Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking

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1 Designer reeptors show role for ventrl pllium input to ventrl tegmentl re in oine seeking Stephen V Mhler 1, Elen M Vzey 1, Jo T Bekley 1, Coly R Keistler 1, Ellen M MGlinhey 1, Jennifer Kufling 1, Steven P Wilson 2, Krl Deisseroth 3, John J Woowr 1 & Gry Aston-Jones 1 npg 21 Nture Ameri, In. All rights reserve. The ventrl pllium is entrlly positione within mesoortiolimi rewr iruits, n its ense projetion to the ventrl tegmentl re () regultes neuronl tivity there. However, the ventrl pllium is heterogeneous struture, n how this omplexity ffets its role within wier rewr iruits is unler. We foun tht projetions to from the rostrl ventrl pllium (), ut not the ul ventrl pllium (), were roustly Fos tivte uring ue-inue reinsttement of oine seeking rt moel of relpse in ition. Moreover, esigner reeptor meite trnsient intivtion of neurons, their terminls in or funtionl onnetivity etween n opmine neurons loke the ility of rug-ssoite ues (ut not oine prime) to reinstte oine seeking. In ontrst, neuronl inhiition loke oine-prime, ut not ue-inue, reinsttement. This oule issoition in ventrl pllium suregionl roles in rug seeking is likely to e importnt for unerstning the mesoortiolimi iruits unerlying rewr seeking n ition. The ventrl pllium is ruil noe in the ventrl stritopllil iruits unerlying rewr-relte ehviors, with mjor reiprol onnetions to the umens, extene mygl, limi thlmus, sustnti nigr (SN) n 1. Ventrl pllium neurons respon to rewrs n their ues in nimls n humns n re neessry for rewr-seeking ehviors, leing to hypotheses tht the ventrl pllium helps trnslte motivtionl sttes into ppetitive ehviors 5 7. However, the ventrl pllium is heterogeneous struture, exhiiting meil-lterl n rostrl-ul vrition in histologil mrkers n onnetivity ptterns 1,,. Notly, the ventromeil ventrl pllium (most of whih is lote suommissurlly, rostrl of regm in rt: ) reeives ense fferents from the nuleus umens shell n projets strongly to the meioorsl thlmus n. The orsolterl ventrl pllium (most of whih is lote sulentiulrlly, ul of regm: ) inste reeives fferents from the umens ore n projets to SN n the suthlmi nuleus, in ition to 1,9. This topogrphy implies the involvement of ventrl pllium suregions in funtionlly istint mesolimi iruits. Funtionl heterogeneity etween ventrl pllium suregions hs lso een reporte. In vitro, n neurons iffer in severl wys: mny neurons hve spiny enrites, glutmtergi fferents, hyperpolrize memrne potentils n no spontneous tion potentils 1. Interestingly, these hrteristis re more typil of umens meium spiny neurons thn lssil ventrl pllium neurons. In ontrst, neurons hve more lssilly pllil hrteristis, inluing spiny enrites, GABAergi inputs, epolrize memrne potentils n spontneous tion potentils. Notly, neuronl firing uring rt oine self-ministrtion vries epening on meiolterl n rostroul loliztion within the ventrl pllium 11, n ventrl pllium rostroul vrition exists in neuronl ytorhiteture, in vivo firing rtes, sensitivity to the rewring effets of eletril stimultion n miroinjete rugs, opioi gonist inue heoni responses to tstes n funtionl mgneti resonne imging (fmri) tivtion to emotionlly rousing pitures However, little is known out how suh heterogeneity ontriutes to the funtions of the ventrl pllium within wier mesolimi iruits. In rts, provies tonilly tive GABA inputs to n SN tht gte the firing of supopultions of opmine neurons n moulte rewr seeking 2. However, ventrl pllium neurons re lso utely tivte y rewr-ssoite ues 6,21,22, n ventrl pllium projetions to re tivte uring ue-inue reinsttement of oine seeking 23. How oes funtionl-ntomil heterogeneity ffet the regultion of y the ventrl pllium n the role of this iruit in rug seeking? We sought to exmine the roles of ventrl pllium suregions n their projetions in relpse to oine seeking. We exmine Fos tivtion of n projetions to n intivte these suregions or their projetions to mirin opminergi regions uring reinsttement of oine seeking using esigner reeptors exlusively tivte y esigner rugs (DREADDs) 2. DREADDs utilize enogenous G-protein signling pthwys ut o not ffet neuronl tivity in the sene of their otherwise inert, exogenously ministere lign, lozpine-n-oxie (). The G i -ouple (hmdi) DREADDs use here therey llow trnsient intivtion of efine neuronl popultions 25. We lso show tht they n e use to mnipulte xonl terminls of DREADD-expressing neurons 1 Deprtment of Neurosienes, Meil University of South Crolin, Chrleston, South Crolin, USA. 2 Deprtment of Phrmology, Physiology n Neurosiene, Shool of Meiine, University of South Crolin, Columi, South Crolin, USA. 3 Deprtment of Bioengineering n Psyhitry n Behviorl Sienes, Stnfor University, Stnfor, Cliforni, USA. Corresponene shoul e resse to S.V.M. (mhler@mus.eu). Reeive 12 Deemer 213; epte 2 Jnury 21; pulishe online 2 Mrh 21; oi:1.13/nn.366 nture NEUROSCIENCE VOLUME 17 NUMBER APRIL

2 npg 21 Nture Ameri, In. All rights reserve. Figure 1 hmdi inhiitory DREADD expression n funtion. () The Syn-hMDi-HA-GFP lentivirus use to express hmdi DREADDs uner neuronl-speifi humn synpsin promoter (Syn) in or. LTR, long terminl repet; IRES, internl riosome entry site; egfp, enhne GFP; WPRE, WHV post-trnsriptionl regultory element; SD, splie onor site; Psi, enpsition signl; GA, trunte GAG sequene; RRE, Rev response element. () Typil stining for HA-tgge hmdi fter ilterl injetions into. Sle r, 1 mm. () HA-tgge hmdi reeptors re expresse on ell oies n proesses (lk stining) in the ventrl pllium (neutrl re ounterstin). Sle r, 5 µm. () Typil HA expression (green) in ontine lrgely within the ventrl pllium orers (efine with sustne P ounterstin in re). (e) Typil HA expression in ontine lrgely within the ventrl pllium orers (s efine in ). Imges in e re representtive of DREADD expression seen in the experimentl groups (with n numers liste in the min text). The with of the imges in n e is 2.65 mm. (f) Inhiition of firing rtes in neurons (n = 2 totl) from nimls with Syn-hMDi-HA-GFP expression in or n lol pplition of 1 µm onto extrellulrly reore ventrl pllium (VP) neurons in vivo (with oule-rrel glss pipette). (g) Typil hnge in ishrge rte of ventrl pllium neuron (wveform inset) fter lol pplition (she region). to intivte prtiulr monosynpti pthwys (for exmple, -) or to isrupt funtionl onnetivity etween speifi neuronl popultions (for exmple, n opmine neurons). We emonstrte oule issoition etween the rostrl