Transplantation with Lewis bone marrow induces the reinstatement of cocaine-seeking behavior in male F344 resistant rats.

B ain, Beha io , and Immuni y 93 (2021) 23–34 A ailable online 3 Decembe 2020 0889-1591/© 2020 Else ie Inc. All igh s ese ed. T ansplan a ion wi h Lewis bone ma ow induces he eins a emen o cocaine-seeking beha io in male F344 esis an a s Ma ía Ampa o Assis a , b , c , * , Da id Díaz d , e , Rosa Fe ado a , Ca melo An onio ´ A ila-Za za e , , Edua do We uaga d , e , Emilio Amb osio a a Depa amen o de Psicobiología, Facul ad de Psicología, Uni e sidad Nacional de Educaci´ on a Dis ancia (UNED), Mad id, Spain b Facul ad de Ciencias M´ edicas, Uni e sidad Nacional de San iago del Es e o (UNSE), San iago del Es e o, A gen ina c Labo a o io de Biología Molecula , Inmunología y Mic obiología, Ins i u o Mul idisciplina io de Salud, Tecnología y Desa ollo (IMSaTeD), CONICET-UNSE, San iago del Es e o, A gen ina d Ins i u o de Neu ociencias de Cas illa y Le´ on (INCyL), Uni e sidad de Salamanca (USAL), Salamanca, Spain e Ins i u o de In es igaci´ on Biom´ edica de Salamanca (IBSAL), Salamanca, Spain G upo de Es adís ica Aplicada, Depa amen o de Es adís icas, USAL, Salamanca, Spain ARTICLE INFO Keywo ds: Cocaine Relapse Lewis a s Fische 344 a s Bone ma ow ansplan a ion T-cells CD4 + CD25 + T-cells IL-17A Cy okines D 5 dopamine gic ecep o s ABSTRACT One o he main challenges o unde s and d ug addic ion is de ining he biological mechanisms ha unde lie indi idual di e ences in ecidi ism. S udies o hese mechanisms ha e mainly ocused on he b ain, ye we demons a e he e a signi ican in luence o he pe iphe al immune sys em on his phenomenon. Lewis (LEW) and Fische 344 (F344) a s ha e di e en immunological p o iles and hey display a dis inc ulne abili y o he ein o cing e ec s o cocaine, wi h F344 mo e esis an o eins a e cocaine-seeking beha io . Bone ma ow om male LEW and F344 a s was ans e ed o male F344 a s (F344/LEW-BM and F344/F344-BM, espec i ely), and hese a s we e ained o sel -adminis e cocaine o e 21 days. Following ex inc ion, hese animals ecei ed a sub- h eshold p ime dose o cocaine o e alua e eins a emen . F344/LEW-BM bu no F344/F344-BM a s eins a ed cocaine-seeking beha io , in conjunc ion wi h changes in hei pe iphe al immune cell popula ions o a p o ile ha co esponded o ha o he LEW dono s. A e cocaine exposu e, highe CD4 + T-cells and lowe CD4 + CD25 + T-cells le els we e obse ed in F344/LEW-BM a s e e ed o con ol, and he splenic exp ession o Il-17a, Tg -β, Tl -2, Tl -4 and Il-1β was al e ed in bo h g oups. We p opose ha pe iphe al T-cells espond o cocaine, wi h CD4 + T-cells in pa icula unde going Th17 pola iza ion and gene a ing long- e m memo y, hese cells eleasing media o s ha igge cen al mechanisms o induce eins a emen a e a second encoun e . This immune esponse may explain he high a es o ecidi ism obse ed despi e long pe iods o de oxi ica ion, shedding ligh on he mechanisms unde lying he ulne abili y and esilience o speci ic indi iduals, and opening new pe spec i es o pe sonalized medicine in he ea men o elapse. 1. In oduc ion Cocaine addic ion is one o he mos o e whelming examples o expe ience-dependen beha io al changes ha may be pe manen , in ol ing li elong memo y. Howe e , his phenomenon canno be ully explained by he neu al al e a ions epo ed ollowing ch onic exposu e o his d ug (Edwa ds and Koob, 2010; E e i and Robbins, 2005; Hyman e al., 2006; Lüsche and Malenka, 2011; Robinson and Kolb, 2004; Thomas e al., 2009). The dele e ious e ec s o psychos imulan s a e no exclusi ely beha io - ela ed, as immune unc ion is also comp omised (Assis e al., 2008; Kube a e al., 2008; Lo Iacono e al., 2018). In ac , while he b ain’s ewa d sys em can in luence he ac i i y o he pe iphe al immune sys em, his may also be di ec ly modula ed by d ugs o abuse, and cocaine may in luence immune cells by p o oking he elease o immuno ansmi e s (e.g. dopamine) and immunomodu- la o s (cy okines) ha ac in au oc ine/pa ac ine loops (A aos e al., 2015; Assis e al., 2011; Ben-Shaanan e al., 2018; Be gquis e al., 1994; Cosen ino e al., 2007; Nis ico e al., 1994; Obe beck, 2006; Pelleg ino and Baye , 1998; Yamada and Nabeshima, 2004). Mo eo e , as cocaine eaches leukocy es be o e c ossing he blood–b ain ba ie (BBB), he * Co esponding au ho a : Labo a o io de Biología Molecula , Inmunología y Mic obiología, IMSaTeD, UNSE-CONICET, Ru a Nacional N ◦9, Km 1125 S/N, Villa El Zanj´ on G4206, San iago del Es e o, A gen ina. E-mail add esses: [email p o ec ed], [email p o ec ed] (M.A. Assis). Con en s lis s a ailable a ScienceDi ec B ain Beha io and Immuni y jou nal homepage: www.else ie .com/loca e/yb bi h ps://doi.o g/10.1016/j.bbi.2020.11.039 Recei ed 12 July 2020; Recei ed in e ised o m 27 Oc obe 2020; Accep ed 22 No embe 2020 B ain Beha io and Immuni y 93 (2021) 23–34 24 e ec o cocaine on immunocy es may e en p ecede i s e ec s in he cen al ne ous sys em (CNS). The e ec s o cocaine on immuno- ansmission may also in luence he CNS esponse o his d ug since he immune sys em egula es lea ning, memo y, neu al plas ici y and neu- ogenesis (Yi miya and Goshen, 2011). Indeed, immunological dys unc ion has been linked wi h neu opa hologies, some o which can be e e ed by bone ma ow (BM) ansplan a ion (Chen e al., 2010; De ecki e al., 2012; Díaz e al., 2019, 2015, 2012; Kwan e al., 2012; Leona d, 2010; Liu e al., 