Visualizing an emotional valence map in the limbic forebrain by TAI-FISH

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1 r t i l e s Visulizing n emotionl vlene mp in the limi forerin y TAI-FISH Jino Xiu 1 3, Qi Zhng 1 3, To Zhou 1,2, Ting-ting Zhou 1,2, Yng Chen 1 & Hiln Hu 1 npg 21 Nture Ameri, In. All rights reserved. A fundmentl prolem in neurosiene is how emotionl vlenes re represented in the rin. We know little out how ppetitive nd versive systems intert nd the extent to whih informtion regrding these two opposite vlues segregte nd onverge. Here we used new method, tyrmide-mplified immunohistohemistry fluoresene in situ hyridiztion, to simultneously visulize the neurl orreltes of two stimuli of ontrsting emotionl vlene ross the limi forerin t single-ell resolution. We disovered hrteristi ptterns of intertion, segregted, onvergent nd intermingled, etween the ppetitive nd versive neurl ensemles in mie. In nuleus umens, we identified mosi tivtion pttern y positive nd negtive emotionl ues, nd unrveled previously unppreited funtionl heterogeneity in the D1- nd D2-type medium-spiny neurons, whih orrespond to the Go nd NoGo pthwys. These results provide insights into the oding of emotionl vlene in the rin nd t s proof of priniple of powerful methodology for simultneous funtionl mpping of two distint ehviors. Emotions olor our lives nd drive ehviors 1,2. Most emotions n e tegorized long two dimensions: vlene (vlue), rnging from negtive to positive, nd sliene (rousl, intensity), rnging from wek to strong 3. Although gret dvnes hve een mde towrd understnding the sensory representtions of the externl world 6, the mnner in whih different emotionl vlenes re represented in the rin hs remined lrgely elusive 7,8. The mjor hllenge is tht mny of the sme rin regions re tivted regrdless of the stimulus eing ppetitive or versive 9. Unfortuntely, funtionl mgneti resonne imging, positron emission tomogrphy or single-stimulus immedite erly gene (IEG) mpping nnot resolve whether the tivtion ours in the sme or different neurl popultions in mny ommonly tivted regions 9,1. Pioneering eletrophysiology studies hve identified interesting ptterns of neurl tivtion to ppetitive nd versive ues t vrious rin loi ; however, these studies smpled hundreds of neurons in one rin struture t time, nd the sptil reltionship of the two neurl ensemles in the entire struture nd whole rin remins unler. To extrt the vlene representtion of n emotion in the whole rin, we devised strtegy to ompre the tivity ptterns evoked y two slient emotionl stimuli with ontrsting vlene (morphine nd foot shok) in the sme mouse rin y improving douleleling tehnique sed on the distint indution time ourse of the mrna nd protein signls of the IEG gene -fos (Fos) 17. -fos expression is generlly low throughout the limi nervous system in resting nimls 18 ; however, following neuronl tivtion, -fos mrna umultes in minutes nd deys in ouple hours, wheres its protein produt ppers slower nd lsts muh longer 19,2. Thus, when two stimuli re sequentilly pplied t n pproprite intervl, neurl iruits tivted y the two stimuli n e represented y the -fos protein nd mrna signls, whih in turn n e reveled y dul leling using fluoresene immunohistohemistry (FIHC) nd fluoresene in situ hyridiztion (FISH) 21 (Fig. 1,). For this dulleling pproh to sueed, the -fos mrna nd protein signls must e temporlly seprle: y the time the mrna of the first stimulus deys, its protein signl should still e resonly high (Fig. 1). However, this riterion ws not stisfied in mny rin regions using regulr immunohistohemistry nd FISH (I-FISH, for exmple, in NA; Fig. 1). To overome this prolem, we used tyrmide-sed step to mplify the immunohistohemil signl, resulting in muh etter temporl seprtion of the mrna nd protein time ourses nd enling mpping in mny more rin regions (Fig. 1). We nmed this tehnique tyrmide-mplified IHC FISH (TAI-FISH). Using TAI-FISH dul leling, we exmined the -fos tivtion ptterns eliited y morphine nd foot shok in the sme mouse rin. We found tht morphine nd foot shok evoked lrgely distint neurl ensemles throughout the limi forerin, despite tivting mny of the sme regions. As morphine nd foot shok hve different sensory properties, we lso introdued dditionl pirs of emotionl stimuli, inluding hoolte nd restrint stress. In the nuleus umens (NA), rewrding nd versive stimulus pirs (morphine/foot shok, morphine/restrint) reruited sptilly intermingled neurons, wheres stimulus pirs of similr vlene (morphine/hoolte, foot shok/restrint) tivted lrgely overlpping neurl groups, suggesting the existene of funtionl vlene mp. This mp prtilly, ut not ompletely, oinides with the mosi distriution of the Go nd NoGo neurons defined y the D1 nd D2 mrkers Our results provide insights into the internl representtion of emotionl vlenes nd the funtionl orgniztion of NA. In ddition, with the new mplifition step llowing etter seprtion of IEG mrna nd protein signls, TAI-FISH provides muh-needed moleulr imging tehnique for mpping the neurl representtions of two distint ehviors ross the whole rin t single-ell resolution. 1 Institute of Neurosiene nd Stte Key Lortory of Neurosiene, Shnghi Institutes for Biologil Sienes, Chinese Ademy of Sienes, Shnghi, P.R. Chin. 2 Grdute Shool of Chinese Ademy of Sienes, University of Chinese Ademy of sienes, Shnghi, P.R. Chin. 3 These uthors ontriuted eqully to this work. Correspondene should e ddressed to H.H. (hiln@ion..n). Reeived 18 Ferury; epted 19 August; pulished online 21 Septemer 21; doi:1.138/nn VOLUME 17 NUMBER 11 NOVEMBER 21 nture NEUROSCIENCE