n ul ventrl pllium in ue s ompre to oine-prime reinsttement, respetively, n show tht ue oine seeking is epenent on projetions to n omplex intertions with opminergi n nonopminergi neurons there. RESULTS Vlition of ventrl pllium DREADDs We use DREADD-se strtegy to remotely ontrol ventrl pllium neurons in vivo n mnipulte the ventrl pllium iruit. We injete synpsin-riven lentivirl vetor yieling expression of the G i -ouple DREADD, hmdi 2, or ontrol virus lking the DREADD gene (Syn-GFP) into or n llowe nimls to survive for t lest 6 weeks efore nlyzing the rin tissue for virus expression. The Syn-hMDi-HA-GFP virus (Fig. 1) use roust hmdi expression within or (visulize with immunoretivity for the hemgglutinin (HA) tg or GFP reporter). These injetions yiele limite expression outsie the orers of the ventrl pllium (<3% leling outsie the ventrl pllium orers; Figs. 1 n 2 n Supplementry Fig. 1). The Syn-hMDi-HA-GFP n Syn-GFP viruses yiele omprle zones of somti GFP or hmdi expression entere t the injetion site (GFP:.2 (.21) mm 3 (men (s.e.m.)); hmdi:.9 (.5) mm 3 ). f 5 LTR +. mm HA SP VP neurons 79% (19) (5) Inhiite Not inhiite SD-Psi- GA-RRE g Spikes per 1 s Syn HA tg hmdi e HA + n Nissl IRES Promoters or filittors.72 mm egfp tg HA + proesses 1 ms WPRE Nissl only 3 LTR Enoe proteins We then use in vivo eletrophysiology to funtionlly vlite hmdi reeptor expression. In isofluorne-nesthetize rts, lol miroinjetion (6 nl, 1 µm) inhiite ventrl pllium neurons (n = 2 ells, 6.3 ± 6.6% (men ± s.e.m., normlize to the pre- firing rte for eh ell) erese in firing, Z = 2.5, P =.11; Fig. 1f,g). Lol miroinjetion of similrly ffete neurons (11/13 ells inhiite) n neurons (/11 inhiite), with 5.2 ±.7% n 63.2 ± 1.6% inhiition, respetively. Pre- firing rtes in (6. ± 2. Hz) were lower thn in (2. ± 5.3 Hz, P =.7, t 12 = 3.26), s reporte previously 1,12. Doule issoition of ventrl pllil roles in reinsttement Next we exmine whether trnsient DREADD-meite inhiition of or woul ttenute either ue or oine-prime (1 mg per kg oy weight) reinsttement of oine seeking fter Time (min) 57 VOLUME 17 NUMBER APRIL 21 nture NEUROSCIENCE

3 npg 21 Nture Ameri, In. All rights reserve. Figure 2 Effets of intivting the rostrl or ul ventrl pllium on reinsttement of oine seeking. () Dily oine infusions n tive or intive lever presses (men ± s.e.m.) for the lst 6 ys of riterion self ministrtion (>1 infusions per y) n tive or intive lever presses for the first 7 ys of extintion trining. () Ative lever presses uring ue-inue reinsttement fter ifferent oses of the DREADD gonist (the vrious oses re istinguishe y r shing: vehile or.1, 1, 1 or 2 mg per kg oy weight) in nimls with ilterl or Syn-hMDi-HA-GFP virus expression, ilterl ventrl pllium ontrol virus (Syn-GFP) expression or no virus expression. Intive lever presses uring reinsttement re shown with overli lk rs. P =.1 for vehile ompre to 1 mg per kg oy weight ; P =.15 for vehile ompre to 2 mg per kg oy weight (nlysis of vrine (ANOVA) with Bonferroni orretion post ho). () Ative n intive lever presses uring oine-prime reinsttement fter vehile or ifferent oses of (1 or 2 mg per kg oy weight) in nimls with or Syn-hMDi-HA-GFP virus expression or ontrol nimls (Syn-GFP or no virus expression). P =. for vehile ompre to 2 mg per kg oy weight (ANOVA with Bonferroni orretion post ho). The t in n re shown s the men ± s.e.m. () The ntomil loliztion of virus expression sites (s visulize with HA or GFP immunoretivity) represente for nimls injete with Syn-hMDi-HA-GFP in (lk res) or (gry res) or with Syn-GFP (gry res; inset). oine self-ministrtion n extintion (Fig. 2). (,.1, 1, 1 or 2 mg per kg oy weight) ttenute ue reinsttement in rts with hmdi expression in ut not in nimls with omprle DREADD expression in (: n = 16 rts, F,39 =., P =.; : n = 17 rts, F,6 =.59, P =.6; Fig. 2). In ontrst, (, 1 or 2 mg per kg oy weight) injetions ttenute oine-prime reinsttement only in nimls with hmdi expression in ut not in (: F 2,29 =.5, P =.6; : F 2,31 = 3., P =.; Fig. 2), reveling oule issoition etween n for reinsttement eliite y ues s ompre to oine. The zones in whih hmdi inhiition ws most effetive t reuing ue reinsttement were entere in, wheres effetive sites for oine-prime reinsttement were entere in (ue, ventrl pllium site rug intertion: F 1,16 = 7.35, P =.15; prime, ventrl pllium site rug intertion: F 1,25 =.5, P =.37). Inhiition of the ventrl pllium ws suffiient for these effets, s ses with miniml (<5%) hmdi expression outsie the ventrl pllium orers showe equivlent reinsttement effets s those with more (<3%) enrohment of expression into jent res (Supplementry Fig. 1). h no effet in nimls with either or infusions Self ministrtion Coine infusions Ative lever Intive lever Extintion Self-ministrtion y Extintion y Vehile.1 mg per kg 1 mg per kg 1 mg per kg 2 mg per kg hmdi Intive Intive Intive Intive Vehile 1 mg per kg 2 mg per kg hmdi Intive Intive Intive Control nimls GFP virus No virus GFP virus injetions (Syn-GFP, lking the hmdi gene; n = 5 rts; ue: F 2,11 =.5, P =.5; prime: F 1, =.5, P =.3) or no virus expression (n = 9 rts; ue: F 2,1 =.7, P =.; prime: F 2,2 =.2, P =.76; ontrol groups omine, ue: F 2,2 =.77, P =.7; prime: F 2,31 =.22, P =.; Fig. 2,). Fos tivtion of - projetions uring reinsttement We next ske whether the role of in ue reinsttement ehvior involves projetions to. First we exmine Fos expression in or -projeting neurons uring ue VP GFP Perentge Fos + -projeting neurons CS+ Ext CS Loo Ative lever presses : r =.7 : r = Perentge Fos + -projeting neurons Figure 3 Rostrl ventrl pllium projetions to re Fos tivte uring ue reinsttement. () Preominntly ipsilterl projetions from n to igrmme in the horizontl plne. () Axon terminls in express the nterogrely trnsporte, HA-tgge hmdi reeptor (lk xonl proesses) fter Syn-hMDi-HA-GFP injetion in ipsilterl (re Nissl ounterstin in ). Coronl view, sle r, 5 µm. The imge is representtive of ventrl pllium xonl DREADD expression in of the experimentl nimls (with n numers liste in the min text). () Perentges (men ± s.e.m.) of -projeting (CT + ) or ells tht express Fos fter ue-inue reinsttement (CS+) or ontrol ehviorl onitions in whih nimls were expose to the extinguishe self-ministrtion environment without ues or oine (Ext), tone or light stimulus not ssoite with oine (CS ) or loomotion-enhning novel environment (Loo). A greter proportion of -projeting neurons in were Fos tivte in CS+ thn in ontrol nimls (P =.1 for CS+ ompre to Ext; P =.5 for CS+ ompre to CS ; P =.25 for CS+ ompre to Loo; one-wy ANOVA with post ho Tukey HSD). No signifint ehvior-speifi tivtion of -projeting neurons ws etete in. The t re shown s the men ± s.e.m. () Correltion of ue-inue reinsttement ehvior with Fos tivtion of -projeting neurons in (P =.2, Person orreltion) n (P =.16, nonsignifint Person orreltion). nture NEUROSCIENCE VOLUME 17 NUMBER APRIL

