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

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. 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