2 r t i l e s Bregm 1.6 mm AStr LA npg CEI BLA e Stimulus II Opt 1 min f Sl AStr CEI BLA 18 Co g 15 min Intervl Min min PKC- mrna/-fos protein 18 nture NEUROSCIENCE Sl Ctx Sl Ctx I-FISH ( tyrmide) TAI-FISH (+tyrmide) min 36 min 36 min 36 1 min -fos protein/mrna 2 d Foot shok indued ple voidne e Lteny to step through (s) Time in pired side (s) phine-indued ple preferene Red (FISH of the 2nd stim.) Green (IHC of the 1st stim.) 6 Sl (n = 13) Merged 2 CEI j 15 1 Intervl Time fter stimultion (h) C tx -S - l M Mo o M r-f r o S M r-m or or -C o C tx -S - l M Mo o M r-f r or S M -M or or -C o + i Stimulus I (n = 16) Ctx (n = 5) (n = 5) CEm CEm min Coleling of -fos nd PKC- 1 Perentge of + PKC- ells h -fos protein We first mpped the time ourses for the -fos mrna nd protein signls indued y morphine or foot shok lone. Mie were given either single morphine injetion or multiple eletril shoks, followed y, 2, 9, 18 or 36 min of rest in their home ge efore eing killed. In unstimulted mie, -fos signls were uniformly low throughout the limi forerin, exept in the prventriulr thlmus nd suprhismti nuleus, onsistent with previous d reserh18. Given the vrition in the tem 2 min 18 1 min porl profile of indued -fos signls ross different rin regions, we determined the optiml time intervls for eh region for oth stimuli to ensure tht, t the time of 2 min fos mrna/-fos protein 18 CEm Ctx 15 1 Numer of -fos ells -fos mrna Mx RESULTS Dul tivity mpping of morphine nd foot shok To study the neurl orreltes of emotionl vlene, we pplied TAI-FISH to visulize neurl responses to morphine nd foot shok, two emotionl stimuli of ontrsting vlene. When morphine nd foot shok were sequentilly pplied with n pproprite temporl intervl, their neurl orreltes ould e represented y the -fos protein nd mrna signls, respetively (Fig. 1 ). To eliit n ppetitive response, we injeted mie with morphine (15 mg per kg of ody weight). At this dosge, single intrperitonel (i.p.) injetion of morphine indued preferene for the injetion hmer in the onditioned ple preferene test (Fig. 1d), onfirming its ppetitive nture25. For versive stimulus, we pplied foot shoks, whih indued roust voidne ehvior in the pssive voidne test (Fig. 1e). -fos mrna/-fos protein 21 Nture Ameri, In. All rights reserved. Figure 1 Use of TAI-FISH dul tivity mpping to revel the neurl ensemles of ppetitive nd versive emotionl stimuli. () Shemti of the priniple of dul tivity mpping sed on differentil time ourses of -fos mrna nd protein expression. () Shemti of the experimentl proedures, showing one dul leling experiment, with morphine eing the first stimulus nd foot shok eing the seond, together with three prllel ontrol experiments. Ctx, ontext;, foot shok;, morphine; Sl, sline. () Importne of tyrmide mplifition of IHC signls. Note tht, in I-FISH, without tyrmide mplifition, the IHC signl of the first stimulus ws either mixed with the FISH signl t 2 min (left) or too wek y 36 min (middle left). With tyrmide mplifition, the IHC, ut not the FISH, signl ws oserved for the first stimulus t 36 min (middle right), enling the seprte representtion of the two stimuli (right). Imges were tken in the NA. Sle rs represent 3 µm. (d) Rewrding effet of morphine (single injetion, 15 mg per kg, i.p.), s indited y signifintly inresed time spent in the morphine-injeted hmer in the onditioned ple preferene ssy (MnnWhitney U test, P <.5). (e) Aversive effet of foot shok, s indited y prolonged lteny to step into shok hmer in onditioned ple voidne ssy (Mnn-Whitney U test, P <.1). Error rs represent s.e.m. 5 -fos+ -fos+ VOLUME 17 NUMBER 11 NOVEMBER 21 Figure 2 Segregted neurl representtions of morphine nd foot shok in CEA. () Shemti illustrting the lotion of CEA. Red ox indites the position of the imges shown in i. BLA, sl lterl mygdl; LA, lterl mygdl; opt, opti trt. ( f) Representtive imges showing doule leling for -fos protein (green) nd mrna (red) in CEA following sequentil stimultion of ontextsline (), foot shok morphine (), morphine foot shok (d), morphine-morphine (e) nd morphineoine (f). The green non-nuler signls were result of kground stining used y dense fiers in CEA, nd were esily distinguishle from the ellulr stining. (g) Sttistis of red (expressing -fos mrna), green (expressing -fos protein) nd yellow (expressing oth) neurons ounted from the setion shown in e nd f. (h,i) Doule stining for -fos protein (green) nd PKC-δ mrna (red) following single stimultion of morphine (h) nd foot shok (i). (j) Perentge of -fos+ neurons tht lso expressed PKC-δ, following single stimultion of morphine or foot shok. Sle rs represent 1 µm nd 3 µm (insets). Error rs represent s.e.m. 1553