4 npg 21 Nture Ameri, In. All rights reserve. Figure Intivting rostrl ventrl pllium fferents moultes ell firing. () hmdi reeptors (green) re expresse on xon terminls in the viinity of TH-lele opmine neurons (re) in. Sle r, 3 µm. The imge is representtive of ventrl pllium xonl DREADD expression in of experimentl nimls (with n numers liste in the Online Methos). () Slie eletrophysiology experiments in whih reore neurons were fille with iotin, lele with viin (re) n ientifie s opminergi y oleling for TH (green). Sle r, 1 µm. () Representtive tres from opmine neurons in seline onitions (top, lk) n fter (ottom, re; vertil sles, 2 pa; horizontl sles, 2 ms). () The effet of on the umultive istriution or men (inset) sipsc mplitue. In the lrger grph, the t re represente s the men ± s.e.m.; the lines in the inset onnet rw vlues for iniviul ells, n the rs show the men ± s.e.m. (e) The shift in IEI umultive istriution (P =.2 for seline ompre to ; two-wy ANOVA) n men sipsc frequeny (P =.6 for seline ompre to (inset); t test) fter. The t in the lrger grph re shown s the men ± s.e.m.; the lines in the inset onnet rw vlues for iniviul ells, n the rs show the men ± s.e.m. (f) In vivo exittion of puttively opminergi type 1 neurons (n = 9 totl) in y lol pplition of in the viinity of hmdi-expressing terminls in. (g) Rte histogrm emonstrting the effet of lol pplition in vivo (6 nl; she region) on the firing of type 1 neuron (wveform inset) in of -hmdi niml. (h) Inhiition of fst-firing, short-wveform type 2 neurons (n = 1 totl) y lol pplition of in vivo in the viinity of hmdi-expressing terminls in. (i) Exmple hnge in the ishrge rte of type 2 neuron (wveform inset) in fter lol pplition in vivo (6 nl; she region) in -hmdi niml. reinsttement or ontrol ehviors (Fig. 3 n Supplementry Fig. 2). We injete the retrogre trer holer toxin β suunit (CT) into the rostrl or ul. We trine nimls (n = 25) to intrvenously self-minister oine plus onurrently presente ues, extinguishe this ehvior n then gve them 2-h ue-inue reinsttement test or ontrol ehviorl test (Online Methos). We euthnize the nimls immeitely fter this test session n o-stine ventrl pllium slies for Fos n CT. In, -projeting (CT + ) neurons were Fos tivte uring ue oine seeking (n = rts) ut not uring exposure to the extinguishe self-ministrtion hmer (n = 7), novel environment (n = 6) or fmilir, non oine ssoite stimulus (n = ; F 3,21 = 6.6, P =.2; Fig. 3). Unlike, projetions to were not signifintly tivte uring ue reinsttement (F 3,21 = 2.3, P =.11; Fig. 3 n Supplementry Fig. 2). Moreover, in iniviul ue oine-seeking nimls, the egree of Fos tivtion of fferents from, ut not, ws positively orrelte with the egree of ue-inue oine-seeking ehvior (: r =.7, P =.2; : r =.5, P =.16; Fig. 3 n Supplementry Fig. 2), Cumultive proility f h 1..5 Bseline Type 1 neurons (1) 9% () Exite Not exite 71% (1) () Inhiite Not inhiite Amplitue i g Spikes per 1 s Spikes per 1 s 6 2 Amplitue (pa) e Cumultive proility Bseline 2 ms Merge initing tht more tivity in efferents to is ssoite with more ue-triggere rug seeking. Vliting inhiition of - projetion with DREADDs The hmdi DREADD is trffike xonlly, s eviene y numerous HA + fiers n terminls tht re relily oserve in the ventrl mirin fter injetion of hmdi vetor in or (Figs. 3 n n Supplementry Fig. 3,e). We exmine whether moultion of G i -ouple signling in ventrl pllium xon terminls in y lol pplition woul lter the tivity of neurons. First we reore spontneous inhiitory postsynpti urrents (sipscs) from opmine neurons (verifie y tyrosine hyroxylse (TH) immunoretivity) in slies from rts tht Rostrl Bseline IEI Meil Bseline Bseline ,2 1,5 Amplitue (pa) Time (ms) Type 2 neurons TH HA 2 ms Time (min) TH Aviin Time (min) 1 5 Frequeny (Hz) 5 VOLUME 17 NUMBER APRIL 21 nture neuroscience

5 npg 21 Nture Ameri, In. All rights reserve. were injete t lest 6 weeks previously with the hmdi vetor in (n = 7 rts; Fig.,). Perfusion with 5 µm h no effet on sipsc mplitue (t 6 =.9, P =.3; Fig. ) ut erese the frequeny (t 6 = 2.5, P =.6) n shifte the umultive istriution of interevent intervls (IEIs) to the right (min effet of IEI urtion: F 21,26 = 22.1, P =.1; tretment: F 1,26 = 1.2, P =.2; Fig. e). In slies from Syn-GFP injete nimls (n = 5 ells), whih lso expresse GFP in neurons n xons, h no effet on sipsc mplitue (t =.7, P =.67) or IEIs (t =.39, P =.; Supplementry Fig. ). These finings inite tht tivtion of hmdi DREADDs on ventrl pllium terminls in erese GABA relese onto opmine neurons. We onfirme this fining in vivo, emonstrting tht intr- miroinjetion of in rts previously injete with Syn-hMDi- HA-GFP in (-hmdi rts) signifintly inrese the firing rte (2.5 ± 5.2% inrese) in of 9 type 1 neurons (those meeting onventionl eletrophysiologil riteri for opmine neurons 26 ; P =.3; t 7 = 2.63; Fig. f,g). Conversely, 1 of 1 fst-spiking, shortwveform type 2 neurons (not onventionlly opmine-like) were inhiite ( 6.5 ± 12.% hnge in ishrge) fter intr- miroinjetion of in -hmdi rts (P =.3; t 9 = 3.93; Fig. h,i). Inhiiting terminls in loks ue reinsttement Next we exmine effets of intivting ventrl pllium terminls in on ue or oine-prime reinsttement ehvior. We miroinjete (in ounterlne orer on seprte ys) -hmdi nimls (n = 2) with (1 mm,.3 µl per sie) or vehile (rtifiil ererospinl flui (CSF)) into (n = 17 rts) or SN (n = 11) 5 min efore ue or prime reinsttement (two tests eh per niml; Fig. 5). We lso injete -hmdi nimls (n = 9) with ( or 1 mm,.3 µl) into n then teste them on ue or prime reinsttement (Fig. 5 n nnule sites in Supplementry Fig. 5). Inhiition of terminls in with lol seletively ttenute ue ut not prime reinsttement (ue: F 1,16 = 27.7, P =.75; prime: F 1,16 =.1, P =.92; Fig. 5), initing tht tivtion of the projetion to is require for rug ues to rive reinsttement of oine seeking. In ontrst, intivting terminls in SN or terminls in h no effets on ue or prime reinsttement (-SN inhiition, ue: F 1,1 =.21, P =.66; prime: F 1,1 =.1, P =.92; - inhiition, ue: F 1, =., P =.39, prime: F 1, =.37, P =.56; Fig. 5,f). This result inites tht lthough is neessry for oine-prime reinsttement (Fig. 2), its trgets re not essentil for this tion. Intivting projetions to in the sene of ues or priming injetions file to ffet rug seeking fter extintion (n = rts; vehile tive lever presses:.7 ± 6.7 (men ± s.e.m.); :.3 ± 1.7; F 1,3 = 3.9, P =.1). In -hmdi (n = 11) or -hmdi (n = 6) nimls, neither intr- (n = 17) nor intr-sn (n = 6) h signifint effet on generl loomotor ehvior in fmilir environment (no min effet of rug or rug time intertions; F s < 1.7, P s >.11; Fig. 5,e,g), initing tht reinsttement effets of - inhiition re more likely motivtionl thn motori in nture. interts with opmine neurons in reinsttement is omplex struture, with opmine, GABA n glutmte projetion neurons, s well s interneurons n other lol onnetivity Beuse ventrl pllium projetions moulte opmine n nonopmine neuron firing in 32 (Fig. ), we ske whether reinsttement ehvior is ffete y ontrhemispheri isonnetion of from opmine neurons. In trnsgeni rts tht express Cre reominse in TH neurons (THøCre + rts) 33, we unilterlly trnsue neurons with the Syn-hMDi-HA-GFP lentivirus esrie ove. We lso injete oule-floxe hmdi enossoite virus (AAV) (DIO-Syn-hMDi-mCherry; Fig. 6) into the ontrlterl (n = 9 rts) or SN (n = ), yieling relile n ntomilly restrite hmdi expression in unilterl opmine neurons (Fig. 6, n Supplementry Fig. 6). A systemi injetion in these nimls therefore funtionlly isonnete inputs from mirin opmine ells ilterlly while spring onnetivity with nonopminergi neurons (Fig. 6). Control groups onsiste of Cre + nimls with unilterl DIO-Syn-hMDi-mCherry injetions ut no virus in (n = 6 rts) or Cre littermtes (n = 1) with unilterl hmdi n ontrlterl injetions of DIO-Syn-hMDi-mCherry (ut no hmdi expression). As shown in Figure 6, ue-inue reinsttement ws mrkely ttenute y isrupting the - opmine iruit in this mnner (F 1, = 37.97, P =.3; Fig. 6e). We foun no effets fter similrly isonneting from SN opmine neurons (F 1,7 =.29, P =.61) or fter unilterlly intivting either (F 1,9 =.6, P =.1) or opmine neurons (F 1,5 =.57, P =.) (Fig. 6e). This result inites tht intt onnetivity etween (not ) SN miroinjetion 6 2 Intive Ext Cue Prime Vehile Intive Bem reks ( 1) Bem reks ( 1) Horizontl loomotion Rering 12 Time in session Intive Intive Ext Cue Prime e Bem reks ( 1) Bem reks ( 1) Horizontl loomotion 9 6 Rering Time in session (min) f 6 2 SN Intive Intive Ext Cue Prime g Bem reks ( 1) Bem reks ( 1) SN Horizontl loomotion 9 6 Rering Time in session (min) Figure 5 Intivting rostrl ventrl pllium projetion to loks ue reinsttement. () Projetions from to or SN or from to were trnsiently intivte y lol mirin miroinjetion of mong ventrl pllium xon terminls expressing hmdi reeptors. () The effet of intivtion of terminls from on ue-inue (Cue) n oine-prime (Prime) reinsttement of oine seeking (n = 17). Also shown re tive lever pressing uring the lst y of extintion trining (Ext, rs t left) n reinsttement fter tretment of with vehile (white rs) or (gry rs). Intive lever presses re represente with lk rs t the ottom. P =.75; two-wy ANOVA. () Effets on horizontl (top grph) n vertil (rering; ottom grph) loomotion in fmilir environment fter intivting projetions to (n = 11). Loomotion (em reks) uring 3 min ins in the 2-h session is shown fter miroinjetion of vehile (lk lines) or (gry lines). (,e) Effets on ue-inue n oine-prime reinsttement (n = 9), s well s horizontl loomotion n rering (n = 6), fter intivtion of efferents to. (f,g) Effets on reinsttement (n = 11) or loomotive ehvior (n = 6) fter intivtion of projetions to SN. All t re shown s the men ± s.e.m. nture NEUROSCIENCE VOLUME 17 NUMBER APRIL 21 51

6 npg 21 Nture Ameri, In. All rights reserve. Figure 6 Funtionl isonnetion of rostrl ventrl pllium projetion to opmine neurons loks ue-inue reinsttement. () A DIO-Syn-hMDi-mCherry AAV onstrut tht ws use to seletively express mcherry-tgge hmdi reeptors in opmine ells in THøCre + trnsgeni rts. ITR, inverte terminl repet. () hmdi expression in representtive niml with DIO-Syn-hMDi-mCherry injetion in. Immunoleling for TH ientifies opmine neurons (green), n mcherry (re) ientifies hmdi-expressing neurons in. Nerly ll hmdi-expressing neurons were opminergi. -injete nimls i not show sustntil hmdi expression lterlly in SN or in the ontrlterl. Sle r, 5 µm. The inset on the right shows hmdi + TH + ell t high mgnifition (imge with, 5 µm). () hmdi expression in representtive niml with DIO-Syn-hMDimCherry injetion in SN. Miniml expression in ws oserve. Sle r, 5 µm. Equivlent expression ws oserve in ehviorlly teste rts;, n = 9; SN, n =. () Unilterl Syn-hMDi-HA-GFP injetions were me in, n DIO-Syn-hMDi-mCherry injetions were me in the ontrlterl or SN of THøCre + rts or of Cre littermtes. When systemi ws ministere, seril onnetivity etween the ventrl pllium n mirin opmine popultions is ompromise ilterlly y unilterl hmdi inhiition of n ontrlterl hmdi inhiition of opmine neurons. (e) Ative n intive lever pressing uring ue-inue reinsttement in nimls with - opmine (DA) ontrlterl isonnet. Pressing uring lte extintion (gry r t left) n ue reinsttement fter vehile (white rs) or (1 mg per kg oy weight; re rs) is shown. Contrlterl isonnet nimls were THøCre + rts tht reeive unilterl Syn-hMDi-HA-GFP injetions n ontrlterl or SN DIO-Syn-hMDi-mCherry injetions. Unilterl intivtion rts (-hmdi) were Cre n reeive unilterl hmdi virus plus ontrlterl opmine hmdi virus (lthough the ltter i not use hmdi expression in these Cre rts). Unilterl opmine intivtion rts ( opmine hmdi) were Cre + n reeive only unilterl opmine hmdi virus. Only ontrlterl n (not SN) opmine popultions is require for onitione ues to trigger oine seeking uring reinsttement. Further exmintions of the nture of the - iruit GABAergi ventrl pllium projetions to n tonilly inhiit opmine neurons 5, n intivting this projetion with DREADDs n isinhiit opmine neurons (Fig. ). We therefore e e TH mcherry TH mcherry ITR Merge Dorsl Dorsl Syn Meil Meil Vehile (1 mg per kg) Extintion Merge Floxe hmdi DREADD AAV 6993 p ske whether isinhiition of opmine neurons oul hve ontriute to the ttenution of reinsttement we oserve fter intivting inputs to with DREADDs. If so, then iret isinhiition of opmine neurons with the GABA A ntgonist gzine shoul lso ttenute reinsttement. Inste we oserve tht intr- gzine (1 µm,.3 µl) strongly inrese ue reinsttement (n = 6 rts; F 2,17 = 9.3, P =.2; vehile s ompre f Intive loxp m Cherry hmdi tg lox 2722 Contrlterl isonnet Intive g WPRE opmine hmdi VP Intive 3 ITR Unilterl ontrols SN