SUPPLEMENTARY INFORMATION
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1 SUPPLEMENTARY INFORMATION oi:.38/ntur58 istn trvll (m) 4 3 Slin multory tim (s) 6 Slin multory ounts 8 6 Slin tim strotypi (s) 5 5 Slin strotypi ounts 6 4 Slin f rsting tim (s) 5 5 Slin g 3 Slin h 8 Slin i Slin vrtil ounts vrtil tim (s) 6 trnsitions 8 4 Supplmntry Figur. Loomotor tivity is not ltr y scpp or., istn trvll is not iffrnt twn groups (slin injt, 36 ± 6, 865 ± 5, n = 8 nimls, p =.45; injt, 86 ± 79, 936 ± 354, n = 5 nimls, p =.848)., multory tim is not iffrnt twn groups (slin injt, 9 ± 7, 9 ± 89, n = 8 nimls, p =.83; injt, 85 ± 6, 89 ± 9, n = 5 nimls, p =.687)., multory ounts r not iffrnt twn groups (slin injt, 4 ± 84, 33 ± 4, n = 8 nimls, p =.688; injt, 38 ±, 36 ± 3, n = 5 nimls, p =.63)., tim strotypi is not iffrnt twn groups (slin injt, 397 ±, 383 ± 5, n = 8 nimls, p =.47; injt, 4 ± 6, 383 ±, n = 3 nimls, p =.)., strotypi ounts r not iffrnt twn groups (slin injt, 536 ±, 56 ± 75, n = 8 nimls, p =.689; injt, 5453 ± 4, 533 ± 9, n = 3 nimls, p =.74). f, rsting tim is not iffrnt twn groups (slin injt, 38 ± 4, 84 ± 36, n = 8 nimls, p =.53; injt, 37 ±, 36 ± 8, n = 5 nimls, p =.368). g, vrtil ounts r not iffrnt twn groups (slin injt, ± 8, ±, n = 8 nimls, p =.466; injt, 7 ±, 97 ± 38, n = nimls, p =.68). h, vrtil tim is not iffrnt twn groups (slin injt, 38 ±, 3 ±, n = 8 nimls, p =.346; injt, 45 ± 3, 7 ± 7, n = nimls, p =.384). i, numr of trnsitions r not iffrnt twn groups (slin injt, 85 ± 7, 74 ± 8, n = 8 nimls, p =.36; injt, 8 ±, 87 ± 9, n = 5 nimls,.759). Summry t r snt s mn ± s..m (*P <.5).
2 RESEARCH SUPPLEMENTARY INFORMATION hom g 3min -tril 4h 3min 4h 3min 4h hom oin hom slin hom 3min -tril slin hitution Rx Rx slin oin I.P. Slin slin oin I.P. OTR - A normliz oin frn I.P. Slin I.P. OTR - A n.s slin oin * slin oin * sutrt oin frn.3.5 n.s. Supplmntry Figur. Coin onition pl frn (CPP) os not rquir OT., Exprimntl tim ours for oin CPP. -, Iniviul (top) n vrg (ottom) rsponss in i.p. slin injt (), vrsus i.p. injt () nimls. Both i.p. slin n i.p. injt nimls, spn mor tim in th oin ontxt following onitioning (i.p. slin, 93 ± 43, 4 ± 54, n = 34 nimls, p =.34 pir t-tst; i.p., 863 ± 46, ± 47, n = 3 nimls, p =.7 pir t-tst)., Comprisons twn i.p. slin vrsus i.p. OTR- A trt nimls rvls no iffrn in normliz oin frn n sutrt oin frn in trt nimls (normliz slin.5 ±.79,.45 ±.74, p =.96 unpir t-tst; sutrt slin.54 ±.69,.65 ±.57, p =.857 unpir t-tst). Summry t r snt s mn ± s..m (*P <.5).