2019a; Miyaoka e al., 2017). Due o di e ences in hei ulne abili y o d ugs o abuse, he inb ed Fische 344 (F344) and Lewis (LEW) a s ains ha e been used o s udy he biological co ela es o addic ion (Cadoni, 2016; Haile e al., 2001; Kos en e al., 1997; Kos en and Amb osio, 2002; Migu´ ens e al., 2011; Sanchez-Ca doso e al., 2007; S´ anchez-Ca doso e al., 2009). The LEW s ain is mo e sensi i e han he F344 a s in e ms o eins a ing cocaine-seeking beha io , e en a e ecei ing sub- h eshold cocaine p iming (K uzich and Xi, 2006; Migu´ ens e al., 2013). In addi ion, di - e ences in he immune sys em o hese a s ha e also been epo ed (Fecho e al., 2007; Macho e al., 2008; Ma apallil e al., 2008; Wilde e al., 2000), whe eby F344 a s ha e ewe mononuclea cells han LEW a s, a lowe p opo ion o CD4 + T-cells and less in ense CD4 s aining (G i in and Whi ac e, 1991). We also ound di e ences be ween he T/ B-cell a io, which in LEW a s is nea ly double ha o F344 a s (Ucha- To ue o, 2012). The beha io al di e ences be ween LEW and F344 a s, and hose in hei immune esponses, make hese s ains an a ac i e model o s udy he e ec s o cocaine on he pe iphe al immune sys em and i s ela- ionship wi h beha io . We p edic ed ha he di e ences in he lymphocy e popula ions, pa icula ly he highe p e alence o CD4 + T- cells in LEW a s, would in luence d ug-e oked long- e m memo y by igge ing mechanisms o immune ecogni ion, explaining he heigh - ened sensi i i y o hese a s o elapse. Acco dingly, o s udy he po- en ial e ec s o he immune sys em on elapse, we ans e ed BM om LEW o i adia ed F344 a s, assessing whe he hese LEW BM cells modi y he cocaine-induced beha io al esponse o F344 a s in a pa adigm o sel -adminis a ion and eins a emen o d ug-seeking beha io . 2. Me hods An abb e ia ed e sion o he me hods employed is p esen ed he e. Fo u he de ails, see he Supplemen a y ma e ial. 2.1. Animals Naï e male F344 a s (n =13) we e andomly ansplan ed wi h BM om male F344 a s (F344/F344-BM, con ol, n =7) o male LEW a s (F344/LEW-BM, expe imen al, n =6), using F344 a s and ansgenic g een luo escen p o ein (GFP + ) LEW a s as dono s. Male a s we e used in acco dance o he p e ious da a epo ed by Migu´ ens e al. (2013). All he a s we e main ained in a empe a u e-con olled i- a ium on a 12 h ligh /da k cycle, wi h ood and wa e ad libi um unless o he wise speci ied. All e o s we e made o minimize animal su e ing and he numbe o animals used, in acco dance wi h Eu opean (di ec i e 2010/63/EU) and Spanish Legisla ion (Law 32/2007, RD 53/2013). All he p o ocols used in his s udy we e app o ed by he local e hical commi ees (UNED and USAL). 2.2. BM ansplan a ion Ionizing adia ion was used o e icien BM cell ansplan a ion (Al a ez-Dolado e al., 2003; Massengale e al., 2005). On pos -na al day 19 (P19), he BM o he ecipien s was abla ed wi h a dose o 7.5 Gy (minimal le hal dose) adia ion using a Gammacell 1000 Eli e gamma i adia ion de ice (MDS No dion, O awa, Canada) wi h a 137 Cs sou ce (Díaz e al., 2012, 2011; Recio e al., 2011). A P20, ecipien s we e ansplan ed wi h BM om F344 o LEW-GFP + dono s (see Fig. 1A). The dono s (8- o 10-weeks-old) we e sac i iced wi h CO 2 , decapi a ed, and BM s em cells we e isola ed om hei ibiae and emu s, as desc ibed p e iously (Díaz e al., 2012). Each ecipien ecei ed 7.5 ×10 6 cells in PBS h ough a ail ein injec ion. 2.3. Beha io Animals we e ained on a ood ein o cemen schedule in ope an chambe s, ollowing by su ge y o implan he ein ca he e s, du ing which blood samples we e ob ained (see Fig. 1A). 2.3.1. Acquisi ion and main enance o sel -adminis a ion: Animals we e ained o e 21 daily sessions o p ess a le e o ecei e cocaine (1 mg/kg/in usion in 100 μ l, i. .), on a FR1, 30 s ime-ou ein o cemen schedule (Migu´ ens e al., 2015, 2011). 2.3.2. Ex inc ion o cocaine sel -adminis a ion: The animals unde wen ex inc ion om day 22 un il hey eached he ex inc ion c i e ion wi hin 3 weeks: h ee consecu i e days pe - o ming ≤20% o he a e age ac i e le e (AL) p esses eco ded on days 19–21. Du ing ex inc ion, cocaine was eplaced wi h he ehicle, injec ed i. . a e p essing he AL unde he same schedule indica ed p e iously. 2.3.3. Reins a emen o d ug-seeking beha io : Following ex inc ion, he eins a emen o d ug-seeking beha io was es ed wi h a sub- h eshold p iming injec ion o cocaine (7.5 mg/kg, i.p.). Du ing he eins a emen session, he ex inc ion condi ions we e main ained and a e he session, he animals we e sac i iced o ob ain b ain, spleen and blood samples. 2.4. Flow cy ome y Fou -colo low cy ome y was pe o med o iden i y GFP + BM- de i ed cells (BMDCs), ocusing on adap i e T- and B-cells and inna e immune cells, such as NK-cells, monocy es and g anulocy es (Table S1). We disc imina ed CD4 + and CD8 + T-cells and also ocused on CD4 + CD25 + T-cells since hey can syn hesize, s o e and elease dopa- mine (Cosen ino e al., 2007). Following e y h ocy e lysis, 1 ×10 6 leukocy es we e incuba ed o 30 min a 4 ◦C in he da k wi h 1 μ g o an i- a an ibodies. A e washing, he cells we e ixed and analyzed. 