Intive - DA -SN DA DA isonnetion of from opmine neurons reue ue reinsttement elow ontrol levels. P =.3; repete-mesures ANOVA. The group n numers re liste in the min text. The t re shown s the men ± s.e.m. hmdi Cells per mm Vehile Gzine (1 µm) Fos + TH + neurons Totl TH + neurons Perentge TH + ells with Fos % 75.3% Veh Gzine (1 µm) Figure 7 GABA A -meite isinhiition of opmine neurons enhnes ue-inue reinsttement. () Exmple of Fos stining fter nery gzine miroinjetion (1 µm,.3 µl). () Exmple TH stining of the sme tissue. () Overly of Fos n TH stining. () Mgnifie view of the Fos n TH overly. Arrows point to olele neurons. Stining typil of gzine-injete rts (n = 5). Sle rs ( ), 1 µm. (e) Fos inution in, n numer of, TH + neurons fter miroinjetion of vehile (n = 3) or gzine (n = 5). (f) The perentge of TH + neurons tht expresse Fos fter injetion of vehile (veh) or gzine. (g) Gzine injetion sites within from ue-inue reinsttement nimls. Gzine ws injete t equivlent sites s in the Fos- n TH-stine setion in. (h) Ative n intive lever pressing uring ue reinsttement fter injetion of vehile or gzine (1 µm; n = 6). In suset of nimls (n = ), gzine (1 µm) roustly inrese pressing oth levers, whih ws proly relte to intense nonspeifi loomotor tivtion. P =.2; one-wy ANOVA. All t re shown s the men ± s.e.m. h 12 gzine injetion sites Ative lever Intive lever Ext 1 1 gzine (µm) 52 VOLUME 17 NUMBER APRIL 21 nture neuroscience

7 npg 21 Nture Ameri, In. All rights reserve. to 1 µm gzine tive lever: t 6 = 2.62, P =.; Fig. 7g,h) n roustly inue Fos in nery opmine neurons (vehile, n = 3; gzine, n = 5 rts; no effet on numer of TH + ells: t 6 =.2, P =.; totl Fos + TH + ells: t. = 5.55, P =.1; perentge Fos + TH + ells: t 6 =., P =.1; Fig. 7 f). This fining inites tht simple isinhiition of opmine neurons is proly not the mehnism y whih inhiiting inputs to reues ue reinsttement. In ition to GABA projetions, sens numerous VGlut2 + efferents to 3 ; we ske whether this glutmtergi projetion is require for ue reinsttement. We use unilterl DREADD-se inhiition n ontrlterl miroinjetion of oktil of the AMPA n NMDA ntgonists 6-yno-7-nitroquinoxline-2,3- ione (CNQX) n ( )-2-mino-5-phosphonovleri i (AP5) (.7 n 1.6 mm, respetively) to ilterlly ompromise glutmte projetions from to. This i not ffet ue reinsttement (n = 5 rts; t = 1.2, P =.29; Supplementry Fig. 7). In ontrst, reinsttement ws ttenute when ll neurons were inhiite (with lofen n musimol,.3 n.3 mm, respetively) ontrlterlly to DREADD-intivte (- isonnet, n = 1 rts, t 9 = 2.1, P =.2; no effet of unilterl lofen n musimol lone, n =, t 7 = 1.3, P =.3; Supplementry Fig. 7). In slies, we lso explore whether inhiiting inputs to woul erese spontneous exittory trnsmission (sepscs) onto opmine neurons. (5 µm) h no effet on sepsc mplitue (t 5 =.22, P =.3) or frequeny (t 5 =.3, P =.6) in TH + ells (n = 6 ells; Supplementry Fig. ). These finings inite tht glutmtergi projetions to o not moulte opmine neurons iretly n re proly not neessry for ue reinsttement. DISCUSSION Here we esrie n - pthwy tivte y, n neessry for, onitione ue-inue reinsttement of oine seeking in rts. The involvement of the ventrl pllium in reinsttement epens ritilly on oth the rostroul position within the ventrl pllium n the mens y whih rug seeking is reinstte (ues or oine prime). Cue-triggere rug seeking speifilly requires oth projetions to n funtionl intertions etween inputs n opmine n nonopmine neurons. In ition to esriing oule issoition etween ventrl pllium suregions in rug seeking, we lso vlite the use of G i -ouple DREADDs to intivte speifi neuronl pthwys through moultion of DREADD-expressing terminls n to funtionlly isonnet rin iruits in vivo. Ventrl pllium roles in rewr seeking Muh previous reserh hs fouse on the portion of the ventrl pllium ul of regm in rts (), whih showe tht the ventrl pllium is n importnt noe in mesoortiolimi rewr iruits. Lesions or phrmologil intivtion of reue rug or foo seeking 13,35 3. Mny of these ventrl pllium neurons re spontneously tive, n their GABAergi projetion to tonilly inhiits popultions of opmine neurons there 5. Ventrl pllium efferents to re lso utely tivte y rewrs n ssoite ues. For exmple, ventrl pllium projetions in rts re Fos-tivte fter ute mphetmine ministrtion, oine self-ministrtion or ue reinsttement of oine seeking 23,39,, n ventrl pllium neurons phsilly fire when slient rewrs n their ues re experiene 6,11. In nonhumn primtes, ventrl pllium neurons lso fire in reltion to rewr ntiiption, n ventrl pllium intivtion ompromises moultion of rewr seeking on the sis of rewr expettion 22. Similrly, the ventrl pllium is fmri-tivte y ues for rug or nturl rewrs in humns 1. Our report therefore supports n sustntilly expns on the known roles for the ventrl pllium in rewr seeking. Heterogeneity within the ventrl pllium Antomilly, oth rostroul n meiolterl heterogeneity exists in ventrl pllium ell morphology, trnsmitter ontent, soures of fferents n efferent trgets 6,1,16,2,3. A lrge proportion of the suommissurl onsists of neurotensin-positive ventromeil suregion, whih reeives fferents from the meil umens shell n sens efferents to the meioorsl thlmus n. In ontrst, lrge proportion of the sulentiulr onsists of linin-positive orsolterl suregion tht reeives umens ore inputs n projets to SN n the suthlmi nuleus 1. (n the jent ul umens shell) hs een esrie s trnsition zone etween the umens n tritionl (ul) ventrl pllium, with onnetivity n morphologil fetures istint from those of the rostrl umens, pllil or extene mygl regions 1,2. Supporting this view, n the ul ventromeil umens shell projet similrly to, n Fos tivtion of oth projetions is orrelte with ue-inue reinsttement ehvior (Fig. 3) 23. Previous eviene supports role for in onitione motivtion n for in the rewring n priming effets of reinforers themselves. For exmple, (ut not ) is Fostivte uring motivte ehviors triggere y Pvlovin ues for speifi slient rewrs n is neessry for these ues to trigger seeking ehviors,5. (not ) is fmri-tivte when people view highly slient, isgusting photogrphs, gin initing role in onitione motivtionl sttes 1. In ontrst, GABA gonists miroinjete into reue oine-prime n stress-inue reinsttement of oine seeking 35,36, in lignment with our oservtion tht DREADD-se inhiition of neurons loke prime reinsttement (lthough to our knowlege ours is the first report of role for the ventrl pllium, n more speifilly,, in ue reinsttement of oine seeking). Aitionlly, (not ) hs een linke to the heoni