypes (Van Vlie e al., 2008). Rema kably, a compa a i e s udy using
AAV i al cons uc s ha we e pseudo yped in a ied AAV se o ypes, ei he o ansduce inhibi o y BLA
neu ons o glu ama e gic neu ons, ound ha he AAV5 se o ype showed much lowe
exp ession/ ansduc ion le els han o he se o ypes (de Solis e al., 2017). A simila se o ype compa ison
wi h an AAV2 genome plasmid and CamKII-d i en exp ession also ound lesse ansduc ion le els o he
AAV5 se o ype (Holehonnu e al., 2014). Conce ning po en ial ansduc ion issues in he NAc, al hough
he AAV5 se o ype has been shown o e ec i ely label s ia al issue (Aschaue e al., 2013; Ma kakis e
al., 2010; Taymans e al., 2007), he o e all ocus appea ed o be on mo e do sal sec ions o he s ia um.
Though nei he luo escence in ensi y (apa om a endency in he BLA) and a ea o exp ession
indica ed any di e ence in i al exp ession be ween animals exposed o oo shock and cocaine, some
animals (o a ied g oups) p esen ed lowe alues o luo escence a ea due o some slices exhibi ing only
minimal labelling, a possible indica o o low i al di usion. The e o e, ansduc ion issues may a leas
ha e con ibu ed o he puzzling esul s ob ained wi h he i al app oach.
Op ogene ic manipula ion o neu ons esponsi e o nega i e alence s imuli
Valence has, by de ini ion, a di ec impac on beha iou , gi en ha i is comp ised by bo h he
alue o a s imulus and consequen eac ions induced on he indi idual (Be idge, 2019). One o he
simples ex e nal esponses o being subjec ed o salien s imuli o ei he posi i e o nega i e alence is
di ec ed mo i a ed mo emen , o ei he app oach ( ewa ding alue) o a oid (a e si e alue) (Tye, 2018).
App oach and a oidance o s imuli a e commonly assessed ia place p e e ence pa adigms such as he
condi ioned place p e e ence es (CPP) and he eal ime place p e e ence (RTPP). In bo h, one can
associa e a pa icula s imulus o a loca ion and in e i i is ewa ding, a e si e o neu al.
Op ogene ics is commonly used in such pa adigms, in o de o e alua e how ce ain neu on ypes
o popula ions in luence physiological and/o beha iou al e ec s (Beyele e al., 2014). In an a emp o
unc ionally alida e ou pAAV5-c- os-ChR2-eYFP ec o , we used op ogene ics o ac i a e nega i e- alence
neu ons in he BLA and assessed i one could elici an a e si e esponse in a neu al con ex . To do so,
we es ed animals in bo h a eal ime place p e e ence (RTPP) and a condi ioned place p e e ence (CPP)

72
pa adigm. Ou da a showed a end o a oidance o op ical ac i a ion o oo shock-ac i a ed neu ons in
he RTPP es (spending mo e ime in he OFF chambe ), sugges ing a nega i e alence-associa ed
beha iou al e ec . This was expec ed due o ea lie s udies, such as he wo k o Nambu i e al., (2015),
in which op ogene ic ac i a ion o BLA-cen omedial amygdala neu ons c ea ed a oidance o he
s imula ion-pai ed chambe o a eal ime place a oidance ask. Howe e , i is impo an o e e ha i
we excluded one animal in which implan a ion coo dina es we e impossible o de e mine, he e ec is no
so e iden , howe e , he low sample size hampe s he in e p e a ion o he da a. In he CPP, neu onal
ac i a ion o oo shock- esponsi e neu ons was no enough o elici a e sion o he ligh -pai ed chambe .
Thus, we we e no able o induce a oidance in a p e iously neu al con ex in he CPP, as one was
expec ing o. This is in con as wi h p e ious li e a u e indica ing ha s imula ing BLA shock- esponsi e
neu ons in an odou -lea ning ask is su icien o induce a oidance o he ligh -condi ioned chambe (Go e
e al., 2015a). A simila ou come was shown by Redondo e al. (2014) when using a doxycycline (dox)-
based op ogene ic place a oidance es wi h ea condi ioning using oo shock. These esul s a e
pa icula ly puzzling when conside ing ha his egion is hea ily in ol ed wi h ea lea ning (Du a ci &
Pa e, 2014; To o e e al., 2015), in which oo shock is o en employed (Bali & Jaggi, 2015), and wi h
encoding oo shock in pa icula (Go e e al., 2015a). This da a, oge he wi h he esul s om
luo escence analysis, seems o sugges ha his i al app oach is no a sui able s a egy o e alua e
alence- ela ed neu onal di e ences in he NAc (and amygdala) a e exposu e o a e si e and appe i i e
s imuli.
Howe e , i should also be s a ed ha in he RTPP and CPP pa adigms, ac o s like op ical
s imula ion se ings and he numbe and leng h o condi ioning sessions a y be ween pa adigms, and as
such, may in luence he condi ioning esul s. Ou op ogene ic s imula ion was achie ed wi h blue ligh
(473nm), a 20Hz in 5ms pulses (10% du y cycle), o bo h RTPP and CPP, wi h he la e being deli e ed
in 60 ligh pulses e e y 5s. Ou RTPP ollowed he eal ime place a oidance app oach o Nambu i e al.,
(2015) in e ms o s imula ion, while ou CPP ollowed he in ac anial op ical sel -s imula ion (ICSS) ligh
s imula ion p o ocol. In Go e e al. (2015a), he Pa lo ian ask p esen ed an odou co- e mina ing wi h
2s s imula ion (20Hz; 20% du y cycle), while Redondo e al. (2014) used 15ms pulses (20Hz, 30% du y
cycle). Applying an al e na i e op ogene ic p o ocol migh cla i y i he absence o e ec is ela ed wi h he
neu onal ac i a ion pa e n o wi h he i al s a egy used.
73
Table 3 – Summa y o he main esul s ob ained by e alua ing neu onal ac i a ion a e exposu e o nega i e- ( oo shock)
and posi i e- alence (cocaine) s imuli ia c- os+ cell densi y (NAc, BLA, CeA), c- os-dependen eYFP exp ession (NAc, BLA) and
o op ogene ically-induced a oidance beha iou in RTPP and CPP pa adigms due o ac i a ion o BLA shock- esponsi e
neu ons.
− no changes;
↑
highe alues/a oidance beha iou no ed; ≈ (
↑
) endency o highe alues/a oidance beha iou no ed
c- os+ cell densi y
eYFP MGV
eYFP luo escence a ea
RTPP
CPP
NAc
-
↑
−
−
+
−
−
−
BLA
-
↑
≈ (↑)
−
≈ (↑)
−
+
−
−
−
CeA
-
≈ (↑)
+
−
5.2. Conclusion
S udies ha e p o ided e idence in ol ing he NAc in esponding o s imuli o posi i e- and
nega i e- alence. Howe e , which speci ic neu ons p ocess each alence (and how ha a ises) s ill
equi es u he explo a ion.
He e, we ha e shown ha elec ic oo shock signi ican ly ac i a es neu ons o NAc co e and o
he BLA egions o he b ain, as e alua ed by endogenous c- os cell coun ing. Su p isingly, cocaine did
no elici signi ican changes in neu onal ac i a ion in hese b ain egions. Se e al hypo heses could
explain he absence o di e ences in he cocaine g oup, such as he ac ha expe imen al condi ions
we e no op imal – a e y high le el o “unspeci ic” c- os ec ui men was obse ed. One could also
conside ha o he IEG could be e e lec NAc (and amygdala) ac i a ion pa e ns.
We also es ed a i al labelling app oach o apply in mo e complex expe imen s o assess which
neu onal popula ions encode posi i e
e sus
nega i e alence, as well as hei po en ial seg ega ion o
o e lap. The i al s a egy showed a endency o highe neu onal ac i a ion in he BLA wi h oo shock,
wi h no di e ences in he NAc. No di e ences wi h cocaine we e ound. This sugges s ha his
me hodology is no sui able o ou goals.