2.5. Immunohis ochemis y Indi ec dual o iple immuno luo escence was pe o med o de ec BMDCs in he b ain o F344/LEW-BM a s. Slices we e incuba ed in PBS con aining no mal donkey se um (5%), T i on X-100 (0.2%) and he p ima y an ibodies: polyclonal goa an i-GFP an ise um in combina ion wi h ei he a polyclonal abbi an i-Iba1 an ise um o a monoclonal abbi an i-CD3 an ibody. A e insing, an ibody binding was de ec ed wi h he co esponding luo escen seconda y an ibodies dilu ed in PBS: Cy2-conjuga ed donkey an i-goa an ibody and ei he Cy3- o Cy5- conjuga ed donkey an ibodies. Be o e he an ibodies we e isualized, he slices we e incuba ed wi h 4′-6-diamidino-2-phenylindole (DAPI) o coun e s ain he cell nuclei, insed, moun ed and co e slipped wi h an i- ading medium. 2.6. qRT-PCR Re e se ansc ip ion (RT) was ca ied ou in a he mal cycle (Ve i i, Applied Biosys ems, Fos e Ci y, CA, USA) on 500 ng o he o al RNA isola ed as de ailed in he Supplemen a y ma e ial. The cDNA ob ained was used as he empla e o he qPCR and ampli ied wi h he p ime s o in e es (Table S2) using he SYBR-G een me hod on a Quan S udio 7 Flex de ice (Applied Biosys ems). The GAPDH gene was used o M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 25 no maliza ion. 2.7. Da a analysis Fo compa ison, wo ailed S uden ’s - es s we e applied unless o he wise speci ied. Leukocy e subpopula ions we e analyzed using a wo-way epea ed measu es analysis o a iance (ANOVA), wi h he BM ansplan (F344 o LEW) as he be ween-subjec ac o and he ea - men (basal o pos -cocaine) as he wi hin-subjec ac o . A Fac o ial Analysis (FA) was pe o med o de ec possible ela ionships among he changes in gene exp ession and he expe imen al g oups. A P incipal Componen Analysis was also used o ex ac componen s when mul iple genes we e analyzed. S a is ical es s we e pe o med using ei he G aphPad.7 o SPSS 22.0 o Windows. 3. Resul s 3.1. T ansplan a ion and in eg a ion o BMDCs In bo h g oups, ansplan a ion o BMDCs was associa ed wi h a simila su i al a e 100 days a e ansplan a ion (90% o F344/F344- BM a s and 85% o F344/LEW-BM a s; Chi squa ed =.096; p =.76; Fig. S1), he ime poin om which he animals we e conside ed o be included in he expe imen ha ing eached 175–225 g body weigh . In hese analyses, we s udied F344/LEW-BM a s in which he e was a con inuous inc ease in he ela i e p opo ions ( e e ed o he ea e as %) o GFP + dono -de i ed blood cells (Fig. 1B, C), as well as F344/F344- BM a s ha main ained he usual leukocy e p o ile o he F344 s ain (see Fig. S2). Thus, a he ime o su ge y he alues o each o he leukocy e subse s we e conside ed as he basal le els, and hey we e compa ed be ween he g oups o con i m he success o he ansplan . A baseline, he F344/LEW-BM a s displayed a simila immune p o ile o ha desc ibed o LEW a s (Fig. S2; G i in and Whi ac e, 1991; Ucha-To ue o, 2012), wi h a highe % o T-cells, a lowe % o B-cells and a highe T/B-cell a io (Figs. 1D-E, and S3) han he F344 con ols. Fu he mo e, F344/LEW-BM a s had highe % o CD4 + T-cells and g anulocy es, ye a lowe % o CD4 + CD25 + T-cells han he con ols (Figs. 1D, S4 and S6). The e we e no di e ences in bo h g oups ega ding he % o CD8 + T-cells, he CD4 + /CD8 + T-cell a io, he % o NK cells o ha o monocy es (Figs. 1D, E, S4–S6). These da a con i m he e icacy o ou p ocedu e in econs i u ing he abla ed BM wi h he ansplan ed cells. 3.2. LEW-BM ans e induces he eins a emen o cocaine-seeking beha io in F344 a s Du ing he sel -adminis a ion pe iod, F344/F344-BM and F344/ LEW-BM a s deli e ed mo e ac i e le e p ess sco es (ALPS) han inac i e le e p ess sco es (ILPS: Fig. 2A and C), wi h no di e ences in he numbe o injec ions pe session (Fig. 2A) o he o al amoun o cocaine consumed (Table S3). No di e ences we e obse ed in he days o ul ill he ex inc ion c i e ion, wi h a simila numbe o ALPS and ILPS in he las ex inc ion session in bo h he g oups (Fig. 2B-C and Table S3). Howe e , du ing he eins a emen session, F344/LEW-BM a s p o- duced mo e ALPS han ILPS, while no di e ences we e obse ed in F344/F344-BM a s (Fig. 2C). In he ligh o his di e ence, we u he compa ed he ALPS pe o med du ing he eins a emen session wi h hose on he las day o ex inc ion o each g oup as desc ibed p e iously (Migu´ ens e al., 2013), which only di e ed signi ican ly in F344/LEW- BM a s ( (5) =4.043, p =.0099; da a no shown). These da a demon- s a ed ha only he F344 a s ansplan ed wi h LEW BM eins a ed cocaine-seeking beha io . Finally, he e we e no signs o g a - s-hos disease (GVHD) in F344/LEW-BM a s h oughou he expe imen : no dia hea, no hai loss and no weigh loss compa ed o he F344/F344- BM a s (Figu e S7). 3.3. In eg a ion o BMDCs in o b ain The in eg a ion o BMDCs in o he b ain o ecipien s depends on he expe imen al condi ions, and i may in ol e glia, neu ons and ound leucocy e-like cells (Díaz e al., 2015; Recio e al., 2011). We sac i iced F344/LEW-BM a s a P155-170, su icien ly long a e ansplan a ion o be able o de ec a conside able numbe o BM-de i ed neu ons in hei b ains (Díaz e al., 2015). Ye in con as o p e ious indings (Díaz e al., 2015; Recio e al., 2011), only mic oglia (Iba1 + ) and ound GFP + cells we e de ec ed, cells ha appea ed o mainly in eg a e in o he meninges and cho oid plexus, wi h only a mino p esence in he pa- enchyma (Figs. 3 and S8). Mo eo e , no p e e ence owa ds in eg a ion in o addic ion- ela ed a eas was obse ed (i.e. p e on al co ex, s ia- um, hippocampus, basal ganglia, amygdala, o en al egmen al a ea: Hyman e al., 2006). Thus, aking in o accoun he small numbe o GFP + elemen s and hei dis ibu ion, no u he cell quan i ica ion was pe o med. In e ms o mic oglial cells, no appa en di e ences in hei dis i- bu ion o mo phology we e de ec ed be ween he wo expe imen al g oups. In addi ion, no speci ic changes in hese cells we e obse ed ela i e o s anda d si ua ions (Fig. S8A). Tha is, he shape, densi y and dis ibu ion o mic oglia we e consis en wi h ea u es o heal hy b ains, di e ing om he amoeboid p o ile o he egional con luence o mic oglia in pa hological si ua ions (Bal an´ as e al., 2013; Díaz e al., 2011). The ound GFP + cells we e cha ac e ized by CD3 immunos aining e ealing a small numbe o T-cells (Figs. 3D and S8B), poo ly in eg a ed in o he cho oid plexus (Fig. S8C). Finally, a ew ound GFP + CD3 − cells we e also de ec ed (Figs. 3D, S8B, and C). 