3 SUPPLEMENTARY INFORMATION RESEARCH lur fitting p outflow tu rug rsrvoir CPu pro iltrl nul gui sl n miroilysis mmrn tuing NA ASh Supplmntry Figur 3. Anlmn rvrs miroilysis pros., Shmti of Anlmn pro for rvrs miroilysis xprimnts in Fig. f-i n 5l-o., Post-ho onfirmtion of pro plmnt n omptny. 3
4 RESEARCH SUPPLEMENTARY INFORMATION Strotxi injtion of RV-GFP into NA Oxytoin ntioy CPu CPu RV-GFP Injt into NA RV-GFP xssion in th PVN NA NA ASh PVN PVN PVN f Supplmntry Figur 4. OT xssing nurons in th prvntriulr nulus (PVN) sn projtions to th NA., Digrm illustrting th injtion of RV-GFP into th NA, follow y ntioy lling of OT prouing nurons in th PVN., Loliztion n xssion of RV-GFP in th NA (sl rs µm). -f, Low mgnifition (,sl r µm) n high mgnifition (-f, sl rs µm) imgs of PVN lls tht hv n rtrogrly ll y RV-GFP tkn up y synpti trminls in th NA (grn, lft pnls) n ll y nt-ot-np ntioy (r, ntr pnls). Co-loliztion (mrg right pnls) inits oxytoinrgi nurons tht projt to th NA; rrowhs highlight iniviul lls tht show ololiztion. (Nulus umns or, NA; nulus umns shll, ASh; ntrior ommissur, ; ut putmn, CPu; thir vntril, ; prvntriulr nulus of th hypothlmus, PVN) 4
5 SUPPLEMENTARY INFORMATION RESEARCH Strotxi injtion of RV-GFP into NA Oxytoin ntioy SON SON SON Supplmntry Figur 5. OT xssing nurons in th Supropti Nulus (SON) o not sn projtions to th NA., Digrm illustrting th injtion of RV-GFP into th NA, follow y ntioy lling of OT prouing nurons in th SON., high mgnifition (sl rs µm) imgs of SON lls tht hv n rtrogrly ll y RV-GFP tkn up y synpti trminls in th NA (grn, lft pnls) or ll y nt- OT-np ntioy (r, ntr pnls). Asn of o-loliztion (mrg right pnls) inits oxytoinrgi nurons o not projt to th NA. 5
6 RESEARCH SUPPLEMENTARY INFORMATION + +. * EPSC (normliz)..5 OT D soil D isolt 5 pa ms 3 45 Tim (minuts) pa ms 6 EPSC t min 5-55 (norm).5 soil isolt + + f. * EPSC (normliz)..5 OT D soil D isolt 5 pa ms 3 45 Tim (minuts) pa 5ms 6 EPSC t min 5-55 (norm).5 soil isolt g + + i. h EPSC (normliz)..5 OT D isolt D isolt 5 pa ms 3 45 Tim (minuts) pa 5ms 6 EPSC t min 5-55 (norm).5 D n.s. D Supplmntry Figur 6. Inrs mgnitu of OT-LTD in oth D n D rptor xssing MSN following isoltion onitioning. -h, Rsnttiv trs (,,g), summry tim ours (,,h), n vrg -trtmnt mgnitu omprisons (,f.i) rvl tht th mgnitu of OT-LTD is signifintly inrs in oth D (-) n D (-f) lls from isoltion vrsus soilly rr nimls (D isoltion,.379 ±.9, n = 6 lls n D soil,.756 ±.78, n = 9 lls, p =.54 unpir t- tst; D isoltion,.386 ±.9, n = 5 n D soil,.78 ±.5, n = lls, p =.845 unpir t-tst); n th mgnitu of EPSC OT-LTD is not iffrnt in D vrsus D MSNs from slis m from isoltion onition nimls (g-i) (p =.957 unpir t-tst). Rsnttiv trs r inn from 5 onsutiv swps tkn t tims init ( n ). Summry t r snt s mn ± s..m (*P <.5). 6