evlution of primry rewrs in nimls n humns 1,19. Here we exmine the funtions of n in ue n prime reinsttement of oine seeking. We showe tht (not ) is neessry for ues to trigger oine seeking, wheres (not ) is neessry for oine prime to eliit reinsttement. Therefore, the ventrl pllium in generl is ritilly involve in promoting relpse-relte rug seeking, ut its rostrl n ul spets ifferentilly meite the motivtionl properties of previously lerne ues s ompre to oine itself. - projetion speifilly meites ue rug seeking To exmine the wier neurl iruits with whih n intert to rive reinsttement, we fouse on ventrl pllium projetions to. First we showe tht ut not projetions to re Fos tivte in proportion to ue reinsttement. To test whether these efferents to re lso neessry for reinsttement, we inue hmdi DREADD expression in or neurons n then miroinjete the DREADD gonist iretly into. This llowe us to speifilly inhiit ventrl pllium projetions ut not other ventrl pllium efferents. When projetions to (ut not the jent SN) were intivte in this mnner, ue-inue reinsttement ws olishe, ut prime reinsttement ws spre. In ontrst, similr inhiition nture NEUROSCIENCE VOLUME 17 NUMBER APRIL 21 53

8 npg 21 Nture Ameri, In. All rights reserve. of projetions to file to ffet either ue or prime reinsttement. Therefore, projetions to re seletively reruite uring ue reinsttement n re neessry for ues to trigger reinsttement, wheres is neessry for oine prime to promote reinsttement through -inepenent iruitry. iniretly moultes opmine neurons in reinsttement We ientifie funtionl iruit ontining n (ut not or SN) opmine neurons tht is neessry for ues to eliit reinsttement y funtionlly isonneting from opmine neurons through hmdi inhiition of in one hemisphere n simultneous hmdi inhiition of opmine neurons in the ontrlterl hemisphere. This seletive, ilterl iruit isruption reue ue-inue reinsttement, initing tht seril onnetivity etween n opmine neurons is require for ues to trigger oine seeking. Ventrl pllium projetions to re preominntly GABAergi 3,5,32, n onsistent with previous reports 32, we oserve tht DREADD-se inhiition of inputs to isinhiite opmine neurons y eresing sipsc frequeny. However, inisriminte isinhiition of opmine neurons is unlikely to ount for the ttenute reinsttement tht we oserve fter - intivtion. When we iretly isinhiite opmine neurons (s eviene y roust Fos inution) with miroinjetions of the GABA A ntgonist gzine, ue reinsttement ws inste inrese (Fig. 7). It is therefore likely tht reinsttement loke y inhiition of inputs to involves ifferentil moultion of heterogeneous neuronl supopultions, possily inluing inhiition of nonopminergi neurons. Notly, this moultion ppers to e le to supersee reinsttement-promoting effets of opmine neuron isinhiition, whih n lso our when the - iruit is intivte. Systemti mpping of inputs to neuronl supopultions will e require to further hrterize the omplexities of this new, ition-relte iruit. In ition to GABA efferents, sens sustntil VGlut2- ontining projetions to 3. However, DREADD-se inhiition of - projetions i not reue spontneous exittory trnsmission to TH + ells, n ontrlterl isonnet se isruption of glutmte projetions to i not ffet ue reinsttement, rguing ginst reinsttement-relte glutmtergi - projetion. Conlusions Here we showe tht projetions to re tivte uring, n re neessry for, ue-inue reinsttement n tht this requires moultion of opmine neurons. However, this moultion proly oes not involve iret GABAergi or glutmtergi projetions onto opmine neurons. Inste, inhiiting inputs to yiele oth exittion of type 1 neurons n inhiition of type 2 neurons in vivo (Fig. ). Type 1 neurons, whih isplye onventionlly opminergi eletrophysiologil hrteristis, were exite y ventrl pllium inhiition, onsistent with our fining tht intivtion of inputs to ttenutes GABA inputs to TH + neurons (Fig. e). However, inisriminte isinhiition of opmine neurons with GABA A ntgonist i not impee reinsttement ut inste roustly filitte it. Therefore, lthough our isonnetion stuy revele tht opmine neurons re neee for ventrl pllium regultion of relpse, the role of the ventrl pllium in reinsttement oes not epen on generl isinhiition of opmine neurons in. One possiility is tht ventrl pllium neurons seletively regulte selet supopultion of opmine neurons uring ue rug seeking. Notly, ontins fst-firing, thin-wveform opmine neurons 6,7, n it is possile tht these oul e the type 2 neurons whose tivity is suppresse fter - intivtion (Fig. h,i). However, this possiility seems unlikely, s inhiitory inputs to opmine neurons reore in vitro here were nerly lwys reue y DREADD-se intivtion of - projetions (Fig. e). Therefore, we hypothesize tht omplex, supopultion-speifi ventrl pllium moultion of neurons is neessry for ues to eliit relpse to oine seeking. These finings ll for itionl reserh to hrterize the reltive onnetivity of ventrl pllium GABA, glutmte, etylholine n peptie-ontining neurons with neuronl supopultions n to further efine the roles for eh in rewr seeking. In summry, we hve esrie new pthwy, the projetion from to, tht is neessry for onitione rug seeking in moel of relpse in ition. Furthermore, this iruit-ehvior reltionship requires moultion of oth nonopmine n opmine neurons. We lso showe tht ventrl pllium suregions hve qulittively ifferent roles in rug-seeking ehvior eliite y either onitione stimuli or the rug itself. Together these finings oth expn the known iruitry of onitione motivtion n lso highlight importnt next iretions for untngling the neurl sustrtes of rewr-seeking ehvior, espeilly in ppetitive isorers suh s rug ition. Methos Methos n ny ssoite referenes re ville in the online version of the pper. Note: Any Supplementry Informtion n Soure Dt files re ville in the online version of the pper. Aknowlegments We thnk P. Do, M.J. Gilstrp n E.C. Lin for ssistne with ehviorl testing n immunohistohemistry n B.L. Roth (Deprtment of Phrmology, University of North Crolin) for DREADD onstruts n onsulttion on DREADDs. ws supplie y US Ntionl Institutes of Helth (NIH) Ntionl Cner Institute (NCI) uner the uspies of NS62-1 n y the Ntionl Institute of Mentl Helth (NIMH) Chemil Synthesis n Drug Supply Progrm. Reserh ws supporte y NIH grnts F32 DA26692, K99 DA35251 (S.V.M.), F31 DA391 (J.T.B.), R21 DA2537 (G.A.-J. n S.P.W.), R1 DA13951 (J.J.W.), R37 DA621 n P5 DA15369 (G.A.