3 r t i l e s npg 21 Nture Ameri, In. All rights reserved. Figure 3 Convergent neurl representtions of morphine nd foot shok in PVN. () Shemti illustrting the lotion of PVN. The red dshed ox indites the field of the imge tken with 1 ojetive. The red solid ox indites the position of the mgnified imges shown in. 3V, third ventrile; f, fornix; sox, supropti deusstion. () Only -fos protein signls (green) were present t 15 min post foot shok nd only -fos mrna signls (red) were present t 2 min post morphine nd 1 min post foot shok. () Representtive imges showing green (-fos protein), red (-fos mrna) nd merged hnnels of I-FISH doule leling from four experiments: foot shok sline, ontext-morphine, foot shok morphine nd foot shok foot shok. (d,e) Perentge of neurons expressing -fos protein (d) nd mrna (e) in PVN in the four experimentl onditions. G, numer of green neurons; R, numer of red neurons; N, numer of NeuN + neurons. (f) Perentge of overlp in the foot shok morphine nd foot shok foot shok doule-leling experiments, s lulted y Y/G (yellow r) or Y/R (ornge r). Y, numer of yellow neurons. (g) Sled Venn digrms showing the numer of green, red nd yellow neurons in PVN in the four experimentl onditions shown in. Sle rs represent 5 µm nd 2 µm (insets). Error rs represent s.e.m. deth, the -fos mrna indued y the first 5 stimulus hd deyed, wheres the -fos protein indued y the seond stimulus hd not yet emerged (Supplementry Figs. 1 3). For doule leling, we then used FISH (in red) nd FIHC (in green) to exmine the mrna nd protein signls in the sme rin slies. We surveyed the whole limi forerin, prtiulrly regions previously implited in the response of morphine or foot shok, inluding NA, lterl septum (LS), ed nuleus of the stri terminlis (BST), prventriulr nuleus of hypothlmus (PVN), entrl mygdl (CEA) nd medil mygdl (MEA). In CEA, MEA nd PVN, mrna nd protein signls were well-segregted, so we pplied stndrd I-FISH without tyrmide mplifition of the IHC signl. In NA, LS nd BST, TAI-FISH ws required nd employed to ssure good seprtion of the mrna nd protein signl time ourses. We redily oserved three sptil ptterns of intertion etween the neurl ensemles for morphine nd foot shok in the ove limi regions: segregted (where the two ensemles were sptilly seprted), onvergent (where the two stimuli tivted the sme neurons) nd intermingled (where they tivted different neurons in the sme region nd signls were sptilly mixed). Segregted pttern in CEA, MEA, AStr nd BSTov In CEA, the nuleus essentil for fer onditioning nd expression 26,27 (Fig. 2), exposure to the ontext of foot shok or sline injetion in the home ge indued few signls (Fig. 2). We then delivered foot shok nd morphine injetion 15 nd 2 min efore killing the mie, respetively, sed on the time ourse of -fos indution in this region (Supplementry Figs. 1 nd 3). Notly, most foot shok evoked green FIHC signls ppered in the medil division of CEA (CEm), wheres most morphine-tivted red FISH signls were loted in the lterl division (CEl), forming lerly segregted pttern (Fig. 2). Bregm.82 mm -fos protein/mrna -fos mrna -fos protein g -Sl Ctx PVN 3V Sox f Sl Ctx min min min min 33 -fos Protein/mRNA 15 1 min 2 min 1 min We then reversed the temporl order of the stimuli, injeting morphine first nd delivering foot shok seond (morphine foot shok onfigurtion). The segregted pttern remined the sme exept tht the olors were swithed (Fig. 2d), suggesting tht the temporl sequene of the two stimuli did not ffet the pttern of signl distriutions. To further verify the tehnique, we injeted the sme (morphine; Fig. 2e) or similr (oine; Fig. 2f ) drug s the seond stimulus fter morphine injetion. In oth the morphine-morphine nd morphineoine onfigurtions, red nd green signls lrgely overlpped in the CEl region, with 91 ± 3% overlp for morphine-morphine nd 77 ± 2% overlp for morphine-oine doule-stimultion (overlp rtio ws lulted s the numer of yellow neurons (Y) divided y the numer of red neurons (R); note tht R inludes neurons tht ppered either red or yellow; Online Methods nd Fig. 2e g). These oservtions suggest tht, in generl, the sme neurl popultions in the CEl were repetedly tivted y the sme or similr rewrding drugs. phine nd oine t vi different routes to inrese dopmine relese; morphine inds to opite reeptors nd inhiits the GABAergi neurons in the ventrl tegmentl re (VTA), wheres oine loks dopmine reuptke t the synpti sites 28. Although opite reeptors re expressed in CEA, the gret similrity tht we oserved in the tivtion pttern etween the two drugs suggests tht the neurl tivtion, t lest in the overlpping prt, ws most likely used y inresed dopmine relese, rther thn lol opite reeptor tivtion outside VTA. Elegnt geneti nd funtionl dissetion of the CEA miroiruit hs unrveled supopultion of neurons in the CEl region, CEl off, whih inhiits output neurons in the CEm nd is essentil for fer 26 d Perentge of NeuN + ells e Perentge of NeuN + ells 8 8 -fos protein (G/N) -Sl Ctx fos mrna (R/N) -Sl Ctx- - - f Y/G Y/R Perentge of overlpping ells Neurons responding to first stimulus Neurons responding to seond stimulus Neurons responding to oth 155 VOLUME 17 NUMBER 11 NOVEMBER 21 nture neuroscience