7 SUPPLEMENTARY INFORMATION RESEARCH Cll-typ spifi ntioy Oxytoin rptor vnus rportr mous DARP OTR-vn ChAT OTR-vn nnos OTR-vn PARV OTR-vn f GFAP OTR-vn Supplmntry Figur 7. OT rptor (OTR) xssing lls in th NA., Digrm illustrting immunolling with ll-typ spifi ntiois in th NA of OTR-Vnus mi., DARP positiv MSNs (lu, lft) n OTR-Vn positiv lls (grn, ntr) o not o-loliz (mrg, right)., CHAT positiv holinrgi intrnurons (lu, lft) n OTR-Vn positiv lls (grn, ntr) o not o-loliz (mrg, right)., nnos positiv inhiitory intrnurons (lu, lft) n OTR-Vn positiv lls (grn, ntr) o not o-loliz (mrg, right)., sust of PARV positiv inhiitory intrnurons (lu, lft) n OTR-Vnus positiv lls (grn, ntr) show o-loliztion (mrg, right). f, sust of GFAP positiv glil lls (lu, lft) n OTR-Vn positiv lls (grn, ntr) show o-loliztion (mrg, right). (Sl rs µm) 7
8 RESEARCH SUPPLEMENTARY INFORMATION g Strotxi injtion of RV-TTomto into NA Oxytoin rptor vnus rportr mous BLA h CoA i ACC vca j OFC Rph k PVN VTA l AON f CA m PV vsu Supplmntry Figur 8. OT rptor xssing lls tht projt to th NA., Digrm illustrting strotxi injtion of RV-TTomto into th NA of OTR-Vnus mi. -m, high mgnifition imgs of lls in th () ntrior ingult ortx, ACC; () oritofrontl ortx, OFC; () prvntriulr nulus of th hypothlmus, PVN; () ntrior olftory nulus, AON; (f) prvntriulr thlmus, PV; (g) soltrl mygl, BLA; (h) ortx of th mygl, CoA; (i) vntrl hippompl CA, vca; (j) orsl Rph nulus, Rph; (k) ul vntrl tgmntl nulus, VTA; (l) ntrl mygl, CA; n vntrl suiulum, vsu tht hv n rtrogrly ll y RV-GFP tkn up y synpti trminls in th NA (r, lft pnls) n tht xss OTR-Vnus (grn, ntr pnls). Co-loliztion (mrg, right pnls) is notly snt in () ACC n () OFC, ut in ll othr rgions (-m), sust of lls show lr o-loliztion (projt to th NA n xss OTR-Vnus), impliting ths nurons s puttiv sour of synpti OTRs in th NA. (Sl rs µm). 8
9 SUPPLEMENTARY INFORMATION RESEARCH + n.s. IPSC (normliz)..5 OT 5 pa ms 4 6 Tim (minuts) 5 min vrg IPSC (norm)..5 OT Supplmntry Figur 9. OT os not ltr inhiitory synpti urrnts (IPSCs) ror from NA MSNs. Rsnttiv trs (), summry tim ours () n vrg -trtmnt mgnitu omprisons () rvl sn of signifint OT-inu hngs in IPSC mplitu (.3 ±.86,.96 ±.456, n = lls, p =.557 pir t-tst). Rsnttiv trs r inn from 5 onsutiv swps tkn t tims init ( n ). Summry t r snt s mn ± s..m (*P <.5). 9
10 RESEARCH SUPPLEMENTARY INFORMATION CPu NA AAV-Cr-GFP NA ASh CPu RV-Cr-GFP NA NA ASh 4V C AAV-Cr-GFP Rph Rph ACC AAV-Cr-GFP ACC LV CC LV vca AAV-Cr-GFP vsub Ctx vsu Supplmntry Figur. Post-ho onfirmtion of virl injtion sits n trnsgn xssion., Loliztion n xssion of () AAV-Cr-GFP n () RV-Cr-GFP strotxi injtions into th NA.,,, Loliztion n xssion of AAV-Cr-GFP strotxi injtions into th () Rph, () ACC n () vsu. (Nulus umns or, NA; nulus umns shll, ASh; ntrior ommissur, ; ut putmn.k.. orsl stritum, CPu; orsl Rph, Rph; fourth vntril, 4V; rllum, C; ntrior ingult ortx, ACC; orpus llosum, CC; ltrl vntril, LV; vntrl suiulum, vsu; vntrl CA rgion of th hippompus, vca; ntorhinl ortx, Ctx; Sl rs 5 µm)