3.4. Cocaine induces di e en e ec s on F344- and LEW-de i ed leukocy es The his o y o exposu e o cocaine (including cocaine sel - adminis a ion and he challenge dose ecei ed ollowing ex inc ion) may di e en ially a ec F344 o LEW-BMDCs. Thus, cocaine ea men ( ac o ea men : basal o pos -cocaine), as well as he e ec o ans- plan ed BM (F344 o LEW), we e analyzed by epea ed measu es ANOVA. Addi ionally, he pos -cocaine da a we e exp essed ela i e o he baseline (Fig. S9). The da a ob ained ega ding cocaine ea men could be g ouped in o i e ca ego ies: I) a simila e ec in bo h g oups; II) an e ec on he F344/F344-BM a s; III) an e ec on he F344/LEW- BM a s; IV) opposi e e ec s be ween he wo g oups; and V) no e ec s. In e ms o ca ego y I, lowe % o B-cells was ound in bo h g oups, such ha he basal di e ences we e main ained (Figs. 4, S3 and S9). In ca ego y II, cocaine dec eased he % o CD4 + T-cells bu inc eased ha o CD8 + T-cells in F344/F344-BM a s, educing he CD4 + /CD8 + T-cell a io. The di e ences in hese pa ame e s obse ed a baseline pe sis ed in he wo g oups a e ea men (Figs. 4, S4 and S9), al hough in- e ac ions o hese ac o s we e de ec ed in all cases and no u he compa isons we e possible. The % o NK cells inc eased a e cocaine ea men in F344/F344-BM a s, al hough his alue was no di e en o ha obse ed in he F344/LEW-BM a s (Figs. 4, S5 and S9). In ca ego y III, a highe % o T-cells was obse ed in F344/LEW-BM a s, causing a ma ked ise in he T/B-cell a io (Figs. 4, S3 and S9), al hough an in e ac ion was de ec ed in he la e . On o he hand, he % o CD4 + CD25 + T-cells in F344/LEW-BM animals in e es ingly ell a e ea men . These h ee pa ame e s di e ed be ween he g oup’s pos - cocaine consump ion (Figs. 4, S4 and S9). In ca ego y IV, an opposi e e ec o cocaine was obse ed on g anulocy es, wi h a highe % in F344/F344-BM a s and a lowe % in F344/LEW-BM a s, in con as o he baseline alues (Figs. 4, S6 and S9). In ca ego y V, cocaine did no a ec he % o monocy es in ei he g oup (Figs. 4, S6 and S9). M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 26 GFP Basal le el Pos -cocaine C B E P0 P19 P20 Bi h I adia ion BM Tx P > 120 Food aining S 10-14 d 10-14 d Cocaine i. . 21 d Su ge y Sel - adminis a ion Veh i. . Ex inc ion R ~10-20 d Cocaine i.p. (7.5 mg/kg) A Blood (Basal Le els) Blood (Pos -cocaine) B ain Spleen D T-cells B-cells CD4+ T-cells CD8+ T-cells CD4+CD25+ T-cells NK cells Monocy es G anulocy es 0 10 20 30 40 50 60 70 80 Pe cen age F344/F344-BM F344/LEW-BM T/B-cells CD4+/CD8+ T-cells 0 1 2 3 4 5 Ra io F344/F344-BM F344/LEW-BM *** ** * ** * * 60 70 80 90 100 % Leucocy es GFP+ Basal le el Pos -cocaine Fig. 1. Timeline o he expe imen al p ocedu es (A) and low cy ome y analysis o bone ma ow-de i ed cells. The e was an inc ease in GFP + leukocy es in he pe iphe al blood o Fische 344 (GFP −/− ) a s ansplan ed wi h bone ma ow om Lewis a s (GFP +/+ ) o e ime (81% ±3.62 basal le els, and 87% ±2.80 pos - cocaine, B), as seen in ep esen a i e his og ams showing GFP + (FL1) leukocy es in pe iphe al blood (C). The alues we e ob ained on he day o su ge y, 10–14 days be o e commencing he sel -adminis a ion p o ocol (basal le el). The samples we e also analyzed o s udy he pe iphe al blood subpopula ions (D): CD3 + CD45RA − lymphocy es (T-cells) ela i e o he o al lymphocy es ( (11) =5.061, p =.0004); CD3 − CD45RA + lymphocy es (B-cells) ela i e o he o al lymphocy es ( (9) =3.946, p =.0034); CD3 + CD4 + lymphocy es (CD4 + T-cells) ela i e o he T-cells ( (10) =2.421, p =.036); CD3 + CD8 + lymphocy es (CD8 + T-cells) ela i e o he T-cells; CD3 + CD4 + CD25 + lymphocy es (CD4 + CD25 + T-cells) ela i e o he T-cells ( (8) =3.356, p =.01); CD3 − CD161a + lymphocy es (NK cells) ela i e o he o al lymphocy es; CD11b/c + monocy es (Monocy es) ela i e o he leukocy es; and CD11b/c + g anulocy es (G anulocy es) ela i e o he leukocy es ( (8) =3.085, p = .015). The a ios be ween he pe iphe al T-cells and B-cells ( (9) =2.398, p =.04), and be ween CD4 + T-cells and CD8 + T-cells, we e also assessed (E). P, pos -na al day; BM Tx, Bone ma ow ansplan a ion; S, Su ge y; Veh, Vehicle; R, Reins a emen o d ug-seeking beha iou ; d, day/s; F344/F344-BM, Fische 344 a s ansplan ed wi h bone ma ow om Fische 344 a s; F344/LEW-BM, Fische 344 a s ansplan ed wi h bone ma ow om Lewis a s. The da a a e exp essed as he mean ±SEM: *p <.05, **p <.01, ***p <.001 unpai ed S uden ’s - es compa ed o he F344/F344-BM con ol g oup. M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 27 3.5. LEW-BM ans e induces a less immunosupp essi e splenic en i onmen in F344 a s a e cocaine, associa ed wi h Th17 pola iza ion and weake inna e signaling Gi en ha he beha io o F344 a s depended on he BMDCs ans e ed, i is no able ha hei immunological pa ame e s a e bio- logically ela ed o he