4 r t i l e s npg 21 Nture Ameri, In. All rights reserved. Figure Intermingled neurl representtions of morphine nd foot shok in NA DMS. () Shemti illustrting the lotion of NA. The red dshed ox indites the field of the imge tken with 1 ojetive for quntifition nlysis. The red solid ox indites the position of the mgnified imges of the NA DMS shown in. CPu, udte putmen;, nterior ommissurl, nterior prt. () Only -fos protein signls (green) were present t 36 min post morphine nd only -fos mrna signls (red) were present t 1 min post foot shok, 2 min post morphine nd 15 min post oine. () Representtive imges showing the green (-fos protein), red (-fos mrna) nd merged hnnels of TAI-FISH doule leling from four experiments: sline-ontext, morphine foot shok, morphine-morphine nd morphineoine. (d,e) Perentge of neurons expressing -fos protein (d) nd mrna (e) in the rostrl NA DMS in the six TAI-FISH experimentl onditions: sline-ontext, sline foot shok, morphine-ontext, morphine foot shok, morphine-morphine nd morphine-oine. Note tht the seond morphine injetion tivted fewer neurons in the morphine-morphine ondition thn the first morphine injetion, potentilly s result of desensitiztion. (f) Perentge of overlp in the morphine foot shok, morphine-morphine nd morphine-oine doule-leling experiments in the rostrl NA DMS. Mesured overlps were lulted s Y/R. Predited hne level of overlps ws onditioning 26,27. To investigte whether morphine-indued -fos + neurons in CEl re of the CEl off type, we exmined the ololiztion of -fos nd protein kinse C-δ (PKC-δ, Prkd), moleulr mrker of CEl off neurons 27. Notly, 95 ± 1% of morphine-indued -fos + neurons expressed PKC-δ, s reveled y doule leling (Fig. 2h j), suggesting tht morphine mostly tivted the OFF neurons in the CEl. Colletively, these results suggest tht morphine nd foot shok tivte distint su-nulei in CEA tht hve opposing roles in fer output. In ddition to CEA, severl other forerin regions lso responded to only one of the stimuli. For exmple, the mygdlostritl trnsition re (AStr) nd MEA responded strongly to foot shok, ut not to morphine (Fig. 2 d nd Supplementry Fig. ). Conversely, the ovl nuleus of BST, BSTov, responded to morphine, ut not to foot shok (Supplementry Fig. 5). Bregm 1.7 mm -fos protein/mrna -fos mrna-fos protein g Sl-Ctx CPu NA ore NA shell -fos protein/mrna - - -Co min 1 min 2 min 15 min Sl Ctx Co 36 1 min 36 1 min 36 2 min min Neurons responding to first stimulus Neurons responding to seond stimulus Neurons responding to oth numer of green neurons (G) divided y the numer of NeuN + neurons (N)) nd 63 ± 2% (R/N) of totl neurons, respetively (Fig. 3 e). For the ontrol, sline injetion in the home ge or exposure to the foot shok ontext indued little signl in the PVN (Fig. 3 e). Notly, 79 ± 3% of the green ells were lso red, inditing tht 79% of foot shok tivted neurons lso responded to morphine (Fig. 3,f). This overlp rtio ws lose to the 78 ± 5% overlp rtio in the foot shok foot shok onfigurtion, in whih two identil foot shok stimuli were pplied sequentilly (Fig. 3,f). These findings suggest tht nerly identil neurl popultions in the PVN were involved in the proessing of oth morphine nd foot shok, in vleneindependent mnner. In ddition to the PVN, neurons tivted y morphine nd foot shok lso displyed prtilly onvergent pttern in the fusiform nuleus of the BST (BSTfu; Supplementry Fig. 6). d Perentge of NeuN + ells e Perentge of NeuN + ells foverlpping (%) Co -fos protein (G/N) Sl-Ctx Sl- -Ctx - - -Co -fos mrna (R/N) Sl-Ctx -Ctx Sl Co Mesured (Y/R) Predited hne level (G/N) - - -Co lulted s G/N (Online Methods). (g) Sled Venn digrms showing the numer of green, red nd yellow neurons in rostrl NA DMS in the four experimentl onditions shown in. e thn neurons per mouse from three mie were ounted for eh group. Pired t test djusted y Benjmini- Hoherg proedure ontrolling the flse disovery rte. P <.5, P <.1, P <.1. Sle rs represent 5 µm. Error rs represent s.e.m. Convergent pttern in PVN The PVN is hypothlmi nuleus known to e tivted y vriety of stressful nd physiologil hnges, nd ontrols the hypothlmipituitry-drenl (HPA) xis nd gluoortioid relese 29 (Fig. 3). At 15 min post foot shok, the -fos mrna signl in the PVN ws diminished, ut the protein signl ws still roust; t 2 min post morphine injetion, the -fos mrna signl peked, ut the protein signl hd not ppered (Fig. 3 nd Supplementry Figs. 1 nd 3). Thus, we hose to dminister foot shok t 15 min nd morphine t 2 min prior to killing the nimls for the I-FISH nlysis in the PVN. In this foot shok morphine onfigurtion, foot shok nd morphine eh indued strong tivtion in the PVN, reruiting 58 ± 3% (the Intermingled pttern in NA nd LSv The NA is n essentil omponent of the rin s rewrd pthwy, loted in the ventrl stritum (Fig. ). On the sis of NA time ourse mpping (Fig. nd Supplementry Figs. 1 nd 3), we injeted morphine 36 min nd delivered foot shok 1 min efore killing the nimls for TAI-FISH nlysis in NA. The -fos signls indued y morphine nd foot shok were mostly loted in the medil shell region of the NA (Fig.,). In the dorsomedil shell of the NA (NA DMS ), morphine nd foot shok eh indued tivtion of similr numers, 8.1 ±.% (G/N) nd 8.6 ± 1.% (R/N), of totl neurons, respetively (Fig. e). Notly, the green nd red signls were distriuted in slt-nd-pepper nture NEUROSCIENCE VOLUME 17 NUMBER 11 NOVEMBER