Op ogene ic ac i a ion o oo shock- esponsi e neu ons in he BLA led o a endency o place
a oidance beha iou in a RTPP pa adigm, a ypical a e sion- ela ed eac ion, bu his e ec was absen
in he CPP es .
74
O e all, ou esul s indica e ha he i al s a egy es ed in his disse a ion is no he bes one o
e alua e alence encoding in he NAc. The e o e, o he ools should be used o label alence- esponsi e
neu ons in he NAc, as b ie ly in oduced in he sec ion below.
5.3. Fu u e pe spec i es
Addi ional s udies a e s ill necessa y o iden i y and cha ac e ize neu onal popula ions encoding
nega i e- and posi i e- alence s imuli in he NAc and s udy neu onal in luence in alence-speci ic
beha iou s. Many ele an me hodological ad ancemen s ha e been de eloped in ecen yea s, allowing
o mo e accu a e and clea iden i ica ion o neu ons.
One o he possible s a egies ha can be used o label alence neu ons is he TRAP sys em
(Ta ge ed Recombina ion in Ac i e Popula ions; (Guen hne e al., 2013)) (wi h he TRAP2 mouse line).
This ansgenic mouse line allows pe manen gene ic access o neu ons ac i a ed by a gi en s imulus
(Allen e al., 2017; DeNa do e al., 2019). This sys em is also c- os-d i en and leads o exp ession o a
amoxi en-inducible C e ecombinase (C eER) in ac i a ed cells. By in oducing a C e-dependen e ec o
gene (ei he ansgenic o i ally-deli e ed), one can empo ally es ic he exp ession o he e ec o gene
o a pa icula momen in ime. This ensu es ha only in neu ons ha a e ac i a ed in he p esence o
amoxi en, C e-based ecombina ion akes place, leading o pe manen exp ession o he e ec o gene. I
he e ec o gene encodes a cons i u i ely luo escen p o ein such as dToma o, we can ha e a ime-
locked, c- os-speci ic labelling o neu onal popula ions ac i a ed by speci ic alence s imuli. This s a egy
has been used wi h success, o example, o label and cha ac e ize wa e dep i a ion- esponsi e neu ons
in he hypo halamic median p eop ic nucleus (Allen e al., 2017), s udy he in luence o p elimbic neu ons
in emo e memo y e ie al (DeNa do e al., 2019), s udy he in ol emen o NAc co e D1- and D2-MSNs
in suc ose- and cocaine-seeking (Bobadilla e al., 2020) and o label quinine- esponsi e disgus -associa ed
neu ons in he CeA (Tanaka e al., 2021).
Op o agging (Pho os imula ion-assis ed Iden i ica ion o Neu onal Popula ions (PINP); (Lima e
al., 2009)) enables he classi ica ion o neu ons in
in i o
elec ophysiological eco dings, ia op ical
a ge ing o speci ic gene ically-iden i ied neu onal popula ions (
e.g.
using C e-d i en opsins) and analysis
o he op ical-e oked esponse. This has been used, o ins ance, o s udy alence in BLA p ojec ions by
Beyele e al. (2018).
Addi ionally, single-cell RNA sequencing (scRNA-seq) ools allow ansc ip ome-wide gene ic
cha ac e iza ion o single cells (Ziegenhain e al., 2017). This can be applied o iden i y ac i a ed neu onal
popula ions (by ocusing on IEG exp ession and po en ial gene ic ma ke s) in an unbiased and high-
75
h oughpu manne . Wu e al. (2017), o example, de eloped Ac -seq (Ac i a ed Cell Popula ion
Sequencing) - an scRNA-seq app oach, and used i o s udy neu onal ac i a ion by acu e s ess in he
medial amygdala.
O e all, his oolbox (and o he s) opens new a enues o a be e unde s anding o neu onal
ac i a ion in speci ic NAc subpopula ions due o exposu e o posi i e and nega i e alence s imuli. This
knowledge on alence encoding migh , in a long e m pe spec i e, iden i y a ge neu onal subpopula ions
o mechanisms ha would con ibu e o de eloping new molecula app oaches o ea men o
emo ional diso de s wi h NAc dis unc ions.
82
o Neu ode elopmen al Diso de s
,
4
(1), 1–43. h ps://doi.o g/10.1186/1866-1955-4-19
Douglass, A. M., Kucukde eli, H., Ponse e, M., Ma ko ic, M., G ündemann, J., S obel, C., Alcala
Mo ales, P. L., Conzelmann, K. K., Lü hi, A., & Klein, R. (2017). Cen al amygdala ci cui s modula e
ood consump ion h ough a posi i e- alence mechanism.
Na u e Neu oscience
,
20
(10), 1384–
1394. h ps://doi.o g/10.1038/nn.4623
Du ieux, P. F., Bea za o, B., Guiducci, S., Buch, T., Waisman, A., Zoli, M., Schi mann, S. N., & De
Ke cho e D’Exae de, A. (2009). D2R s ia opallidal neu ons inhibi bo h locomo o and d ug ewa d
p ocesses.
Na u e Neu oscience
,
12
(4), 393–395. h ps://doi.o g/10.1038/nn.2286
Du a ci, S., & Pa e, D. (2014). Amygdala mic oci cui s con olling lea ned ea .
Neu on
,
82
(5), 966–980.
h ps://doi.o g/10.1016/j.neu on.2014.04.042
Fanselow, M. S. (1994). Neu al o ganiza ion o he de ensi e beha io sys em esponsible o ea .
Psychonomic Bulle in & Re iew
,
1
(4), 429–438. h ps://doi.o g/10.3758/BF03210947
Fanselow, M. S. (2018). The ole o lea ning in h ea imminence and de ensi e beha io s.
Cu en
Opinion in Beha io al Sciences
,
24
(5), 44–49. h ps://doi.o g/10.1016/j.cobeha.2018.03.003
Fanselow, M. S., & Les e , L. S. (1988). A Func ional Beha io is ic App oach o A e si ely Mo i a ed
Beha io : P eda o y Imminence as a De e minan o he Topog aphy o De ensi e Beha io . In
E olu ion and Lea ning
(Issue Janua y 1988, pp. 185–211).
h ps://doi.o g/10.4324/9780203781647-14
Fau e, A., Richa d, J. M., & Be idge, K. C. (2010). Desi e and d ead om he nucleus accumbens:
Co ical glu ama e and subco ical GABA di e en ially gene a e mo i a ion and hedonic impac in
he a .
PLoS ONE
,
5
(6). h ps://doi.o g/10.1371/jou nal.pone.0011223
Felix-O iz, A. C., & Tye, K. M. (2014). Amygdala inpu s o he en al hippocampus bidi ec ionally
modula e social beha io .
Jou nal o Neu oscience
,
34
(2), 586–595.
h ps://doi.o g/10.1523/JNEUROSCI.4257-13.2014
Fe e i, V., Mal ese, F., Con a ini, G., Nig o, M., Bona ia, A., Huang, H., Gigliucci, V., Mo elli, G.,
Scheggia, D., Managò, F., Cas ellani, G., Le e e, A., Cancedda, L., Chini, B., G ine ich, V., &
Papaleo, F. (2019). Oxy ocin Signaling in he Cen al Amygdala Modula es Emo ion Disc imina ion
in Mice.
Cu en Biology
,
29
(12), 1938-1953.e6. h ps://doi.o g/10.1016/j.cub.2019.04.070
Fio illo, C. D., Toble , P. N., & Schul z, W. (2003). Disc e e coding o ewa d p obabili y and unce ain y
by dopamine neu ons.
Science
,
299
(5614), 1898–1902.
h ps://doi.o g/10.1126/science.1077349
Fulle , T. A., Russchen, F. T., & P ice, J. L. (1987). Sou ces o p esump i e glu ame gic/aspa e gic
a e en s o he a en al s ia opallidal egion.