eins a emen da a, albei in a complex manne . This complexi y is one eason why we pe o med a mul i-dimensional analysis ha allowed us examine he ela ionship be ween all o hese complex ac o s. Fu he mo e, as he exp ession o mul iple ela ed genes was s udied simul aneously, we used a FA as a mul i-dimensional s a is ical es . This FA analysis did no e eal any ela ionship be ween he immune cell pa ame e s and he eins a emen da a, and he e o e, we analyzed he immune- ela ed genes. Bea ing in mind he complexi y o his mul i a ia e analysis and o a oid con usion, only he signi ican esul s ob ained a e desc ibed below. In his sense, conside ing he dec ease in he CD4 + CD25 + T-cells le els in LEW-BM ansplan ed a s, we e alua ed he splenic exp ession o i e genes ela ed o hese cells (Table S2). Ini ially, using he FA, we e i ied ha he i s wo componen s explained 88.35% o he a iance (Table 1.AI), which enabled he dimensionali y o he da a o be educed om 5 (numbe o genes o ini ial dimensions) o 2 (componen s). Componen 1 was ela ed o he exp ession o D 5 , Foxp3 and C la-4, whe eas he second componen was mainly explained by Il-10 and Tg - β1 exp ession (Fig. 5A and Table 1.AII). Thus, componen 1 con i med a close ela ionship amongs non-sec e ed p o eins, while componen 2 was ela ed o sec e ed cy okines. Once hese genes had been so ed in o g oups o ela ed a iables, a Mann-Whi ney U es con i med he e we e no di e ences among he genes o componen 1, unlike he sec e ed cy okines due o he inc ease in Tg -β1 exp ession in F344/F344-BM ela i e o F344/LEW-BM a s (Fig. 5C). The lowe numbe s o CD4 + CD25 + T-cells and he weake TGF-β1 exp ession sugges ed weake immunosupp essi e modula ion, which migh boos he expansion o e ec o T-cells in F344/LEW-BM a s. Thus, we e alua ed he splenic exp ession o genes ela ed o T helpe 17 (Th17) cells (Table S2). Following a simila FA, wo i s componen s explained 93.68% o he a iance in he da a (Table 1.BI), which again enabled he dimensionali y o he da a o be educed om 5 o 2. Componen 1 was ela ed o Il-1β, Il-23 and Tg -β1 exp ession, while he second componen was explained by Il-17a and Il-6 exp ession (Fig. 5B and Table 1.BII). A Mann-Whi ney U es e ealed highe Tg -β1 and IL- 1β exp ession in F344/F344-BM a s (Fig. 5C), p obably ela ed o an associa ion wi h CD4 + CD25 + T cells (see abo e) and inna e immuni y (see below), espec i ely. Rega ding he second componen , he e was s onge exp ession o Il-17a in F344/LEW-BM a s, sugges ing a p e- dominan Th17 p o ile in hese animals (Fig. 5C). An addi ional analysis plo ed he gene exp ession da a co esponding o he expe imen al a s in he i s ac o ial plane, which suppo ed ou hypo hesis o Th17 A B C 0 5 10 15 20 25 F344/F344-BM F344/LEW-BM Le e p esses *** ** Sel -adminis a ion F344/F344-BM F344/LEW-BM Ex inc ion Ac i e Le e Inac i e Le e F344/F344-BM F344/LEW-BM ** Reins a emen Fig. 2. Cocaine sel -adminis a ion, ex inc ion and eins a emen . A, I adia ed Fische 344 a s ansplan ed wi h Fische 344 bone ma ow (F344/F344-BM, squa es, n =7) o wi h Lewis bone ma ow (F344/LEW-BM, ci cles, n =6) we e ained o sel -adminis e cocaine (1 mg/kg/in usion) du ing 21 days unde a ixed a io 1 (FR1) schedule. The daily sessions las ed 2 h. No signi ican di e ences we e obse ed in cocaine sel -adminis a ion (A) o ex inc ion beha io (B) be ween F344/F344-BM and F344/LEW-BM a s. C, In he las sel -adminis a ion session, bo h g oups showed signi ican di e ences in he numbe o ac i e and he inac i e le e p esses (C, le panel (12) =4.703, p =.0005, F344/F344-BM; (10) =4.584, p =.001, F344/LEW-BM: **p <.01, ***p <.005 unpai ed S uden ’s - es ), di e ences ha had disappea ed by he las ex inc ion session (C, middle panel). Howe e , he cocaine p iming injec ion (7.5 kg/mg, i.p.) in F344/LEW-BM a s was associa ed wi h mo e esponses using he le e p e iously associa ed wi h cocaine (C, igh panel: (10) =3.883, p =.003, **p <.01, unpai ed S uden ’s - es ). Reins a emen o cocaine-seeking beha io was no obse ed in F344/F344-BM a s, as e lec ed by he lack o s a is ically signi ican di e ences in his g oup be ween he numbe o ac i e and inac i e le e esponses ( (12) =1.657, p =.1233, unpai ed S uden ’s - es ). M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 28 pola iza ion (Fig. S10). Finally, inna e immuni y was e alua ed by conside ing Tl -2, Tl -4 and Il-1β exp ession using he classic non-pa ame ic Mann-Whi ney U es . These h ee genes we e exp essed mo e s ongly in F344/F344-BM animals (Fig. 5C), highligh ing a s ong educ ion in he inna e signaling a e LEW BM ans e . Fu he mo e, he ela i e splenic weigh was highe in F344/LEW-BM a s (7.25 ±0.77 mg/g) han in hei con ol coun e pa s (4.12 ±0.39 mg/g; Fig. S11). 