-J.). This projet ws supporte y the Ntionl Center for Reserh Resoures n the Offie of the Diretor of the Ntionl Institutes of Helth through grnt numer C6 RR1555. AUTHOR CONTRIBUTIONS S.V.M. ttine funing, esigne n onute experiments, performe surgeries, nlyze t n wrote the mnusript. E.M.V. esigne n onute nesthetize eletrophysiology experiments, nlyze these t n wrote the mnusript. J.T.B. esigne n onute slie eletrophysiology experiments, nlyze these t n wrote the mnusript. C.R.K. onute ehviorl experiments n wrote the mnusript. E.M.M. performe surgeries for n onute ehviorl n immunohistohemil experiments. J.K. onute immunohistohemil n onfol mirosopy experiments. S.P.W. ttine funing n ontriute the Syn-GFP virl onstrut. K.D. ontriute the THøCre trnsgeni rt line. J.J.W. ttine funing, esigne n onute slie eletrophysiology experiments n wrote the mnusript. G.A-J. ttine funing, esigne experiments n wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Reprints n permissions informtion is ville online t reprints/inex.html. 1. Zhm, D.S. & Heimer, L. Two trnspllil pthwys originting in the rt nuleus umens. J. Comp. Neurol. 32, 37 6 (199). 5 VOLUME 17 NUMBER APRIL 21 nture neuroscience

9 npg 21 Nture Ameri, In. All rights reserve. 2. Zhm, D.S., Willims, E. & Wohltmnn, C. Ventrl stritoplliothlmi projetion: IV. Reltive involvements of neurohemilly istint suterritories in the ventrl pllium n jent prts of the rostroventrl forerin. J. Comp. Neurol. 36, (1996). 3. Klivs, P.W., Churhill, L. & Klitenik, M.A. GABA n enkephlin projetion from the nuleus umens n ventrl pllium to the ventrl tegmentl re. Neurosiene 57, (1993).. Her, S.N. & Nut, W.J. Rmifitions of the glous pllius in the rt s inite y ptterns of immunohistohemistry. Neurosiene 9, (193). 5. Floreso, S.B., West, A.R., Ash, B., Moore, H. & Gre, A.A. Afferent moultion of opmine neuron firing ifferentilly regultes toni n phsi opmine trnsmission. Nt. Neurosi. 6, (23). 6. Smith, K.S., Tinell, A.J., Alrige, J.W. & Berrige, K.C. Ventrl pllium roles in rewr n motivtion. Behv. Brin Res. 196, (29). 7. Mogenson, G.J., Bruzynski, S.M., Wu, M., Yng, C.Y. & Yim, C.Y. 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Ventrl pllil neuronl responses to opmine reeptor stimultion in the nuleus umens. Brin Res. 9, (199). 13. MBrie, W.J., Murphy, J.M. & Ikemoto, S. Loliztion of rin reinforement mehnisms: intrrnil self-ministrtion n intrrnil ple-onitioning stuies. Behv. Brin Res. 11, (1999). 1. Pngis, G., Miliressis, E., Angnostkis, Y. & Spyrki, C. Ventrl pllium selfstimultion: movele eletroe mpping stuy. Behv. Brin Res. 6, (1995). 15. Bengtson, C.P. & Osorne, P.B. Eletrophysiologil properties of holinergi n nonholinergi neurons in the ventrl pllil region of the nuleus slis in rt rin slies. J. Neurophysiol. 3, (2). 16. Zhm, D.S. & Heimer, L. Ventrl stritopllil prts of the sl gngli in the rt: I. Neurohemil omprtmenttion s reflete y the istriutions of neurotensin n sustne P immunoretivity. J. Comp. Neurol. 272, (19). 17. Johnson, P.I., Stellr, J.R. & Pul, A.D. Regionl rewr ifferenes within the ventrl pllium re revele y miroinjetions of µ opite reeptor gonist. Neurophrmology 32, (1993). 1. Cler, A.J. et l. Disgust sensitivity preits the insul n pllil response to pitures of isgusting foos. Eur. J. Neurosi. 25, (27). 19. Smith, K.S. & Berrige, K.C. The ventrl pllium n heoni rewr: neurohemil mps of surose liking n foo intke. J. Neurosi. 25, (25). 2. Gre, A.A., Floreso, S.B., Goto, Y. & Loge, D.J. Regultion of firing of opminergi neurons n ontrol of gol-irete ehviors. Trens Neurosi. 3, (27). 21. Tinell, A.J., Berrige, K.C. & Alrige, J.W. Ventrl pllil representtion of pvlovin ues n rewr: popultion n rte oes. J. Neurosi. 2, (2). 22. Thin, Y. & Hikosk, O. The primte ventrl pllium enoes expete rewr vlue n regultes motor tion. Neuron 76, (212). 23. Mhler, S.V. & Aston-Jones, G.S. Fos tivtion of seletive fferents to ventrl tegmentl re uring ue-inue reinsttement of oine seeking in rts. J. Neurosi. 32, (212). 2. Armruster, B.N., Li, X., Push, M.H., Herlitze, S. & Roth, B.L. Evolving the lok to fit the key to rete fmily of G protein ouple reeptors potently tivte y n inert lign. Pro. Ntl. A. Si. USA 1, (27). 25. Ferguson, S.M. & Neumier, J.F. Grteful DREADDs: engineere reeptors revel how neurl iruits regulte ehvior. Neuropsyhophrmology 37, (212). 26. Ungless, M.A. & Gre, A.A. Are you or ren t you? Chllenges ssoite with physiologilly ientifying opmine neurons. Trens Neurosi. 35, 22 3 (212). 27. Ikemoto, S. Dopmine rewr iruitry: two projetion systems from the ventrl mirin to the nuleus umens-olftory tuerle omplex. Brin Res. Rev. 56, 27 7 (27). 2. Morles, M. & Pikel, V.M. Insights to rug ition erive from ultrstruturl views of the mesoortiolimi system. Ann. NY A. Si. 12, 71 (212). 29. Fiels, H.L., Hjelmst, G.O., Mrgolis, E.B. & Niol, S.M. Ventrl tegmentl re neurons in lerne ppetitive ehvior n positive reinforement. Annu. Rev. Neurosi. 3, (27). 3. vn Zessen, R., Phillips, J.L., Buygin, E.A. & Stuer, G.D. Ativtion of GABA neurons isrupts rewr onsumption. Neuron 73, (212). 31. Stmtkis, A.M. et l. 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10 npg 21 Nture Ameri, In. All rights reserve. ONLINE METHODS Sujets. 159 mle Sprgue-Dwley rts (25 35 g; Chrles River) n 5 outre mle Long-Evns THøCre rts (hemizygous Cre +, n = 35; Cre littermtes, n = 1) 33 were single or pir house in 12 h, 12 h reverse light yle vivrium (ll tests were onute in the rk perio). No sttistil methos were use to preetermine smple sizes, ut they were similr to those reporte in previous pulitions 15,19,23,9. All proeures were pprove y the Meil University of South Crolin s Institutionl Animl Cre n Use Committee. Surgil proeures. Animls were nesthetize with ketmine, xylzine n meloxim (56.5,.7 n 1. mg per kg oy weight, respetively) n implnte with inwelling jugulr theters. They then reeive 2 3 nl injetions of.5% CT unilterlly in or 1 µl unilterl or ilterl virus injetions into, or. CT n ventrl pllium lentivirus injetions were me y pneumti pressure using glss pipette (for CT, 15 2 µm tip; for lentivirus, 3-5 µm tip) over 5 min n left in ple for 15 2 min. In THøCre nimls, mirin injetions of n AAV floxe DIO-Syn-hMDi-mCherry virus were me over 1 min through Hmilton miroinjetion syringe. For ilterl or gzine or unilterl glutmte ntgonist or GABA gonist miroinjetions in ehving nimls, guie nnule were implnte 2 mm orsl to or SN. Virl onstruts. To trnsue ventrl pllium neurons with hmdi DREADDs, we use synpsin promoter riven lentivirl vetor with GFP reporter ustom pkge (VSV-G envelope) y the University of Pennsylvni vetor ore. To