Jou nal o Compa a i e Neu ology
,
258
(3), 317–
338. h ps://doi.o g/10.1002/cne.902580302
Ganga ossa, G., Espalle gues, J., D’Exae de, A. de K., El Mes ikawy, S., Ge en, C. R., He é, D., Gi aul ,
J. A., & Valjen , E. (2013). Dis ibu ion and compa men al o ganiza ion o GABAe gic medium-sized
spiny neu ons in he mouse Nucleus Accumbens.
F on ie s in Neu al Ci cui s
,
7
(FEBRUARY 2013),
1–20. h ps://doi.o g/10.3389/ nci .2013.00022
Ge en, C. R. (1992). The neos ia al mosaic: Mul iple le els o compa men al o ganiza ion.
Jou nal o
Neu al T ansmission, Supplemen
,
36
, 43–59. h ps://doi.o g/10.1007/978-3-7091-9211-5_4

83
Ge en, C. R., & Young, W. S. (1988). Dis ibu ion o s ia onig al and s ia opallidal pep ide gic neu ons
in bo h pa ch and ma ix compa men s: an in si u hyb idiza ion his ochemis y and luo escen
e og ade acing s udy.
B ain Resea ch
,
460
(1), 161–167. h ps://doi.o g/10.1016/0006-
8993(88)91217-6
Ge le , T. S., Chan, C. S., & Su meie , D. J. (2008). Dicho omous ana omical p ope ies o adul s ia al
medium spiny neu ons.
Jou nal o Neu oscience
,
28
(43), 10814–10824.
h ps://doi.o g/10.1523/JNEUROSCI.2660-08.2008
Ghi za, U. E., Fabb ica o e, A. T., P okopenko, V., Pawlak, A. P., & Wes , M. O. (2003). Pe sis en cue-
e oked ac i i y o accumbens neu ons a e p olonged abs inence om sel -adminis e ed cocaine.
Jou nal o Neu oscience
,
23
(19), 7239–7245. h ps://doi.o g/10.1523/jneu osci.23-19-
07239.2003
Ghi za, U. E., P okopenko, V. F., Wes , M. O., & Fabb ica o e, A. T. (2006). Highe magni ude accumbal
phasic i ing changes among co e neu ons exhibi ing onic i ing inc eases du ing cocaine sel -
adminis a ion.
Neu oscience
,
137
(3), 1075–1085.
h ps://doi.o g/10.1016/j.neu oscience.2005.10.026
Gio anniello, J., Yu, K., Fu lan, A., Nach ab, G. T., Sha ma, R., Chen, X., & Li, B. (2020). A cen al
amygdala-globus pallidus ci cui con eys uncondi ioned s imulus- ela ed in o ma ion and con ols
ea lea ning.
Jou nal o Neu oscience
,
40
(47), 9043–9054.
h ps://doi.o g/10.1523/JNEUROSCI.2090-20.2020
Golden, S. A., Jin, M., Heins, C., Venni o, M., Michaelides, M., & Shaham, Y. (2019). Nucleus Accumbens
D d1-Exp essing Neu ons Con ol Agg ession Sel -Adminis a ion and Agg ession Seeking in Mice.
The Jou nal o Neu oscience
,
39
(13), 2482–2496. h ps://doi.o g/10.1523/JNEUROSCI.2409-
18.2019
Gómez-Gómez, Y. M., Sánchez-Apa icio, P., Mejía-Chá ez, S., Ga cía-Ga cía, F., Pascual-Ma hey, L. I.,
Aguile a-Reyes, U., Galicia, O., & Veneb a-Muñoz, A. (2019). C-Fos immuno eac i i y in he
hypo halamus and ewa d sys em o young a s a e social no el y exposu e.
Neu oRepo
,
30
(7),
510–515. h ps://doi.o g/10.1097/WNR.0000000000001236
Go e, F., Schwa z, E. C., B ange s, B. C., Aladi, S., S ujenske, J. M., Likh ik, E., Russo, M. J., Go don, J.
A., Salzman, C. D., & Axel, R. (2015). Neu al Rep esen a ions o Uncondi ioned S imuli in
Basola e al Amygdala Media e Inna e and Lea ned Responses.
Cell
,
162
(1), 134–145.
h ps://doi.o g/10.1016/j.cell.2015.06.027
Go e, F., Schwa z, E. C., & Salzman, C. D. (2015). Manipula ing neu al ac i i y in physiologically
classi ied neu ons: iumphs and challenges.
Philosophical T ansac ions o he Royal Socie y B:
Biological Sciences
,
370
(1677), 20140216. h ps://doi.o g/10.1098/ s b.2014.0216
G a eland, G. A., & Di iglia, M. (1985). The equency and dis ibu ion o medium-sized neu ons wi h
inden ed nuclei in he p ima e and oden neos ia um.
B ain Resea ch
,
327
(1–2), 307–311.
h ps://doi.o g/10.1016/0006-8993(85)91524-0
G aybiel, A. M., & Ragsdale, C. W. (1978). His ochemically dis inc compa men s in he s ia um o
human, monkey, and ca demons a ed by ace yl hiocholines e ase s aining.
P oceedings o he
Na ional Academy o Sciences o he Uni ed S a es o Ame ica
,
75
(11), 5723–5726.
h ps://doi.o g/10.1073/pnas.75.11.5723
G ill, H. J., & No g en, R. (1978). The as e eac i i y es . I. Mime ic esponses o gus a o y s imuli in
84
neu ologically no mal a s.
B ain Resea ch
,
143
(2), 263–279. h ps://doi.o g/10.1016/0006-
8993(78)90568-1
G oenewegen, H. J., & Russchen, F. T. (1984). O ganiza ion o he e e en p ojec ions o he nucleus
accumbens o pallidal, hypo halamic, and mesencephalic s uc u es: A acing and
immunohis ochemical s udy in he ca .
Jou nal o Compa a i e Neu ology
,
223
(3), 347–367.
h ps://doi.o g/10.1002/cne.902230303
Gu, Y., Pipe , W. T., B anigan, L. A., Vazey, E. M., As on-Jones, G., Lin, L., LeDoux, J. E., & Sea s, R. M.
(2020). A b ains em-cen al amygdala ci cui unde lies de ensi e esponses o lea ned h ea s.
Molecula Psychia y
,
25
(3), 640–654. h ps://doi.o g/10.1038/s41380-019-0599-6
Guen hne , C. J., Miyamichi, K., Yang, H. H., Helle , H. C., & Luo, L. (2013). Pe manen Gene ic Access
o T ansien ly Ac i e Neu ons ia TRAP: Ta ge ed Recombina ion in Ac i e Popula ions.
Neu on
,
78
(5), 773–784. h ps://doi.o g/10.1016/j.neu on.2013.03.025
Gunaydin, L. A., G osenick, L., Finkels ein, J. C., Kau a , I. V., Fenno, L. E., Adhika i, A., Lammel, S.,
Mi zabeko , J. J., Ai an, R. D., Zalocusky, K. A., Tye, K. M., Anikee a, P., Malenka, R. C., &
Deisse o h, K. (2014). Na u al neu al p ojec ion dynamics unde lying social beha io .
Cell
,
157
(7),
1535–1551. h ps://doi.o g/10.1016/j.cell.2014.05.017
Hall, J., Pa kinson, J. A., Conno , T. M., Dickinson, A., & E e i , B. J. (2001). In ol emen o he cen al
nucleus o he amygdala and nucleus accumbens co e in media ing pa lo ian in luences on
ins umen al beha iou .
Eu opean Jou nal o Neu oscience
,
13
(10), 1984–1992.
h ps://doi.o g/10.1046/j.0953-816X.2001.01577.x
Hamel, L., Thanga asa, T., Samadi, O., & I o, R. (2017). Caudal nucleus accumbens co e is c i ical in
he egula ion o cue-elici ed app oach-a oidance decisions.