4. Discussion We demons a e he e ha LEW BM ansplan a ion in o F344 a s p oduces a shi in he a ’s immune cell p o ile and in hei eins a e- men o cocaine-seeking beha io , bo h esembling ha o LEW a s (Migu´ ens e al., 2013; Ucha-To ue o, 2012). LEW-BM ans e augmen ed he numbe o T-cells and he T/B-cell a io, which was u he enhanced ollowing cocaine exposu e, as epo ed p e iously (Gan e al., 1998; Lax e al., 2018; Le andowski e al., 2016). Ou indings sugges ha T-cells, and pa icula ly he CD4 + T-cell esponse igge ed by cocaine e-exposu e, may unde lie his beha io al change. A CD4 + T-cell esponse pola ized o a Th17 p o ile could be ac i a ed by ecognizing cocaine, a o ed by a dampened in luence o immunosup- p essi e elemen s like CD4 + CD25 + T-cells, TGF-β1 and IL-10. In addi- ion, s onge D 5 dopamine gic signaling in lymphocy es migh also pa icipa e in his phenomenon. The e ec s o cocaine in he CNS ha e been widely s udied (Ri z e al., 1990) and i is known o in luence immuni y h ough pe iphe al pa hways (Bhowmick e al., 2009; Ma asco e al., 2014; Nis ico e al., 1994). Mo eo e , as ca echolamine ecep o s and memb ane anspo e s a e widesp ead in immune cells (Amen a e al., 2001, 1999; Cosen ino e al., 2002; Ma and Gaskill, 2020; Mignini e al., 2009; Ricci and Amen a, 1994), immunocy es may be di ec ly s imula ed by sus- ained ca echolamine le els on exposu e o cocaine. Cocaine may also be ecognized as a o eign subs ance by leukocy es, ac i a ing adap i e and inna e immuni y. Indeed, we p e iously ound ha cocaine sel - adminis a ion p oduces a s iking inc ease in spleen size, as desc ibed elsewhe e (Kube a e al., 2008), and in he splenic leukocy e coun in LEW a s, augmen ing he T/B-cell a io as seen he e (Assis e al., 2020). These e en s esemble he splenic p oli e a i e T-cell esponse igge ed by blood-bo ne subs ances (e.g. cocaine), which is cap u ed by splenic an igen-p esen ing cells (APCs) o be p esen ed o and ecognized by CD4 + T-cells. This immune ecogni ion o cocaine by T-cells could ha e long-las ing consequences, especially in e ms o elapse. The lowe le els o CD4 + CD25 + T-cells de ec ed in F344/LEW-BM a s we e consis en wi h hei weake Tg -β1 exp ession, accompanied by a educ ion in Il-10 and a endency owa ds s onge splenic D 5 exp ession. These da a is consis en wi h D1- ype ecep o ac i a ion limi ing he syn hesis o hese immunosupp essi e cy okines (Cosen ino e al., 2007; Fe ei a e al., 2014; Kipnis e al., 2004; Le i e, 2016). Since cocaine may heigh en pe iphe al dopamine gic one, enhanced D 5 signaling in LEW-de i ed cells could u he inhibi he syn hesis o hese cy okines by CD4 + CD25 + T-cells (“inhibi ion o inhibi ion”: Cosen ino e al., 2007; Kipnis e al., 2004). In line wi h cocaine inducing a less immunosupp essi e s a e, he e is less IL-10 a e cocaine ein- s a emen , in conjunc ion wi h an inc ease in CD4 + and CD8 + T-cells (Fox e al., 2012; Gan e al., 1998; Kube a e al., 2008; Mo ei a e al., 2016). Indeed, he CD4 + /CD8 + T-cell a io in Wis a a s inc eased ollowing cocaine consump ion (Jankowski e al., 2010), a simila Fig. 3. Analysis o he ansplan -de i ed cells in he b ain o F344/LEW-BM a s. A, The as majo i y o ansplan -de i ed cells (GFP + , g een) appea ed in he meninges (a owheads), some o which we e mic oglia (Iba1, ed). B, The cho oid plexus has a high densi y o ansplan -de i ed mic oglial cells: GFP + (g een) and Iba1 + ( ed). C, D, A ew bone ma ow-de i ed cells (GFP + ) we e e iden in he hippocampus, some o which we e mic oglial cells (Iba1 + , ed: C) and o he s we e ound elemen s, ei he T-cells (CD3 + , ed: D) o o he immune-like cells (a ow). In A, B and D, all he cell nuclei we e coun e s ained wi h DAPI (blue). Scale ba 200 µm o A, 100 µm o B, and 50 µm o C and D. (Fo in e p e a ion o he e e ences o colo in his igu e legend, he eade is e e ed o he web e sion o his a icle.) M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 29 esponse as ha o F344/LEW-BM a s. Thus, clinical and expe imen al da a suppo ou hypo hesis ha cocaine igge s an e ec o CD4 + T-cell esponse, boos ed by a less immunosupp essi e en i onmen in LEW- de i ed lymphocy es. We had expec ed o de ec di e ences in Foxp3 and C la-4 mRNA exp ession be ween he expe imen al g oups, al hough a ecen pa a- digm shi ega ding T-cell lineage de elopmen highligh s he plas ici y o T eg cells and hei po en ial o pa adoxically con e in o highly p o- B A Fig. 4. Flow cy ome y analysis o BMDCs om F344 a s ansplan ed wi h F344 (F344/F344-BM) o Lewis (F344/LEW-BM) BM a e he eins a emen session. The alues we e compa ed wi h hose ob ained on he day o su ge y, be o e s a ing he sel -adminis a ion p o ocol (Basal Le els: BL), o e alua e he esponse o a his o y o cocaine ea men (sel -adminis a ion and cocaine challenge dose: Pos -cocaine, PC). The samples we e analyzed o s udy he pe iphe al blood sub- popula ions (A): CD3 + CD45RA − lymphocy es (T-cells) ela i e o he o al lymphocy es ( epea ed measu es ANOVA BL/PC F =5.631, p =.042; (11) =5.061, p = .0004, BL; and (9) =3.036, p =.0141, PC; epea ed measu es ANOVA o a g oups F =16.726, p =.003; (5) =3.987, p =.0105, F344/LEW-BM a s); CD3 − CD45RA + lymphocy es (B-cells) ela i e o he o al lymphocy es ( epea ed measu es ANOVA BL/PC F =36.020, p =.000; (9) =3.946, p =.0034, BL; and (9) =3.637, p =.0054, PC; epea ed measu es ANOVA o a g oups F =19.733, p =.002; (4) =3.586, p =.0231, F344/F344-BM a s; and (5) =5, p =.0041, F344/ LEW-BM a s); CD3 + CD4 + lymphocy es (CD4 + T-cells) ela i e o he T-cells ( epea ed measu es ANOVA showed in e ac ion o ac o s F =4.600, p =.064); CD3 + CD8 + lymphocy es (CD8 + T-cells) ela i e o he T-cells ( epea ed measu es ANOVA showed in e ac ion o ac o s F =9.690, p =.014); CD3 + CD4 + CD25 + lymphocy es (CD4 + CD25 + T-cells) ela i e o he T-cells ( epea ed measu es ANOVA BL/PC F =9.600, p =.015; (8) =3.356, p =.01, BL; and (9) =2.612, p =.0282, PC; epea ed measu es ANOVA o a g oups F =12.110, p =.008; (4) =4.572, p =.0102, F344/LEW-BM a s); CD3 − CD161a + lymphocy es (NK cells) ela i e o he o al lymphocy es; CD11b/c + monocy es (Monocy es) ela i e o he leukocy es; and CD11b/c + g anulocy es (G anulocy es) ela i e o he leukocy es ( epea ed measu es ANOVA showed in e ac ion o ac o s F =14.127, p =.006). Mo eo e , he a ios be ween he pe iphe al T-cells and B-cells ( epea ed measu es ANOVA showed in e ac ion o ac o s F =5.420, p =.045), and he CD4 + T-cells and CD8 + T-cells ( epea ed measu es ANOVA showed in e ac ion o ac o s F =4.738, p = .061) we e ob ained (B). The da a a e exp essed as he mean ±SEM: *p <.05, **p <.01, ***p <.001 unpai ed S uden ’s - es compa ed o he F344/F344-BM con ol g oup; # p <.05, ## p <.01 pai ed S uden ’s - es compa ed o he BL. M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 30 in lamma o y Th17 cells (Bo enschen e al., 2011; Remedios e al., 2018). FoxP3 + cells om indi iduals wi h au oimmune diseases can easily di e en ia e in o IL-17A p oducing cells as FoxP3 exp ession is p og essi ely los (“T eg ins abili y”: Du e al., 2014). These IL- 17A + FoxP3 + T-cells may also e ain he exp ession o o he T eg- ela ed p o eins like CTLA-4 (Du e al., 2014). We ound highe Il-17a splenic exp ession in F344/LEW-BM a s coinciden wi h high le els o Il-6 bu less Il-23 and Il-1b mRNA ( he exp ession o which a ies oge he wi h Tg -β1 and is lowe in F344/LEW-BM a s). These da a sugges ha a Th17 subse , p obably induced by an IL-6-dependen mechanism, may be he e ec o T-cells esponsible o ecognizing cocaine a e LEW-BM ans e . Some Th17 cells may de i e om FoxP3 + cells ha also exp ess CTLA-4, and ha p og essi ely ans o m in o IL-17A-p oducing cells in F344/LEW-BM a s. Ou da a s ongly ag ee wi h e idence ha dopa- mine s imula es he expansion o IL-17-p oducing T-cells in au oimmune diseases h ough i s abili y o induce IL-6 p oduc ion by monocy es and CD4 + T-cells (Fe ei a e al., 2014). Thus, his Th17 pola iza ion in LEW- BMDCs migh be due o T eg ins abili y and/o s onge D 5 signalling, possibly also explaining he high incidence o au oimmuni y in LEW a s (Wilde e al., 2000). A Th-17 p o ile could also be induced in F344/ LEW-BM a s by mino his ocompa ibili y an igens, which di e ed in bo h s ains, al hough he low mo ali y a e (simila in bo h g oups) and he absence o signs o GVHD incline us o ule ou his possibili y, e en mo e so i we conside he ulne abili y o LEW a s o au oim- muni y desc ibed p e iously. Indeed, we sugges ha he T eg/Th17 balance (immunosupp ession/p o-in lamma ion) is skewed owa ds an ac i a ed s a e in LEW-de i ed lymphocy es, con as ing wi h he s a us o F344-de i ed cells. This is consis en wi h LEW a s ha ing ewe inhibi o y cy okines han F344 a s, wi h hei immunocy es main ained mo e e icien ly in a p oli e a i e s a e (Ma apallil e al., 2008). Finally, as cocaine u he educed he CD4 + CD25 + T-cells in F344/LEW-BM a s, hese da a could also explain he mild au oimmune esponse e- po ed a e cocaine exposu e (T ima chi e al., 2013). Rega ding inna e immuni y, se e al s udies ha e ocused on cocaine and mic oglia (Cla k e al., 2013; Hu chinson and Wa kins, 2014). Al hough no associa ion was ound be ween his d ug and mic oglial s imula ion (Na end an e al., 2014), addic ion was ecen ly p oposed o be a consequence o inna e immune ac i a ion in he b ain (C ews e al., 2011). This heo y and ou T-cell hypo hesis a e no mu ually exclusi e, since bo h inna e and adap i e immuni y migh ac in pa allel, in lu- encing each o he o ul ima ely modula e beha io . This idea is sup- po ed by ecen e idence ega ding IL-17A as an ac i a o o mic oglia in Pa kinson’s disease (Liu e al., 2019b). Cocaine can ac i a e TLR-4 and TLR-2 on mic oglia (Liao e al., 2016; No hcu e al., 2015), and pe haps also on pe iphe al inna e immunocy es (including APCs). Ou da a demons a es s onge Tl -4, Tl -2 and Il-1β mRNA exp ession in F344-BMDCs, which oge he wi h he inc eased g anulocy e le els sugges s ha he F344 s ain may moun a s onge inna e esponse o cocaine, while i s adap i e immuni y migh be limi ed by enhanced immunosupp ession. Conco dan ly, inc eased TLR-4 exp ession by monocy es p omo es CD4 + CD25 + T-cell di e en ia ion (Hao e al., 2017). In pa allel, he ac i a ion o TLRs on APCs imp o es he e ec- i eness o an igen p esen a ion (Abbas e al., 2017). Thus, i cocaine is hap enized and p esen ed by APCs o T-cells in he spleen, his p ocess could be exagge a ed by cocaine ac i a ing TLR-4 and TLR-2 in pa allel, e en in LEW-de i ed cells exp essing lowe le els o TLRs. Such a mechanism migh unde lie he enhanced an igen p esen a ion p o oked by cocaine and epo ed some 20 yea s ago (Shen e al., 1999). In conjunc ion, splenic ac i a ion induced by cocaine (in ol ing TLRs, APCs and CD4 + T-cells) migh explain he splenomegaly we and o he s ha e obse ed (Khan e al., 2017; Kube a e al., 2008). Indeed, F344/ LEW-BM a s de elop a ela i ely hea ie spleen han hei con ol coun e pa s. Toge he hese da a ein o ce he idea ha he balance owa ds an inna e immune esponse would p edomina e o e he adap i e esponse in F344/F344-BM a s. By con as , he la e would p edomina e in F344/LEW-BM a s, unde lying immune-media o elease ha induces beha io al eins a emen . The in luence ha CD4 + T-cells can exe on beha io a ises om cy okines ha c oss he BBB and/o by hei ansmig a ion in o he CNS (Filiano e al., 2017; P inz and P ille , 2017), which can a ec se e al Table 1 Reduc ion o dimensionali y using he PCA as ex ac ion me hod (I, le ) and he associa ed