5 Figure 5 Neurl representtions of morphine nd foot shok in the whole field of NA reveled y TAI-FISH. () Representtive imge of TAI-FISH dul-leling for morphine foot shok in the whole field of rostrl NA. DMS, dorsomedil shell; VMS, ventromedil shell; LS, lterl shell. White dshed line indites the oundry of the NA. This imge is one single mirosopi field tken with 1 ojetive, illustrting the potentil of TAI-FISH to mp lrge rin res. () Representtive imges of TAI-FISH leling for morphine foot shok in the NADMS t middle nd udl setions. Red oxes indite position of the mgnified imges of the NADMS shown on the right. Sle rs represent 5 µm. () Quntifition of neurons expressing the -fos protein (green), -fos mrna (red) or oth (yellow) in different prts of the rostrl NA (left), or from the rostrl to udl NADMS (right). Note tht green rs (G/N) lso represent the predited hne level of overlp. e thn 3 neurons per mouse from three mie were ounted for eh group. Pired t test djusted y Benjmini-Hoherg proedure ontrolling the flse disovery rte. P <.5; n.s., not signifint, P =.7. Error rs represent s.e.m. 36 Bregm 1.7 mm (Rostrl) 1 min 1 Bregm 1.18 mm DMS (middle) Bregm.86 mm DMS (udl) DMS Core LS VMS 2 µm 12 -fos mrna (R/N) -fos protein (G/N) Mesured overlp rtio (Y/R) Perentge of ells 8 n.s. 15 Perentge of ells 1 5 Drd1+, -fos+ Drd2+, -fos R M 1556 D2 62% D2 21% Perentge + of totl -fos ells -fos protein/ Drd1 mrna pttern with n overlp rtio of only 1.7 ± 1.% (Fig. ), whih ws signifintly lower thn LS Core Rostrl Middle Cudl DMS VMS predited hne level (8.1 ±.%, P =.17; Fig. f; predited hne level of overlp ws lulted s G/N, Online Methods). In ontrst, the overlps in the with one of these two sugroups. Co-stining of -fos FIHC with the morphine-morphine, nd morphine-oine onfigurtion rehed D1 or D2 FISH reveled tht the mjority of morphine-indued -fos+ 93 ± 2% nd 68 ± 5%, respetively (Fig. g). The intermingled neurons expressed D1 (8 ±.8% D1+ nd 21 ± 2.1% D2+; Fig. 6,), pttern remined the sme from the rostrl to the udl side of perentge similr to tht previously reported for oine-indued the NADMS (Fig. 5 ). In the ventromedil shell (NAVMS), more -fos+ neurons using Drd1-GFP nd Drd2-GFP mie31. Among the neurons responded to foot shok nd the overlp (6.6 ± 1.%) ws foot shok indued -fos+ neurons, D2+ neurons predominted, ut lose to hne level (.9 ±.5%, P =.7; Fig. 5 ). These results D1+ neurons lso ontriuted sustntil portion (62 ±.9% D2+ suggest tht, in the NADMS, two distint, intermingled popul- nd 39 ±.9% D1+; Fig. 6,). Similr rtios were present from rostrl tions of neurons enoded the response to morphine nd foot shok. to udl NADMS (Fig. 6). In ddition to the NA, neurons tivted y morphine nd foot shok were lso intermingled in the posterior ventrl prt of the LS (LSv), Representtions of multiple emotionl ues in NA n re tht proesses plesnt senstions nd stress responses3 Does the intermingled pttern of tivtion in the NADMS reflet the (Supplementry Fig. 7). representtion of emotionl vlues or other fetures (for exmple, spena neurons re divided into two morphologilly indistinguish- ifi sensory properties) ssoited with morphine or foot shok? To le, ut intermingled, supopultions, D1- nd D2-MSNs, whih define distinguish etween these possiilities, we further tested dul leling the Go nd NoGo pthwys for tion22 2. We sked whether the with n dditionl ppetitive stimulus, hoolte, nd n dditionl morphine-indued nd foot shok indued -fos+ neurons segregted versive stimulus, restrint, whih re experiened through mrkedly different sensory modlities, nd determined their optiml pplition time for dul leling (Fig. 7 nd Supplementry Fig. 3). First, phinefoot shok indued indued when piring morphine with the nturl rewrd, hoolte (morphine -fos -fos hoolte), mie were injeted with morphine nd then fed hoolte 36 min 9 min D1 D1 15 min efore eing killed. The hoolte-indued -fos mrna signls 39% 8% -fos protein/ Drd2 mrna npg 21 Nture Ameri, In. All rights reserved. r t i l e s C R M C Figure 6 Heterogeneity in D1- nd D2-MSNs in the NA in response to morphine nd foot shok. () Representtive imges showing oleling of Drd1 or Drd2 mrna with -fos protein in the NADMS, s indued y morphine or foot shok. Arrowheds indite ololized signls. Arrows indite single -fos protein signls. Sle rs represent 3 µm. () Perentge of morphine-indued -fos+ (left) nd foot shok indued -fos+ (right) neurons tht expressed D1 (red) or D2 (lue). Counting ws performed for the whole NADMS. () Coleling of -fos signls with D1 nd D2 mrkers long the rostrl-udl xis in the NA DMS. R, rostrl; M, middle; C, udl. Error rs represent s.e.m. VOLUME 17 NUMBER 11 NOVEMBER 21 nture NEUROSCIENCE

6 r t i l e s npg 21 Nture Ameri, In. All rights reserved. Figure 7 Representtions of multiple positive nd negtive emotionl ues in NA. () Only -fos mrna signls (red) were present t 15 min post hoolte (Choo) nd 15 min post restrint (Res), nd only -fos protein signls (green) were present t 2 min fter the strt of restrint in NA DMS. () Representtive imges showing green (-fos protein), red (-fos mrna) nd merged hnnels of TAI-FISH doule leling in the NA DMS following sequentil stimultion of morphine-hoolte, morphine-restrint nd restrint foot shok. White rrowheds indite merged signls. ( e) Perentge of neurons expressing -fos protein () nd mrna (d), nd perentge of overlp (e) in the NA DMS in the morphine-hoolte, morphine-restrint nd restrint foot shok TAI- FISH experiment. (f) Sled Venn digrms showing the numer of green, red nd yellow neurons in the whole NA DMS in the four experimentl onditions shown