ENeu o
,
4
(1), 1–14.
h ps://doi.o g/10.1523/ENEURO.0330-16.2017
Ha daway, J. A., Halladay, L. R., Mazzone, C. M., Pa i, D., Bloodgood, D. W., Kim, M., Jensen, J., DiBe o,
J. F., Boy , K. M., Shiddapu , A., E ani, A., Hon, O. J., Nei a, S., S anhope, C. M., Sugam, J. A.,
Saddo is, M. P., Tip on, G., McElligo , Z., Jhou, T. C., … Kash, T. L. (2019). Cen al Amygdala
P ep onocicep in-Exp essing Neu ons Media e Pala able Food Consump ion and Rewa d.
Neu on
,
102
(5), 1037-1052.e7. h ps://doi.o g/10.1016/j.neu on.2019.03.037
Ha is, G. C., & As on-Jones, G. (1994). In ol emen o D2 dopamine ecep o s in he nucleus accumbens
in he opia e wi hd awal synd ome. In
Na u e
(Vol. 371, Issue 6493, pp. 155–157).
h ps://doi.o g/10.1038/371155a0
Hassani, O. K., C omwell, H. C., & Schul z, W. (2001). In luence o expec a ion o di e en ewa ds on
beha io - ela ed neu onal ac i i y in he s ia um.
Jou nal o Neu ophysiology
,
85
(6), 2477–2489.
h ps://doi.o g/10.1152/jn.2001.85.6.2477
Ha ield, T., Han, J. S., Conley, M., Gallaghe , M., & Holland, P. (1996). Neu o oxic lesions o basola e al,
bu no cen al, amygdala in e e e wi h pa lo ian second-o de condi ioning and ein o ce
de alua ion e ec s.
Jou nal o Neu oscience
,
16
(16), 5256–5265.
h ps://doi.o g/10.1523/jneu osci.16-16-05256.1996
Heidb ede , C. A., & G oenewegen, H. J. (2003). The medial p e on al co ex in he a : E idence o a
do so- en al dis inc ion based upon unc ional and ana omical cha ac e is ics.
Neu oscience and
Biobeha io al Re iews
,
27
(6), 555–579. h ps://doi.o g/10.1016/j.neubio e .2003.09.003
85
Heime , L., & Alheid, G. F. (1991). Piecing oge he he Puzzle o Basal Fo eb ain Ana omy. In
The Basal
Fo eb ain
(Vol. 295, pp. 1–42). h ps://doi.o g/10.1007/978-1-4757-0145-6_1
Heime , L., Zahm, D. S., Chu chill, L., Kali as, P. W., & Wohl mann, C. (1991). Speci ici y in he p ojec ion
pa e ns o accumbal co e and shell in he a .
Neu oscience
,
41
(1), 89–125.
h ps://doi.o g/10.1016/0306-4522(91)90202-Y
Hikida, T., Kimu a, K., Wada, N., Funabiki, K., & Nakanishi Shige ada, S. (2010). Dis inc Roles o
Synap ic T ansmission in Di ec and Indi ec S ia al Pa hways o Rewa d and A e si e Beha io .
Neu on
,
66
(6), 896–907. h ps://doi.o g/10.1016/j.neu on.2010.05.011
Hikida, T., Mo i a, M., & Macphe son, T. (2016). Neu al mechanisms o he nucleus accumbens ci cui
in ewa d and a e si e lea ning.
Neu oscience Resea ch
,
108
, 1–5.
h ps://doi.o g/10.1016/j.neu es.2016.01.004
Hikida, T., Yawa a, S., Yamaguchi, T., Danjo, T., Sasaoka, T., Wang, Y., & Nakanishi, S. (2013). Pa hway-
speci ic modula ion o nucleus accumbens in ewa d and a e si e beha io ia selec i e ansmi e
ecep o s.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica
,
110
(1),
342–347. h ps://doi.o g/10.1073/pnas.1220358110
Hnasko, T. S., Hjelms ad, G. O., Fields, H. L., & Edwa ds, R. H. (2012). Ven al egmen al a ea glu ama e
neu ons: Elec ophysiological p ope ies and p ojec ions.
Jou nal o Neu oscience
,
32
(43), 15076–
15085. h ps://doi.o g/10.1523/JNEUROSCI.3128-12.2012
Holehonnu , R., Luong, J. A., Cha u edi, D., Ho, A., Lella, S. K., Hosek, M. P., & Ploski, J. E. (2014).
Adeno-associa ed i al se o ypes p oduce di e ing i e s and di e en ially ansduce neu ons wi hin
he a basal and la e al amygdala.
BMC Neu oscience
,
15
(1), 1–14.
h ps://doi.o g/10.1186/1471-2202-15-28
Holland, P. C., & Gallaghe , M. (2003). Double dissocia ion o he e ec s o lesions o basola e al and
cen al amygdala on condi ioned s imulus-po en ia ed eeding and Pa lo ian-ins umen al ans e .
Eu opean Jou nal o Neu oscience
,
17
(8), 1680–1694. h ps://doi.o g/10.1046/j.1460-
9568.2003.02585.x
Holland, P. C., & Gallaghe , M. (2004). Amygdala- on al in e ac ions and ewa d expec ancy.
Cu en
Opinion in Neu obiology
,
14
(2), 148–155. h ps://doi.o g/10.1016/j.conb.2004.03.007
Hollande , J. A., & Ca elli, R. M. (2007). Cocaine-associa ed s imuli inc ease cocaine seeking and ac i a e
accumbens co e neu ons a e abs inence.
Jou nal o Neu oscience
,
27
(13), 3535–3539.
h ps://doi.o g/10.1523/JNEUROSCI.3667-06.2007
Holle man, J. R., T emblay, L., & Schul z, W. (1998). In luence o ewa d expec a ion on beha io - ela ed
neu onal ac i i y in p ima e s ia um.
Jou nal o Neu ophysiology
,
80
(2), 947–963.
h ps://doi.o g/10.1152/jn.1998.80.2.947
Huang, W. C., Zucca, A., Le y, J., & Page, D. T. (2020). Social Beha io Is Modula ed by Valence-Encoding
mPFC-Amygdala Sub-ci cui y.
Cell Repo s
,
32
(2), 107899.
h ps://doi.o g/10.1016/j.cel ep.2020.107899
Ikemo o, S., Glazie , B. S., Mu phy, J. M., & McB ide, W. J. (1997). Role o dopamine D1 and D2 ecep o s
in he nucleus accumbens in media ing ewa d.
Jou nal o Neu oscience
,
17
(21), 8580–8587.
h ps://doi.o g/10.1523/jneu osci.17-21-08580.1997
I o, R., Robbins, T. W., & E e i , B. J. (2004). Di e en ial con ol o e cocaine-seeking beha io by nucleus
86
accumbens co e and shell.
Na u e Neu oscience
,
7
(4), 389–397.
h ps://doi.o g/10.1038/nn1217
I zhak, Y., & Ma in, J. L. (2002). Cocaine-induced condi ioned place p e e ence in mice: Induc ion,
ex inc ion and eins a emen by ela ed psychos imulan s.
Neu opsychopha macology
,
26
(1), 130–
134. h ps://doi.o g/10.1016/S0893-133X(01)00303-7
Janak, P. H., Chang, J. Y., & Woodwa d, D. J. (1999). Neu onal spike ac i i y in he nucleus accumbens
o beha ing a s du ing e hanol sel -adminis a ion.
B ain Resea ch
,
817
(1–2), 172–184.
h ps://doi.o g/10.1016/S0006-8993(98)01245-1
Johansson, B., Linds öm, K., & F edholm, B. B. (1994). Di e ences in he egional and cellula
localiza ion o c- os messenge RNA induced by amphe amine, cocaine and ca eine in he a .