11 SUPPLEMENTARY INFORMATION RESEARCH Strotxi injtion of RV-TTomto into NA Strotxi injtion of AAV-BFP into ithr Rph, ACC, or vsub Oxytoin rptor vnus rportr mous Strotxi injtion of Oxytoin rptor AAV-BFP into NA vnus rportr nti-prvlumin mous ntioy f NA NA NA g Rph Rph Rph Rph h NA NA Strotxi injtion of RV-TTomto into NA Oxytoin rptor vnus rportr mous NA ACC ACC ACC ACC i vsub vsub vsub vsub NA NA NA Supplmntry Figur. Confirmtion of virl tropism for OTR-xssing n NA-projting lls., Digrm illustrting strotxi injtion of RV-TTomto into th NA of OTR-Vnus mi follow y AAV-BFP xssion in ithr orsl Rph nulus, Rph, ntrior ingult ortx, ACC or vntrl suiulum, vsu shown in (-). -, high mgnifition imgs of lls in th () RpH () ACC or () vsub tht hv n rtrogrly ll y RV-GFP tkn up y synpti trminls in th NA (r, lft pnls), ll y lol inftion with AAV-BFP (lu, ntr lft), n tht xss OTR-Vnus (grn, ntr right pnls). Clr ololiztion (mrg, right pnls) twn RV-TTomto, OTR-Vnus n AAV-BFP is vint in ( n ) n o-loliztion twn OTR-Vnus n AAV-BFP is vint ()., Digrm illustrting strotxi injtion of AAV-BFP into th NA of OTR-Vnus mi or WT mi follow y susqunt immunohistohmistry with nti-prvlumin ntioy shown in (f-g). f, Lol inftion with AAV-BFP (lu, lft) in lls tht xss OTR-Vnus (grn, ntr). Co-loliztion (mrg, right) inits AAV tropism for OTR xssing lls in th NA. g, Lol inftion with AAV-BFP (lu, lft) in lls tht xss Prvlumin (r, ntr). Co-loliztion (mrg, right) inits AAV tropism for Prvlumin xssing lls in th NA. h, Digrm illustrting strotxi injtion of RV-TTomto into th NA of OTR-Vnus mi shown in (i). i, Lol inftion with RV-TTomto (r, lft) in lls tht xss OTR-Vnus (grn, ntr). Co-loliztion (mrg, right) inits RV tropism for OTR xssing lls in th NA. (Sl rs µm).
12 RESEARCH SUPPLEMENTARY INFORMATION AAV-Cr-GFP injtion 4h 4h 7 hom g -tril soil onitioning isolt onitioning -tril Strotxi injtion of AAV-Cr-GFP into ACC Strotxi injtion of AAV-Cr-GFP into vsub isolt soil isolt soil UNINJECTED OTR WT * isolt soil isolt soil ACC OTR AAV-Cr-GFP * isolt soil isolt soil vsub OTR AAV-Cr-GFP * normliz soil frn sutrt soil frn n.s. CTL ACC vsub n.s. CTL ACC vsub Supplmntry Figur. ACC n vsu OT rptors (OTRs) r not rquir for scpp., Digrm illustrting tim ours of virl injtions into th ACC n vsu for scpp xprimnts in -.,, Iniviul (top) n vrg (ottom) frn sors init tht un-injt OTR KO nimls (), OTR KO nimls injt with AAV-Cr-GFP into th ACC () n OTR KO nimls injt with AAV-Cr-GFP into th vsu () hv inrs frn for th soil ing u ftr onitioning (un-injt OTR KO, 935 ± 8, 5 ± 68, n =, p =.44 pir t-tst; OTR KO AAV-Cr-GFP ACC, 935 ± 78, 6 ± 6, n =, p =.34; OTR KO AAV-Cr-GFP vsu, 97 ± 9, 85 ± 85, n =, p =.43)., Comprisons twn un-injt OTR KO vrsus OTR KO nimls injt with AAV-Cr-GFP into th ACC or vsu rvls no iffrn in normliz soil frn n sutrt soil frn (normliz uninjt.5 ±.95, OTR KO AAV-Cr-GFP ACC.36 ±.66, OTR KO AAV-Cr-GFP vsu.7 ±.4, p =.8, ANOVA; sutrt uninjt. ±.78, OTR KO AAV-Cr-GFP ACC.5 ±.4, OTR KO AAV-Cr-GFP vsu.99 ±.87, p =.9, ANOVA). Summry t r snt s mn ± s..m (*P <.5).