in. Counting ws performed for the whole NA DMS. e thn neurons per mouse from 3 mie were ounted for eh group. Pired t test djusted y Benjmini-Hoherg proedure ontrolling the flse disovery rte. P <.5, P <.1, P <.1. Sle rs represent 5 µm. Error rs represent s.e.m. were generlly weker nd fewer thn those indued y morphine (Fig. 7). However, there ws gret degree of overlp (61 ± 2.6%) etween the hoolte- nd morphine-tivted ell groups in NA DMS (Figs. 7 f nd 8). In ontrst, when piring morphine with nother versive stimulus, restrint (morphine-restrint), the overlp ws only.6 ±.8% (Figs. 7,e,f nd 8). Finlly, in the onfigurtion in whih two versive stimuli were sequentilly pplied (restrint foot shok), the overlp rtio rehed 6 ± 1.5% (Figs. 7 f nd 8). These results reveled tht emotionl stimuli of the sme vlene tivted lrgely overlpping neurl ensemles in the NA, wheres stimuli of different vlene tivted distint neurl popultions (Fig. 8). DISCUSSION We pplied TAI-FISH to simultneously visulize the neurl sustrtes of morphine nd foot shok in the sme mouse rin. We found tht the neurl representtion of these emotionl stimuli were stle nd stereotypi. In ll the rin strutures exmined, sequentil pplitions of the sme emotionl stimulus lwys tivted highly overlpping neurl groups (Figs. 2e,g, 3,f nd,f), suggesting tht the rin uses dedited neurl ensemles to enode emotionl stimuli. The sme priniple hs een demonstrted for the hippompl enoding of sptil informtion 32. We lso found tht the two emotionl stimuli evoked lrgely distint neurl ensemles throughout the limi forerin despite tivting mny of the sme regions (Fig. 8 nd Supplementry Fig. 8). This ntomil mp inludes distint ptterns of intertion, segregted (for exmple, in CEA), onvergent (for exmple, in the PVN) nd intermingled (for exmple, in the NA nd posterior LSv), etween the two neurl ensemles. Notly, in the NA DMS, neurons responding to two stimuli of different vlene (morphine versus foot shok) were lmost equl in numer (~8% of the neurons in the NA DMS ), ut were sptilly intermingled (Fig. ). In ontrst, there ws lrge overlp etween neurl ensemles representing the sme vlene, morphine versus hoolte or foot shok versus restrint, in the NA DMS (Figs. 7 nd 8). These oservtions reveled the existene of funtionl vlene mp in the NA. An extensive mount of work hs foused on the funtion of NA in ppetitive motivtion nd positive reinforement, s well s in versive motivtion 28, How does the NA differentilly enode rewrding nd versive stimuli? Considering tht D1-MSNs define the Go pthwy tht filittes tion, nd D2-MSNs define the NoGo pthwy tht suppresses tion 22 2, n ppeling model is tht the D1- nd D2-MSNs in the NA differentilly ode ppetitive nd versive informtion,1. We found tht D1- nd D2-MSNs ould f -fos protein/mrna Neurons responding to first stimulus Neurons responding to seond stimulus Neurons responding to oth -Choo -Res Res- 88 Choo Res Res 15 min 15 min 2 21 min Choo Res Res -fos protein/mrna -fos mrna -fos protein Perentge of NeuN + ells min min min -fos protein (G/N) -Choo -Res Res- d Perentge of NeuN + ells 6 2 -Choo -fos mrna (R/N) -Res Res respond to oth morphine nd foot shok stimuli, ut with different preferene: the numer of D1-MSNs responding to morphine ws roughly twie tht of those responding to foot shok (8% versus 39%, s the totl numer of morphine- nd foot shok indued were similr), wheres the numer of D2-MSNs responding to foot shok ws threefold greter thn those responding to morphine (62% versus 21%) in NA DMS (Fig. 6). This symmetril distriution would enle positive nd negtive emotionl stimuli to differentilly tivte the Go nd NoGo tion pthwys, respetively, nd diret opposite ehviorl outputs. However, we found tht D1-MSNs overll ontriuted to sustntil portion (out %) of -fos + neurons tivted y the versive foot shok stimulus. This frtion ws muh e Overlpping (%) Mesured (Y/R) Predited hne level (G/N) -Choo -Res Res- nture NEUROSCIENCE VOLUME 17 NUMBER 11 NOVEMBER

7 r t i l e s npg 21 Nture Ameri, In. All rights reserved. Figure 8 An emotionl vlene mp in the forerin. () Summry of ptterns of intertion etween neurl representtions of morphine nd foot shok in different regions of the limi forerin, s reveled y TAI-FISH (one dot represents n = 5 neurons ounted from representtive setions in eh orresponding region). () Sled Venn digrm summrizing the intertion of multiple positive nd negtive emotion representtions in the NA DMS. The numers of -fos + neurons ounted from the whole NA DMS (smpled from 3 µm 3 µm re of eh rostro-udl level, n = 3 mie) re leled in the irle. Stimuli of similr emotionl vlenes hd muh lrger overlp thn those of opposite vlenes. greter thn, nd ould not e ounted for y, the 1 17% of MSNs oexpressing D1 nd D2 reeptors 31,2. Thus, the mp identified here suggests dditionl omplexity in the funtionl orgniztion of the NA nd rises the interesting question on the funtion of the foot shok tivted D1-MSNs. Wht is the funtionl relevne underlying the intermingled pttern of vlene oding in the NA DMS? This mosi orgniztion is reminisent of the erly-stge funtionl mp of severl sensory representtions, inluding the distriution of on nd off ells in the retin 3, where lterl inhiition is n importnt feture of the neurl network rhiteture, serving to enhne