Neu oscience
,
59
(4), 837–849. h ps://doi.o g/10.1016/0306-4522(94)90288-7
Kayyal, H., Yiannakas, A., Chand an, S. K., Khamaisy, M., Sha ma, V., & Rosenblum, K. (2019). Ac i i y
o insula o basola e al amygdala p ojec ing neu ons is necessa y and su icien o as e alence
ep esen a ion.
Jou nal o Neu oscience
,
39
(47), 9369–9382.
h ps://doi.o g/10.1523/JNEUROSCI.0752-19.2019
Kelley, A. E., & Be idge, K. C. (2002). The Neu oscience o Na u al Rewa ds: Rele ance o Addic i e
D ugs.
Jou nal o Neu oscience
,
22
(9), 3306–3311. h ps://doi.o g/10.1523/jneu osci.22-09-
03306.2002
Kelley, A. E., & Domesick, V. B. (1982). The dis ibu ion o he p ojec ion om he hippocampal o ma ion
o he nucleus accumbens in he a : An an e og ade and e og ade-ho se adish pe oxidase s udy.
Neu oscience
,
7
(10), 2321–2335. h ps://doi.o g/10.1016/0306-4522(82)90198-1
Killc oss, S., Robbins, T. W., & E e i , B. J. (1997). Di e en ypes o ea -condi ioned beha iou media ed
by sepa a e nuclei wi hin amygdala.
Na u e
,
388
(6640), 377–380.
h ps://doi.o g/10.1038/41097
Kim, J., Pigna elli, M., Xu, S., I oha a, S., & Tonegawa, S. (2016). An agonis ic nega i e and posi i e
neu ons o he basola e al amygdala.
Na u e Neu oscience
,
19
(12), 1636–1646.
h ps://doi.o g/10.1038/nn.4414
Kim, J., Zhang, X., Mu alidha , S., LeBlanc, S. A., & Tonegawa, S. (2017). Basola e al o Cen al Amygdala
Neu al Ci cui s o Appe i i e Beha io s.
Neu on
,
93
(6), 1464-1479.e5.
h ps://doi.o g/10.1016/j.neu on.2017.02.034
Kim, M. J., Mizumo i, S. J. Y., & Be ns ein, I. L. (2010). Neu onal ep esen a ion o condi ioned as e in
he basola e al amygdala o a s.
Neu obiology o Lea ning and Memo y
,
93
(3), 406–414.
h ps://doi.o g/10.1016/j.nlm.2009.12.007
Ki ouac, G. J. (2015). Placing he pa a en icula nucleus o he halamus wi hin he b ain ci cui s ha
con ol beha io .
Neu oscience and Biobeha io al Re iews
,
56
, 315–329.
h ps://doi.o g/10.1016/j.neubio e .2015.08.005
Knowland, D., & Lim, B. K. (2018). Ci cui -based amewo ks o dep essi e beha io s: The ole o ewa d
ci cui y and beyond.
Pha macology Biochemis y and Beha io
,
174
(July 2017), 42–52.
h ps://doi.o g/10.1016/j.pbb.2017.12.010
Koekkoek, L. L., Masís-Va gas, A., Kool, T., Eggels, L., an de Gun, L. L., Lamuadni, K., Slomp, M.,
Diepenb oek, C., Kalsbeek, A., & la Fleu , S. E. (2021). Suc ose d inking mimics e ec s o nucleus
87
accumbens µ-opioid ecep o s imula ion on a in ake and b ain c-Fos-exp ession.
Nu i ional
Neu oscience
,
0
(0), 1–13. h ps://doi.o g/10.1080/1028415X.2021.1975365
Koo, J. W., Lobo, M. K., Chaudhu y, D., Labon é, B., F iedman, A., Helle , E., Peña, C. J., Han, M. H., &
Nes le , E. J. (2014). Loss o BDNF signaling in D1R-exp essing NAc neu ons enhances mo phine
ewa d by educing GABA inhibi ion.
Neu opsychopha macology
,
39
(11), 2646–2653.
h ps://doi.o g/10.1038/npp.2014.118
Ko ács, K. J. (2008). Measu emen o immedia e-ea ly gene ac i a ion- c- os and beyond.
Jou nal o
Neu oendoc inology
,
20
(6), 665–672. h ps://doi.o g/10.1111/j.1365-2826.2008.01734.x
K a i z, A. V., Moo man, D. E., Simpson, A., & Peoples, L. L. (2006). Session-long modula ions o
accumbal i ing du ing suc ose- ein o ced ope an beha io .
Synapse
,
60
(6), 420–428.
h ps://doi.o g/10.1002/syn.20311
K a i z, A. V., Tye, L. D., & K ei ze , A. C. (2012). Dis inc oles o di ec and indi ec pa hway s ia al
neu ons in ein o cemen .
Na u e Neu oscience
,
15
(6), 816–818.
h ps://doi.o g/10.1038/nn.3100
K e ek, J. E., & P ice, J. L. (1978a). A desc ip ion o he amygdaloid complex in he a and ca wi h
obse a ions on in a‐amygdaloid axonal connec ions.
Jou nal o Compa a i e Neu ology
,
178
(2),
255–279. h ps://doi.o g/10.1002/cne.901780205
K e ek, J. E., & P ice, J. L. (1978b). Amygdaloid p ojec ions o subco ical s uc u es wi hin he basal
o eb ain and b ains em in he a and ca .
Jou nal o Compa a i e Neu ology
,
178
(2), 225–253.
h ps://doi.o g/10.1002/cne.901780204
K uko , T. L. (1999). c- os Exp ession as a Ma ke o Func ional Ac i i y in he B ain:
Immunohis ochemis y. In
Cell Neu obiology Techniques
(pp. 213–230). Humana P ess.
h ps://doi.o g/10.1385/0-89603-510-7:213
Kupchik, Y. M., B own, R. M., Heinsb oek, J. A., Lobo, M. K., Schwa z, D. J., & Kali as, P. W. (2015).
Coding he di ec /indi ec pa hways by D1 and D2 ecep o s is no alid o accumbens p ojec ions.
Na u e Neu oscience
,
18
(9), 1230–1232. h ps://doi.o g/10.1038/nn.4068
Lanciego, J. L., Gonzalo, N., Cas le, M., Sanchez-Escoba , C., Ayme ich, M. S., & Obeso, J. A. (2004).
Thalamic inne a ion o s ia al and sub halamic neu ons p ojec ing o he a en opeduncula
nucleus.
Eu opean Jou nal o Neu oscience
,
19
(5), 1267–1277. h ps://doi.o g/10.1111/j.1460-
9568.2004.03244.x
Lang, P. J., & Da is, M. (2006). Emo ion, mo i a ion, and he b ain: Re lex ounda ions in animal and
human esea ch. In
P og ess in B ain Resea ch
(Vol. 156, pp. 3–29).
h ps://doi.o g/10.1016/S0079-6123(06)56001-7
La iole e, S. R., & G ace, A. A. (2006). Cannabinoids po en ia e emo ional lea ning plas ici y in neu ons
o he medial p e on al co ex h ough basola e al amygdala inpu s.
Jou nal o Neu oscience
,
26
(24), 6458–6468. h ps://doi.o g/10.1523/JNEUROSCI.0707-06.2006
Lebow, M. A., & Chen, A. (2016). O e shadowed by he amygdala: The bed nucleus o he s ia e minalis
eme ges as key o psychia ic diso de s.
Molecula Psychia y
,
21
(4), 450–463.
h ps://doi.o g/10.1038/mp.2016.1
LeDoux, J. E., Iwa a, J., Cicche i, P., & Reis, D. J. (1988). Di e en p ojec ions o he cen al amygdaloid
nucleus media e au onomic and beha io al co ela es o condi ioned ea .
Jou nal o Neu oscience
,

88
8
(7), 2517–2529. h ps://doi.o g/10.1523/jneu osci.08-07-02517.1988
Lee, I. S., Necka, E. A., & A las, L. Y. (2020). Dis inguishing pain om nocicep ion, salience, and a ousal:
How au onomic ne ous sys em ac i i y can imp o e neu oimaging es s o speci ici y.