o a ed componen ma ices showing he esul s o he FA (II, igh ), o he CD4 + CD25 + T-cell ela ed genes (A) and Th17- ela ed genes (B). No e ha he wo i s componen s (shadowed lines, I, le ) explain he 88.345 % and he 93.674 % o he a iance ( ha is o say, much mo e han he 75 %) o A and B, espec i ely, hus allowing he educ ion o ou da a o a bidimensional space (plane) in bo h analyses. Each o a ed componen ma ix explains he con ibu ion o each a iable o he a iance o hese wo i s componen s o he bidimensional space (shadowed lines, II, igh ). No e ha he i e genes in A and B ( a iables; II, igh side) a e clea ly so ed in bo h componen s (i.e. con ibu ion alues close o 1 o one componen and close o 0 o he ano he ). Con ibu ions lowe han 0.3 ha e been so ened on he o a ed componen ma ices. Comp., componen ; cumul., cumula i e. I. To al a iance explained II. Ro a ed componen ma ices a A Comp. Ini ial au o alues Ex ac ion sums o squa ed loadings Ro a ion sums o squa ed loadings Va iables (CD4+CD25+T-cell ela ed genes) To al % o a iance Cumul. % To al % o a iance Cumul. % To al % o a iance Cumul. % C la-4 D5Foxp3 Il-10 Tg - 1 12.799 55.977 55.977 2.799 55.977 55.977 2.523 50.458 50.458 0.922 0.934 0.833 -0.117 -0.096 21.618 32.367 88.345 1.618 32.367 88.345 1.894 37.887 88.345 0.017 -0.207 -0.135 0.955 0.959 30.320 6.402 94.747 40.161 3.212 97.959 50.102 2.041 100.000 B Comp. Ini ial au o alues Ex ac ion sums o squa ed loadings Ro a ion sums o squa ed loadings Va iables (Th17- ela ed genes) To al % o a iance Cumul. % To al % o a iance Cumul. % To al % o a iance Cumul. %Il- Il-6 Il-17a Il-23 Tg - 1 12.765 55.309 55.309 2.765 55.309 55.309 2.733 54.658 54.658 0.973 0.198 -0.120 0.958 0.903 21.918 38.365 93.674 1.918 38.365 93.674 1.951 39.016 93.674 -0.022 0.979 0.992 0.041 0.077 30.259 5.185 98.858 40.055 1.105 99.964 50.002 0.036 100.000 M.A. Assis e al. B ain Beha io and Immuni y 93 (2021) 23–34 31 b ain p ocesses (e.g. social beha iou , spa ial lea ning and memo y) and may in ol e plas ici y in T-cell esponses (Filiano e al., 2016; Kipnis and Filiano, 2018; Ko n and Kallies, 2017; Sub amanian e al., 2001; Wilson e al., 2010; Yi miya and Goshen, 2011). The ec ui men o T- cells o he CNS is pa ially unde s ood as hey can mig a e ac oss he cho oid epi helium and c oss he BBB when i is al e ed, while ascula channels connec ing he skull BM and b ain ha e also ecen ly been desc ibed (He isson e al., 2018; Ko n and Kallies, 2017; S azielle e al., 2016; Wilson e al., 2010). Fu he mo e, psychos imulan s can al e BBB pe meabili y (Da idson e al., 2018; Kousik e al., 2012). Ne e heless, we ound GFP + cells o be sca ce in he b ain o F344/LEW-BM a s and hence, ou indings sugges a mo e p ominen pe iphe al immune in- luence on eins a emen (e.g. IL-17A eleased pe iphe ally) a he han an e ec o T-cells ha ha e ansmig a ed in o he b ain. In e es ingly, IL-17A was ecen ly ela ed o dopamine gic dys unc ions in Pa kinson’s disease (Liu e al., 2019b). A pe iphe al in luence associa ed wi h cocaine elapse was p oposed when cocaine me hiodide, which canno c oss he BBB, induced eins a emen in animals ha had p e iously sel - adminis e ed cocaine (Wang e al., 2013a; Wise e al., 2008). This componen was a ibu ed o in e ocep i e keys ha “p ecede and p edic he ewa ding ac ion o cocaine in expe ienced use s” and ha can al e he ac i i y o en al egmen al a ea (Mejías-Apon e and Kiya kin, 2012; Wise e al., 2008). In he ligh o he cu en indings, hese e ec s migh be explained by CD4 + T-cells eleasing media o s a e ecognizing cocaine me hiodide pe iphe ally. Fu he s udies will be necessa y o con i m he neu o-immune connexions unde lying hese phenomena. Repea ed cocaine exposu e p oduces las ing changes in neu al ci - cui s ha unde lie addic ion (E e i and Robbins, 2016; Guillem and Ahmed, 2018; Higue a-Ma as e al., 2011). One consequence o his plas ici y is he beha io al and incen i e-mo i a ional sensi iza ion ha ollows e-exposu e o cocaine (Bickel e al., 2018; Robinson and Be - idge, 2008, 1993; Robinson and Kolb, 2004; Vezina, 2004; Wang e al., 2013b), which is dis inc in LEW and F344 a s (Kos en e al., 1994). In e es ingly, he phenomenon o sensi iza ion is no exclusi e o he Fig. 5. Analysis o gene exp ession. A, Fi s ac o ial plane in o a ed space displays he ela ionship amongs he cha ac e is ic genes o CD4 + CD25 + T-cells. No e ha C la-4, D 5 and Foxp3 (genes encoding wo memb ane ecep o s and a ansc ip ion ac o , i.e. non-sec e ed p o eins) ha e a clea dis ibu ion in he i s componen o he FA, whe eas Tg -β1 and Il-10 (sec e ed cy okines) a e dis ibu ed in he second componen . B, Fi s ac o ial plane in he o a ed space showing he ela ionship amongs ep esen a i e genes o Th17 cells. He e, Il-1β, Il-23 and Tg -β1 (genes associa ed wi h Th17 pola iza ion ha sha e ac i i ies wi h o he immunological p ocesses) ha e a clea dis ibu ion in he i s componen o he FA, whe eas Il-6 and Il-17a (genes in ol ed in Th17 pola iza ion and sec e ion, espec i ely) a e dis ibu ed in he second componen . C, The g aphs showing he change in exp ession o he ele en genes analysed, so ed by immunological cell ype o p ocess. No e ha Tl -2 (p =.022), Tl -4 (p =.035), Tg -β1, (p =.035) and Il-1β (p =.035) a e all exp essed mo e s ongly in F344/F344-BM a s, whe eas Il- 17a (p =.014) is exp essed mo e s ongly by F344/LEW-BM a s (*p <.05, Mann-Whi ney U es ). M.A. Assis e al.