ontrst. Indeed, the xon ollterls of MSNs in the NA n inhiit neighoring MSNs through GABAergi synpses, providing n ntomil sis for the lterl inhiition. It is therefore interesting to speulte tht the intermingled pttern of vlene representtion in the NA my llow neurons of opposite emotionl vlues to inhiit eh other nd ompete, direting tion outputs in more disrete fshion. Choie of emotionl stimuli We hose to use unonditioned stimuli (US, stimuli tht n nturlly trigger emotionl responses) insted of onditioned stimuli (CS, previously neutrl stimuli tht eome ple of evoking emotionl responses fter ssoition with US) in our proedure for severl resons. First, US generlly eliit stronger response thn CS. Seond, no trining is required for the protool. Third, giving CS without the tul US ould trigger rewrd-error signl 5. Finlly, US-indued signls re independent of previous lerning, therey eliminting the effets of dpttion nd memory. This my explin why -fos signls tivted y foot shok (in the CEm; Fig. 2) distriuted differently from those tivted y rell of fer memory ( type of CS; in the CEl 6 ). However, there re potentil ompounding effets ssoited with US tht should e utioned. For exmple, morphine, s drug, my eliit omplex effets in ddition to n ppetitive senstion. At very low dosge (.5 mg per kg), morphine n e versive 25,7. phine my lso hve lsting effet on emotion. To ddress these onerns, we first rried out onditioned ple preferene test to onfirm tht morphine ws ppetitive t the dosge injeted (15 mg per kg; Fig. 1d). Next, to onfirm tht morphine s potentil lingering effet did not lter the negtive vlene ssoited with foot shok, we tested reltime ple preferene for foot shok t 6 h fter morphine injetion. Foot shok still indued strong ple version in the morphineinjeted group, similr to the sline-injeted group (Supplementry Fig. 9), suggesting tht erlier morphine did not invert or redue the senstion of foot shok s negtive stimulus. Furthermore, foot shok indued -fos signls were indistinguishle if mie were treted with sline or with morphine erlier (Fig. d). Strengths nd limittions of TAI-FISH Behviorl neurosiene hs long lled for moleulr imging tehnique tht n ompre tivity ptterns of different physiologil sttes t the ellulr level ross the whole rin 7. Severl innovtive Segregted Convergent Intermingled phine-indued signl NA DMS BST ov BST fu LSv IEG-sed funtionl imging methods hve een developed to exmine the neurl orreltes of two distint stimuli: I-FISH 17, ellulr omprtment nlysis of temporl tivity y FISH (tfish, employing the distint time ourse of nuler versus ytoplsmi loliztion of IEG mrna 32,8 ), nd TetTg 9 nd TRAP mie 5, whih use geneti mouse models. CtFISH requires high-mgnifition (for exmple, 6 ) imging to differentite the suellulr loliztion. In ontrst, I-FISH n e performed with low mgnifition (for exmple, 1 ), mking it esier to mp lrge rin res or even the whole rin 21 (Fig. 5). However, the strit seprtion of the IEG protein nd mrna time ourses is not lwys stisfied in different rin regions or for different stimuli (for exmple, in the NA when morphine ws pplied s the first stimulus; Fig. 1), whih my hve impeded the generl pplition of I-FISH. TAI-FISH improves I-FISH y introduing tyrmidemplifition step for FIHC. This step extended the time ourse of the protein signl (Fig. 1), enling etter seprtion of the mrna nd protein time ourses nd gretly improving the pity of the method. Indeed, exmintion of hlf of the rin strutures in this study (NA, LSv nd BST) would not hve een possile without the FIHC tyrmide mplifition. eover, the time intervl etween the two stimuli for TAI-FISH (severl hours) is sustntilly longer thn tht for tfish (~3 min), whih ould e useful feture when the interferene etween the two ehviors or stimuli needs to e minimized. TAI-FISH does hve limittions tht re intrinsi to IEG imging pprohes. First, it is unle to detet neurons elow the detetion threshold or inhiited y stimulus, resulting in prtil mp. Seond, not ll neurons n express ll IEGs undntly. For exmple, unlike reports using the IEG Ar s proe, we deteted little -fos signls in the hippompus y morphine or foot shok. Third, tivity under ontrol onditions n e high in some rin res. For exmple, the medil prefrontl ortex nd nterior LS showed sustntil -fos signls y sline injetion nd ontext lone, nd were therefore not inluded in the nlysis. Finlly, the mrna signl of the first stimulus n e long-lsting (for exmple, in the sl lterl mygdl nd dorsl rphe, presumly s result of reurrent tivtion; Supplementry Fig. 2) or the protein signl of the first stimulus n dey too fst, preluding the seprtion of the mrna nd protein time ourses. Nevertheless, it is hopeful tht some of these prolems n e overome y using different IEG genes, suh s Ar, zif268 or Homer1, whih hve different indution time ourses nd expression profiles. In ddition, TAI-FISH n e used in onjuntion with PVN MEA Foot shok indued signl AStr CEI CEm phine Choolte Restrint Foot shok Stimulus pir of similr vlene Stimulus pir of distint vlene 1558 VOLUME 17 NUMBER 11 NOVEMBER 21 nture neuroscience