Neu oImage
,
204
(Janua y 2019), 116254. h ps://doi.o g/10.1016/j.neu oimage.2019.116254
Lee, S. C., Ami , A., Headley, D. B., Hau le , D., & Pa e, D. (2016). Basola e al amygdala nucleus
esponses o appe i i e condi ioned s imuli co ela e wi h a ia ions in condi ioned beha iou .
Na u e
Communica ions
,
7
, 1–7. h ps://doi.o g/10.1038/ncomms12275
LeGa es, T. A., K a a, M. D., Tooley, J. R., F ancis, T. C., Lobo, M. K., C eed, M. C., & Thompson, S. M.
(2018). Rewa d beha iou is egula ed by he s eng h o hippocampus–nucleus accumbens
synapses.
Na u e
,
564
(7735), 258–262. h ps://doi.o g/10.1038/s41586-018-0740-8
Lex, A., & Haube , W. (2008). Dopamine D1 and D2 ecep o s in he nucleus accumbens co e and shell
media e Pa lo ian-ins umen al ans e .
Lea ning and Memo y
,
15
(7), 483–491.
h ps://doi.o g/10.1101/lm.978708
Li, G., Wang, Y., Yan, M., Xu, Y., Song, X., Li, Q., Zhang, J., Ma, H., & Wu, Y. (2015). Inhibi ion o ac in
polyme iza ion in he NAc shell inhibi s mo phine-induced CPP by dis up ing i s econsolida ion.
Scien i ic Repo s
,
5
(Augus ), 1–9. h ps://doi.o g/10.1038/s ep16283
Li, H., Penzo, M. A., Taniguchi, H., Kopec, C. D., Huang, Z. J., & Li, B. (2013). Expe ience-dependen
modi ica ion o a cen al amygdala ea ci cui .
Na u e Neu oscience
,
16
(3), 332–339.
h ps://doi.o g/10.1038/nn.3322
Liao, R. M. (2008). De elopmen o condi ioned place p e e ence induced by in a-accumbens in usion o
amphe amine is a enua ed by co-in usion o dopamine D1 and D2 ecep o an agonis s.
Pha macology Biochemis y and Beha io
,
89
(3), 367–373.
h ps://doi.o g/10.1016/j.pbb.2008.01.009
Lima, S. Q., H omádka, T., Znamenskiy, P., & Zado , A. M. (2009). PINP: A new me hod o agging
neu onal popula ions o iden i ica ion du ing in i o elec ophysiological eco ding.
PLoS ONE
,
4
(7).
h ps://doi.o g/10.1371/jou nal.pone.0006099
Liu, J., Nickolenko, J., & Sha p, F. R. (1994). Mo phine induces c- os and junB in s ia um and nucleus
accumbens ia D1 and N-me hyl-D-aspa a e ecep o s.
P oceedings o he Na ional Academy o
Sciences o he Uni ed S a es o Ame ica
,
91
(18), 8537–8541.
h ps://doi.o g/10.1073/pnas.91.18.8537
Li neh, U., & Paz, R. (2012). A e si e-bias and s age-selec i i y in neu ons o he p ima e amygdala du ing
acquisi ion, ex inc ion, and o e nigh e en ion.
Jou nal o Neu oscience
,
32
(25), 8598–8610.
h ps://doi.o g/10.1523/JNEUROSCI.0323-12.2012
Lobo, M. K., Co ing on, H. E., Chaudhu y, D., F iedman, A. K., Sun, H., Damez-We no, D., Die z, D. M.,
Zaman, S., Koo, J. W., Kennedy, P. J., Mouzon, E., Mog i, M., Ne e, R. L., Deisse o h, K., Han, M.-
H., & Nes le , E. J. (2010). Cell Type-Speci ic Loss o BDNF Signaling Mimics Op ogene ic Con ol
o Cocaine Rewa d.
Science
,
330
(6002), 385–390. h ps://doi.o g/10.1126/science.1188472
Lo iaux, A. L., Roi man, J. D., & Roi man, M. F. (2011). Nucleus accumbens shell, bu no co e, acks
mo i a ional alue o sal .
Jou nal o Neu ophysiology
,
106
(3), 1537–1544.
h ps://doi.o g/10.1152/jn.00153.2011
Lu, X. Y., Ghasemzadeh, M. B., & Kali as, P. W. (1997). Exp ession o D1 ecep o , D2 ecep o ,
89
subs ance P and enkephalin messenge RNAs in he neu ons p ojec ing om he nucleus
accumbens.
Neu oscience
,
82
(3), 767–780. h ps://doi.o g/10.1016/S0306-4522(97)00327-8
Mal aez, M., G een ield, V. Y., Wang, A. S., Yo i a, A. M., Feng, L., Linke , K. E., Monbouque e, H. G., &
Wassum, K. M. (2015). Basola e al amygdala apid glu ama e elease encodes an ou come-speci ic
ep esen a ion i al o ewa d-p edic i e cues o selec i ely in igo a e ewa d-seeking ac ions.
Scien i ic Repo s
,
5
(June), 1–17. h ps://doi.o g/10.1038/s ep12511
Managò, F., Cas ellano, C., Oli e io, A., Mele, A., & De Leonibus, E. (2009). Role o dopamine ecep o s
sub ypes, D1-like and D2-like, wi hin he nucleus accumbens sub egions, co e and shell, on memo y
consolida ion in he one- ial inhibi o y a oidance ask.
Lea ning and Memo y
,
16
(1), 46–52.
h ps://doi.o g/10.1101/lm.1177509
Ma kakis, E. A., Vi es, K. P., Bobe , J., Leich le, S., Le an h, C., Beecham, J., Elswo h, J. D., Ro h, R.
H., Samulski, R. J., & Redmond, D. E. (2010). Compa a i e T ansduc ion E iciency o AAV Vec o
Se o ypes 1–6 in he Subs an ia Nig a and S ia um o he P ima e B ain.
Molecula The apy
,
18
(3),
588–593. h ps://doi.o g/10.1038/m .2009.286
McCullough, K. M., Mo ison, F. G., Ha mann, J., Ca lezon, W. A., & Ressle , K. J. (2018). Quan i ied
coexp ession analysis o cen al amygdala subpopula ions.
ENeu o
,
5
(1), 1–12.
h ps://doi.o g/10.1523/ENEURO.0010-18.2018
Mcdonald, A. J. (1991). O ganiza ion o amygdaloid p ojec ions o he p e on al co ex and associa ed
s ia um in he a .
Neu oscience
,
44
(1), 1–14. h ps://doi.o g/10.1016/0306-4522(91)90247-L
Mcdonald, A. J. (1998). Co ical pa hways o he mammalian amygdala.
P og ess in Neu obiology
,
55
(3),
257–332. h ps://doi.o g/10.1016/S0301-0082(98)00003-3
Mendl, M., Bu man, O. H. P., & Paul, E. S. (2010). An in eg a i e and unc ional amewo k o he s udy
o animal emo ion and mood.
P oceedings o he Royal Socie y B: Biological Sciences
,
277
(1696),
2895–2904. h ps://doi.o g/10.1098/ spb.2010.0303
Miñano, F. J., Mene es Sancho, M. S., Sancib ián, M., Salinas, P., & Mye s, R. D. (1992). GABAA
ecep o s in he amygdala: ole in eeding in as ed and sa ia ed a s.
B ain Resea ch
,
586
(1), 104–
110. h ps://doi.o g/10.1016/0006-8993(92)91377-Q
Mogenson, G. J., Jones, D. L., & Yim, C. Y. (1980). F om mo i a ion o ac ion: Func ional in e ace
be ween he limbic sys em and he mo o sys em.