13 SUPPLEMENTARY INFORMATION RESEARCH Strotxi injtion of RV-TTomto into NA Tryptophn Hyroxyls (5-HT nuron) ntioy Oxytoin rptor vnus rportr mous 4V 4V 4V 4V Rph Rph Rph Rph Supplmntry Figur 3. OT rptor-xssing lls in th Rph sn srotonrgi projtions to th NA., Digrm illustrting strotxi injtion of RV-TTomto into th NA of OTR-Vnus rportr mi, follow y susqunt immunohistohmistry with nti-tryptophn hyroxyls ntiois to ll srotonrgi nurons of th Rph.,, Low mgnifition (, sl r µm) n high mgnifition (, sl r µm) imgs of nurons in th Rph tht sn projtions to th NA (r, lft), xss OTR (grn, mil lft) n prou 5-HT (lu, mil right). Arrowhs n mrg img (right) shows sust of nurons tht show ololiztion. (orsl rph, Rph; fourth vntril, 4V). 3
14 RESEARCH SUPPLEMENTARY INFORMATION + pa 5 ms AAV-Cr-GFP RPh OTR * EPSC (normliz)..5 CP AAV-Cr-GFP Rph / OTR 4 6 Tim (minuts) 5 min vrg EPSC (norm)..5 CP Supplmntry Figur 4. Molulr ltion of OTRs in orsl rph os not isrupt 5HTR gonist inu LTD., Digrm illustrting tim ours of virl injtions into th Rph for LTD xprimnts in -. Rsnttiv trs (), summry tim ours () n vrg -trtmnt mgnitu omprisons () rvl 5HT inu-ltd in EPSCs ror from OTR nimls whos OTRs h n molulrly lt y AAV-Cr-GFP Rph injtion (Rph-AAV-Cr-GFP injt OTR,.58 ±.35,.3566 ±.89, n = 5 lls, p =.3 pir t-tst). Rsnttiv trs r inn from 5 onsutiv swps tkn t tims init ( n ). Summry t r snt s mn ± s..m (*P <.5). 4
15 SUPPLEMENTARY INFORMATION RESEARCH umlitv proilty intrvnt intrvl (s) umlitv proilty 5 pa.5s mplitu (pa) frquny (Hz) 4 n.s. mplitu (pa) n.s. CTL NAS CTL NAS Supplmntry Figur 5. 5HTR-A lon os not ltr mini EPSC frquny or mplitu., Rsnttiv minitur EPSCs (mepscs) ror in ontrol nurons (purpl) n nurons trt with NAS-8 (r, µm, minuts)., Cumultiv proility (top) n vrg (ottom) mepsc frquny is not rs in NAS-trt lls om to ontrol lls (ontrol 3.3±.456, n = lls, NAS ±.49, n = lls; p =.9346 unpir t-tst)., Cumultiv proility (top) n vrg (ottom) mepsc mplitu is not iffrnt in NAS-trt lls om to ontrol lls (ontrol 6.3 ±.5, n = lls, NAS ±.886, n = lls; p =.586 unpir t-tst). Summry t r snt s mn ± s..m (*P <.5). 5
16 RESEARCH SUPPLEMENTARY INFORMATION Rph inputs OTR 5-HT nuron ortil, thlmi, myglr inputs glutmt nuron 5-HTR PVN inputs OT nuron NA mium spiny nuron LTD Supplmntry Figur 6. Digrm illustrting mol for inution of OT-inu LTD in NA. OT prouing nurons from th PVN rls OT into th NA. Ativtion of OTRs on synpti trminls of 5-HT nurons from th Rph uss th rls of 5-HT into th NA. Ativtion of 5HT rptors on glutmtrgi trminls from vrious rin rgions (ortx, mygl, thlmus) rs synpti funtion t xittory synpss onto MSNs. 6
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