ssist with ientifition, the hmdi hs n N-terminl HA tg n n IRES GFP reporter (Syn-hMDi-HA-GFP; Fig. 1). A Syn-GFP lentivirus (lking the DREADD gene) ws use to ontrol for nonspeifi virus effets (Fig. 2, n Supplementry Fig. 5). In THøCre trnsgeni rts 33, n AAV ontining oule-floxe, inverte open reing frme hmdi-mcherry sequene (DIO-Syn-hMDimCherry; University of North Crolin Vetor Core) ws miroinjete into the ventrl mirin to express hmdi reeptors seletively in TH neurons of or SN (Fig. 6). Loliztion of DREADD expression. hmdi reeptor expression in ell oies n proesses ws visulize with immunohistohemistry for HA, GFP or mcherry tgs (esrie elow). Continment of trnsution sites within or, or within or SN opmine ells, ws onfirme y o-stining for sustne P (SP; efining orers of the ventrl pllium) or TH (efining n SN) n rin tls 5. A synpsin promoter virus ws employe in the ventrl pllium, so some expression ws oserve outsie the orers of or in most nimls. Animls with more thn 3% of DREADD expression oserve outsie or were exlue from nlyses (n = 7). For nimls with virus injetions, no more thn 2% of DREADD-expressing tissue enrohe into (i.e., ul of regm) or vie vers. Animls injete with the Syn-GFP ontrol virus in the ventrl pllium h omprle GFP expression ptterns to the hmdi-injete nimls. For THøCre rts, hmdi expression ws roust in either or SN n ws restrite lmost exlusively to TH + ell oies n proesses (perentge mcherry + neurons oexpressing TH (men ± s.e.m.): 97.5 ±.7%; Fig. 6, n Supplementry Fig. 6). mcherry expression ws not oserve in the ontrlterl hemisphere, n expression i not exten sustntilly into SN of -injete nimls or into of SNinjete nimls (Fig. 6,). Eletrophysiologil vlition of DREADD-se inhiition of ventrl pllium neurons n efferents. Brin slies from nimls injete in the ventrl pllium with Syn-hMDi-HA-GFP (n = 6 rts) or Syn-GFP (n = 2) were prepre s previously esrie 9. Slie physiology t olletion n nlyses were not performe lin to the experimentl group. Horizontl setions ontining were trnsferre to 32 3 C hmer ontining rogen-ule CSF solution. Slies were trnsferre to reoring hmer n perfuse with 32 C CSF t 2 ml per min. Neurons were visully ientifie uner infrre light using Dot grient ontrst imging. Reoring pipettes with resistne of 1 3 MΩ were fille with internl solution ontining (in mm) CsCl (12), -(2-hyroxyethyl)- 1-piperzineethnesulfoni i (1), MgCl 2 (2), ethylene glyol tetreti i (1), N 2 ATP (2), NGTP (.3) n.2% ioytin; 295 mosm, ph 7.3. To isolte sipscs, reoring CSF ws supplemente with AMPA n NMDA ntgonists (NBQX n AP5; 1 µm n 1 µm, respetively; Am), n for sepsc reorings, pirotoxin (1 µm; Toris) ws e to the CSF. Neurons meil to the nuleus of the essory opti trt were trgete for pth lmp (in the viinity of ense, GFP-expressing fferents), n whole-ell moe ws hieve while holing the ell t 7 mv. Dt were quire using n Axon MultiClmp 7B mplifier n n ITC-1 igitl interfe (HEKA Instruments) ontrolle y AxogrphX softwre. Reorings were filtere t khz n quire t 1 khz, n sipscs were etete n nlyze using the templte-mthing event-etetion lgorithm in AxogrphX. Detetion prmeters were set t mplitue > pa, n quire events were visully inspete efore verging. We reore t lest 15 spontneous urrents uring seline perio n fter 1 min of 5 µm perfusion. Series resistne (R s ) ws monitore throughout the reoring, n n experiment ws isontinue if the R s exeee 25 MΩ or hnge more thn 25%. In vivo eletrophysiology t olletion n nlyses were performe in isofluorne-nesthetize rts y n experimenter lin to experimentl onitions. Glss reoring pipettes (5 12 MΩ) were fille with 2% pontmine sky lue in.5 M soium ette. A glss injetion pipette (2-µm tip) glue ~15 µm ehin the reoring tip elivere 6 nl of 1 µm to the ventrl pllium or neurons with rief pneumti pulses. Multiple pplitions ourre in eh niml in ifferent ells, eh seprte y >3 min. Signls were mplifie n filtere (.3 5 khz) using Moel 16 Neuroproe Amplifier (A-M Systems) n BMA 2 Biomplifier (CWE) efore igitl onversion n reoring to Spike 2 (v5.21) with Miro 11 MkII interfe (CED). Boy temperture ws mintine t C throughout the reorings. Reore ventrl pllium neurons were lote ner previous virus injetions n were ientifie y their firing rtes n wveforms, s reporte previously 12. For onfirmtion of reoring lotions, mrkings 1 mm ove n immeitely elow reoring trks in eh ventrl pllium n site were me y iontophoreti ye eposits immeitely efore perfusion. type 1 neurons were ientifie y eletrophysiologil riteri tritionlly esriing opmine neurons 26, inluing (i) iphsi or triphsi wveform >2.5 ms in urtion, (ii) >1.1 ms from spike onset to negtive trough n (iii) spontneous firing rte <1 Hz. All ells inlue in nlyses were from onfirme reorings in the ventrl pllium or. Drugs n trt trers. Coine HCl (NIDA) ws issolve in.9% sterile sline. CT (Sigm) ws issolve t.5% in.1 M PBS. ws supplie y NIH NCI uner the uspies of NS62-1. For systemi ministrtion, (,.1, 1, 1 or 2 mg per kg oy weight) ws issolve in 5% imethyl sulfoxie n then ilute to 1 ml per kg oy weight volume with.9% sline. For intrrnil ministrtion, ws issolve in CSF to 1 µm (in vivo eletrophysiology) or 1 mm (ehviorl miroinjetion) or 5 µm in vitro. Gzine (1 or 1 µm), lofen n musimol (.3 n.3 mm, respetively), n CNQX n AP5 (.7 n 1.6 mm, respetively; ll from Sigm) were issolve in CSF for.3 µl miroinjetions. Behviorl trining n testing proeures. Behviorl trining n testing took ple in stnr Me Assoites opernt hmers esrie elsewhere 23. Rts unerwent ten ily 2-h self-ministrtion sessions (>1 oine infusions per y,.2 mg, 5 µl infusion). Pressing one lever yiele 3.6-s oine infusion, tone n light ove the tive lever (FR1 sheule), followe y 2-s timeout uring whih pressing ws reore ut i not yiel oine or ues (Fig. 2). Intive lever presses were reore ut h no onsequenes. Animls then reeive t lest 7 of extintion trining (until the riterion of <25 presses for 2 onseutive ys ws rehe) uring whih lever presses yiele neither oine nor ues. During 2-h ue-inue reinsttement tests, tive lever presses yiele oine ues ut no oine. For oine priming tests, 1 mg per kg oy weight oine ws ministere intrperitonelly (i.p.) immeitely efore the 2-h test uring whih lever presses yiele neither oine nor ues. For the CT n Fos experiment, nimls were perfuse immeitely fter one of the following 2-h test sessions: CS+ reinsttement, n itionl extintion session, exposure to novel environment or exposure to isrete CS (further etils re liste in ref. 23). nture NEUROSCIENCE oi:1.13/nn.366