P og ess in Neu obiology
,
14
(2–3), 69–97.
h ps://doi.o g/10.1016/0301-0082(80)90018-0
Mogenson, G. J., Swanson, L. W., & Wu, M. (1983). Neu al p ojec ions om nucleus accumbens o
globus pallidus, subs an ia innomina a, and la e al p eop ic-la e al hypo halamic a ea: An
ana omical and elec ophysiological in es iga ion in he a .
Jou nal o Neu oscience
,
3
(1), 189–
202. h ps://doi.o g/10.1523/jneu osci.03-01-00189.1983
Mo gane, P. J., Galle , J. R., & Mokle , D. J. (2005). A e iew o sys ems and ne wo ks o he limbic
o eb ain/limbic midb ain.
P og ess in Neu obiology
,
75
(2), 143–160.
h ps://doi.o g/10.1016/j.pneu obio.2005.01.001
Mülle , R., B a o, R., Bu ckha d , J., & Cu an, T. (1984). Induc ion o c- os gene and p o ein by g ow h
ac o s p ecedes ac i a ion o c-myc.
Na u e
,
312
(5996), 716–720.
h ps://doi.o g/10.1038/312716a0
Mu amo o, K., Ono, T., Nishijo, H., & Fukuda, M. (1993). Ra amygdaloid neu on esponses du ing
90
audi o y disc imina ion.
Neu oscience
,
52
(3), 621–636. h ps://doi.o g/10.1016/0306-
4522(93)90411-8
Nambu i, P., Al-Hasani, R., Calhoon, G. G., B uchas, M. R., & Tye, K. M. (2016). A chi ec u al
Rep esen a ion o Valence in he Limbic Sys em.
Neu opsychopha macology
,
41
(7), 1697–1715.
h ps://doi.o g/10.1038/npp.2015.358
Nambu i, P., Beyele , A., Yo ozu, S., Calhoon, G. G., Halbe , S. A., Wichmann, R., Holden, S. S., Me ens,
K. L., Anah a , M., Felix-O iz, A. C., Wicke sham, I. R., G ay, J. M., & Tye, K. M. (2015). A ci cui
mechanism o di e en ia ing posi i e and nega i e associa ions.
Na u e
,
520
(7549), 675–678.
h ps://doi.o g/10.1038/na u e14366
Nam a , P., Za abian, S., Naza i-Se enjeh, F., Sadeghzadeh, F., & Haghpa as , A. (2019). In ol emen
o D1- and D2-like dopamine ecep o s wi hin he a nucleus accumbens in he main enance o
mo phine ewa ding p ope ies in he a s.
Beha io al Neu oscience
,
133
(6), 556–562.
h ps://doi.o g/10.1037/bne0000336
Na subo i, A., Tsu sui-Kimu a, I., Nishida, H., Bouchekioua, Y., Sekiya, H., Uchigashima, M., Wa anabe,
M., De Ke cho e D’Exae de, A., Mimu a, M., Taka a, N., & Tanaka, K. F. (2017). Ven ola e al
s ia al medium spiny neu ons posi i ely egula e ood-incen i e, goal-di ec ed beha io
independen ly o D1 and D2 selec i i y.
Jou nal o Neu oscience
,
37
(10), 2723–2733.
h ps://doi.o g/10.1523/JNEUROSCI.3377-16.2017
Nau a, W. J. H., Smi h, G. P., Faull, R. L. M., & Domesick, V. B. (1978). E e en connec ions and nig al
a e en s o he nucleus accumbens sep i in he a .
Neu oscience
,
3
(4–5), 385–401.
h ps://doi.o g/10.1016/0306-4522(78)90041-6
Neugebaue , V. (2015). Amygdala pain mechanisms.
Handbook o Expe imen al Pha macology
,
227
,
261–284. h ps://doi.o g/10.1007/978-3-662-46450-2_13
Neugebaue , V., Mazzi elli, M., C agg, B., Ji, G., Na a ilo a, E., & Po eca, F. (2020). Amygdala,
neu opep ides, and ch onic pain- ela ed a ec i e beha io s.
Neu opha macology
,
170
(Decembe
2019), 108052. h ps://doi.o g/10.1016/j.neu opha m.2020.108052
Nicola, S. M., Yun, I. A., Wakabayashi, K. T., & Fields, H. L. (2004a). Cue-E oked Fi ing o Nucleus
Accumbens Neu ons Encodes Mo i a ional Signi icance du ing a Disc imina i e S imulus Task.
Jou nal o Neu ophysiology
,
91
(4), 1840–1865. h ps://doi.o g/10.1152/jn.00657.2003
Nicola, S. M., Yun, I. A., Wakabayashi, K. T., & Fields, H. L. (2004b). Fi ing o Nucleus Accumbens
Neu ons du ing he Consumma o y Phase o a Disc imina i e S imulus Task Depends on P e ious
Rewa d P edic i e Cues.
Jou nal o Neu ophysiology
,
91
(4), 1866–1882.
h ps://doi.o g/10.1152/jn.00658.2003
Nieh, E. H., Kim, S. Y., Nambu i, P., & Tye, K. M. (2013). Op ogene ic dissec ion o neu al ci cui s
unde lying emo ional alence and mo i a ed beha io s.
B ain Resea ch
,
1511
, 73–92.
h ps://doi.o g/10.1016/j.b ain es.2012.11.001
Nowend, K. L., A izzi, M., Ca lson, B. B., & Salamone, J. D. (2001). D1 o D2 an agonism in nucleus
accumbens co e o do somedial shell supp esses le e p essing o ood bu leads o compensa o y
inc eases in chow consump ion.
Pha macology Biochemis y and Beha io
,
69
(3–4), 373–382.
h ps://doi.o g/10.1016/S0091-3057(01)00524-X
O’Neill, P. K., Go e, F., & Salzman, C. D. (2018). Basola e al amygdala ci cui y in posi i e and nega i e
91
alence.
Cu en Opinion in Neu obiology
,
49
, 175–183.
h ps://doi.o g/10.1016/j.conb.2018.02.012
Pa kinson, J. A., Olms ead, M. C., Bu ns, L. H., Robbins, T. W., & E e i , B. J. (1999). Dissocia ion in
e ec s o lesions o he nucleus accumbens co e and shell on appe i i e Pa lo ian app oach
beha io and he po en ia ion o condi ioned ein o cemen and locomo o ac i i y by D-
amphe amine.
Jou nal o Neu oscience
,
19
(6), 2401–2411.
h ps://doi.o g/10.1523/jneu osci.19-06-02401.1999
Pa kinson, J. A., Willoughby, P. J., Robbins, T. W., & E e i , B. J. (2000). Disconnec ion o he an e io
cingula e co ex and nucleus accumbens co e impai s pa lo ian app oach beha io : Fu he
e idence o limbic co ical- en al s ia opallidal sys ems.
Beha io al Neu oscience
,
114
(1), 42–
63. h ps://doi.o g/10.1037/0735-7044.114.1.42
Pa el, S., Robe s, J., Moo man, J., & Rea ill, C. (1995). Localiza ion o se o onin-4 ecep o s in he
s ia onig al pa hway in a b ain.
Neu oscience
,
69
(4), 1159–1167.
h ps://doi.o g/10.1016/0306-4522(95)00314-9
Pa on, J. J., Belo a, M. A., Mo ison, S. E., & Salzman, C. D. (2006). The p ima e amygdala ep esen s
he posi i e and nega i e alue o isual s imuli du ing lea ning.
Na u e
,
439
(7078), 865–870.
h ps://doi.o g/10.1038/na u e04490
Pa lo , I. P. (2010). Condi ioned e lexes: An in es iga ion o he physiological ac i i y o he ce eb al
co ex.
Annals o Neu osciences
,
17
(3), 153. h ps://doi.o g/10.5214/ans.0972-7531.1017309
Paxinos, G., & F anklin, K. B. J. (2001).
The Mouse B ain in S e eo axic Coo dina es.
(Academic P ess
(ed.); 2nd ed.).
Paxinos, G., & Wa son, C. (2007).
The Ra B ain in S e eo axic Coo dina es.
(Academic P ess (ed.); 6 h
ed.).
Peoples, L. L., Uzwiak, A. J., Guye e, F. X., & Wes , M. O. (1998). Tonic inhibi ion o single nucleus
accumbens neu ons in he a : A p edominan bu no exclusi e i ing pa e n induced by cocaine
sel - adminis a ion sessions.
Neu oscience
,
86
(1), 13–22. h ps://doi.o g/10.1016/S0306-
4522(98)00116-X
Peoples, L. L., & Wes , M. O. (1996). Phasic i ing o single neu ons in he a nucleus accumbens
co ela ed wi h he iming o in a enous cocaine sel -adminis a ion.
Jou nal o Neu oscience
,
16
(10), 3459–3473. h ps://doi.o g/10.1523/jneu osci.16-10-03459.1996
Phillipson, O. T., & G i i hs, A. C. (1985). The opog aphic o de o inpu s o nucleus accumbens in he
a .
Neu oscience
,
16
(2), 275–296. h ps://doi.o g/10.1016/0306-4522(85)90002-8
Pian adosi, P. T., Yea es, D. C. M., Wilkins, M., & Flo esco, S. B. (2017). Con ibu ions o basola e al
amygdala and nucleus accumbens sub egions o media ing mo i a ional con lic du ing punished
ewa d-seeking.
Neu obiology o Lea ning and Memo y
,
140
, 92–105.
h ps://doi.o g/10.1016/j.nlm.2017.02.017
Po e -S ansky, K. A., Seile , J. L., Day, J. J., & A agona, B. J. (2013). De elopmen o beha io al
p e e ences o he op imal choice ollowing unexpec ed ewa d omission is media ed by a educ ion
o D2-like ecep o one in he nucleus accumbens.
Eu opean Jou nal o Neu oscience
,
38
(4),
2572–2588. h ps://doi.o g/10.1111/ejn.12253
P ensa, L., Richa d, S., & Pa en , A. (2003). Chemical ana omy o he human en al s ia um and
98
Cell RNA-Seq.
Neu on
,
96
(2), 313-329.e6. h ps://doi.o g/10.1016/j.neu on.2017.09.026
Wu, Y., Li, Y., Yang, X., & Sui, N. (2014). Di e en ial e ec o be a-ad ene gic ecep o an agonism in
basola e al amygdala on econsolida ion o a e si e and appe i i e memo ies associa ed wi h
mo phine in a s.
Addic ion Biology
,
19
(1), 5–15. h ps://doi.o g/10.1111/j.1369-
1600.2012.00443.x
Yanagimo o, K., & Maeda, H. (2003). The nucleus accumbens uni ac i i ies ela ed o he emo ional
signi icance o complex en i onmen al s imuli in eely mo ing ca s.
Neu oscience Resea ch
,
46
(2),
183–189. h ps://doi.o g/10.1016/S0168-0102(03)00058-0
Yasoshima, Y., Shimu a, T., & Yamamo o, T. (1995). Single uni esponses o he amygdala a e
condi ioned as e a e sion in conscious a s.
Neu oRepo
,
6
(17), 2424–2428.
h ps://doi.o g/10.1097/00001756-199511270-00034
Yawa a, S., Yamaguchi, T., Danjo, T., Hikida, T., & Nakanishi, S. (2012). Pa hway-speci ic con ol o
ewa d lea ning and i s lexibili y ia selec i e dopamine ecep o s in he nucleus accumbens.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica
,
109
(31), 12764–
12769. h ps://doi.o g/10.1073/pnas.1210797109
Ye, L., Allen, W. E., Thompson, K. R., Tian, Q., Hsueh, B., Ramak ishnan, C., Wang, A. C., Jennings, J.
H., Adhika i, A., Halpe n, C. H., Wi en, I. B., Ba h, A. L., Luo, L., McNab, J. A., & Deisse o h, K.
(2016). Wi ing and Molecula Fea u es o P e on al Ensembles Rep esen ing Dis inc Expe iences.
Cell
,
165
(7), 1776–1788. h ps://doi.o g/10.1016/j.cell.2016.05.010
Yee, M., Maal-Ba ed, G., Ting-A-Kee, R., Chwalek, M., Mackay-Clacke , I., Be gamini, M., G iede , T. E.,
& an de Kooy, D. (2020). Seg ega ion o ca eine ewa d and a e sion in he a nucleus
accumbens shell e sus co e.
Eu opean Jou nal o Neu oscience
,
52
(3), 3074–3086.
h ps://doi.o g/10.1111/ejn.14718
Yin, H. H., & Knowl on, B. J. (2006). The ole o he basal ganglia in habi o ma ion.
Na u e Re iews
Neu oscience
,
7
(6), 464–476. h ps://doi.o g/10.1038/n n1919
Young, A. M. J. (2004). Inc eased ex acellula dopamine in nucleus accumbens in esponse o
uncondi ioned and condi ioned a e si e s imuli: S udies using 1 min mic odialysis in a s.
Jou nal
o Neu oscience Me hods
,
138
(1–2), 57–63. h ps://doi.o g/10.1016/j.jneume h.2004.03.003
Young, E. A., D eumon , S. E., & Cunningham, C. L. (2014). Role o nucleus accumbens dopamine
ecep o sub ypes in he lea ning and exp ession o alcohol-seeking beha io .
Neu obiology o
Lea ning and Memo y
,
108
(1), 28–37. h ps://doi.o g/10.1016/j.nlm.2013.05.004
Yu, K., da Sil a, P. G., Albeanu, D. F., & Li, B. (2016). Cen al amygdala soma os a in neu ons ga e
passi e and ac i e de ensi e beha io s.
Jou nal o Neu oscience
,
36
(24), 6488–6496.
h ps://doi.o g/10.1523/JNEUROSCI.4419-15.2016
Zábo sky, L., & Cullinan, W. E. (1992). P ojec ions om he nucleus accumbens o choline gic neu ons
o he en al pallidum: a co ela ed ligh and elec on mic oscopic double-immunolabeling s udy in
a .
B ain Resea ch
,
570
(1–2), 92–101. h ps://doi.o g/10.1016/0006-8993(92)90568-T
Zábo szky, L., Alheid, G. F., Bein eld, M. C., Eiden, L. E., Heime , L., & Palko i s, M. (1985).
Cholecys okinin inne a ion o he en al s ia um: A mo phological and adioimmunological s udy.
Neu oscience
,
14
(2), 427–453. h ps://doi.o g/10.1016/0306-4522(85)90302-1
Zahm, D. S., & B og, J. S. (1992). On he signi icance o sub e i o ies in he “accumbens” pa o he

99
a en al s ia um.
Neu oscience
,
50
(4), 751–767. h ps://doi.o g/10.1016/0306-
4522(92)90202-D
Zangenehpou , S., & Chaudhu i, A. (2002). Di e en ial induc ion and decay cu es o c- os and zi 268
e ealed h ough dual ac i i y maps.
Molecula B ain Resea ch
,
109
(1–2), 221–225.
h ps://doi.o g/10.1016/S0169-328X(02)00556-9
Zhou, L., Fu u a, T., & Kaneko, T. (2003). Chemical o ganiza ion o p ojec ion neu ons in he a
accumbens nucleus and ol ac o y ube cle.
Neu oscience
,
120
(3), 783–798.
h ps://doi.o g/10.1016/S0306-4522(03)00326-9
Ziegenhain, C., Vie h, B., Pa ekh, S., Reinius, B., Guillaume -Adkins, A., Sme s, M., Leonha d , H., Heyn,
H., Hellmann, I., & Ena d, W. (2017). Compa a i e Analysis o Single-Cell RNA Sequencing Me hods.
Molecula Cell
,
65
(4), 631-643.e4. h ps://doi.o g/10.1016/j.molcel.2017.01.023