8 r t i l e s npg 21 Nture Ameri, In. All rights reserved. tfish to give more omplete mp, or even triple-epoh mp when needed. In the VTA region, there ws strong kground fluoresent stining of the xon fiers. It would e worth trying non fluoresenesed hemil stining methods (for exmple, NBT/BCIP for mrna nd DAB for protein) to re-exmine this re. In onlusion, TAI-FISH permits glol whole-rin dul-tivity mpping in wild-type mie with single-ell resolution. It provides powerful methodology, omplementing eletrophysiologil nd other funtionl imging methods, for identifying neurl networks responsile for the internl representtions of different ehviorl nd emotionl sttes. The vlene mp tht we identified my llow the exiting possiility of speifilly mnipulting the ppetitive nd versive ehviors. Methods Methods nd ny ssoited referenes re ville in the online version of the pper. Note: Any Supplementry Informtion nd Soure Dt files re ville in the online version of the pper. Aknowledgments We thnk J. Feldmn for ritil review of the mnusript, S. Srh, H. Kessels, A. Roe nd M. Poo for omments on the mnusript, nd S. Song, J. Hung, B. Lu nd memers of the Hu lortory for stimulting disussions. This work ws supported y the Chinese 973 Progrm (211CBA), the Strtegi Priority Reserh Progrm (B) of the Chinese Ademy of Sienes (XDB23), the One Hundred Tlents Progrm nd the Outstnding Youth Grnt (to H.H.). AUTHOR CONTRIBUTIONS J.X. designed the study nd performed the TAI-FISH experiments. Q.Z. nd To Zhou ontriuted to the TAI-FISH experiments. Ting-ting Zhou tested the time ourse for hoolte stimultion. Y.C. performed the sttistil nlysis. H.H. oneived the ide, designed the study nd wrote the mnusript with input from J.X., Q.Z. nd To Zhou. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. LeDoux, J.E. 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9 npg 21 Nture Ameri, In. All rights reserved. ONLINE METHODS Animls. All niml studies were pproved y the institutionl Animl Cre nd Use Committee of the Institute of Neurosiene, Chinese Ademy of Sienes. 3-month-old mle C57BL/6 mie were used for this study nd housed four per ge in room with 12-h light/drk yle (7:.m. to 7: p.m.), in stle onditions of temperture (22 C) nd humidity (6%), with food nd wter d liitum. Behviorl studies were onduted etween 1: nd 5: p.m. For morphologil studies, mie were divided into two per ge d efore the strt of the experiment to ensure they reeived the sme tretment, were killed within 1 s of the tretment termintion nd were perfused y two people simultneously within ~1 min (to redue ross-influene on gemtes). All mie were killed etween 3:3 nd 5:3 p.m. fter eh tretment. With these preutions tken, we found tht sl levels of -fos expression in unstimulted mie were uniformly low throughout the limi forerin, exept in the prventriulr thlmus nd suprhismti nuleus, despite some kground stining in vrious ortil regions (mostly in the piriform ortex, nd some wek signls in the visul nd motor orties). Drugs nd tretments. phine-hcl (15 mg per kg, i.p., Shenyng Phrmeutil) nd oine-hcl (2 mg per kg, i.p., Shenyng Phrmeutil) were dissolved in.9% NCl (wt/vol, sline). 6 g of rumled milk hoolte (Dove), similr in size to rie grins, were sttered in the home ge 15 min efore mie were srified. All mie egn eting the hoolte within 3 5 min. For versive tretments, 15 rndomly rrnged foot shoks (1 ma, 2 s) were dministered on mie within 1 min in fer onditioning hmer (Coulourn Instruments). Restrint stress ws performed in the home ge y pling mie in short 9-m-long trnsprent Plexigls tue (3.2-m inside dimeter), whih lsted for 3 (s first stimulus) or 15 min (s seond stimulus). Tissue preprtion. Mie were rpidly nesthetized with hlorl hydrte (1%, wt/vol, i.p.) nd sequentilly perfused trnsrdilly with ie-old.1 M phosphte-uffered sline (PBS, ph 7.) nd prformldehyde (% in PBS, wt/vol, Sigm). Brins were postfixed for 8 h in the sme fixtive solution nd stored t C. After ryoprotetion with 3% surose (wt/vol) in PBS, three serils of -µm-thik oronl setions were ut on ryostt (CM195, Lei) nd immeditely sujeted to histohemil stining. FISH nd FIHC. Brin slies were sequentilly sujeted to FISH nd FIHC stining. For FISH, ll solutions used for the hyridiztion were prepred using RNse-free regents nd diethylpyroronte (DEPC)-treted doule deionized wter (ddh 2 O). Throughout the hyridiztion proess, ll instruments were RNse-deontminted using RNse ZAP solution (Amion). Proes ginst -fos, PKC-δ, Drd1 nd Drd2 were onstruted ording to the desription on Allen Brin Atls wesite ( All proes were loned into pgem-t vetor (Promeg). The RNA proes leled y digoxygenin-utp (Rohe) were generted y in vitro trnsription nd dissolved t 1 µg ml 1 in the hyridiztion solution (5% formmide (vol/ vol), 5 SSC,.3 mg ml 1 yest trna, 1 µg ml 1 heprin, 1 Denhrdt s solution,.1% Tween 2 (vol/vol),.1% 3-[(3-holmidopropyl) dimethylmmonio]-1-propnesulfonte (CHAPS, wt/vol) nd 5 mm EDTA). For FISH, free-floting setions were wshed with.1 M PBS for 1 min nd treted with 2% H 2 O 2 (vol/vol) in.1 M PBS for 1 min, followed y nother round of wsh with PBS for 1 min t 25~28 C. They were then treted with.3% Triton X-1 (vol/vol) in.1 M PBS for 2 min nd etylted y.25% eti nhydride (vol/vol) in.1 M triethnolmine (ph 8.) for 1 min, followed y two rounds of wshing with PBS. Afterwrd, the setions were pled in the hyridiztion solution without proes for 1 h nd inuted in the hyridiztion solution with the orresponding proes for h t 6 C. After hyridiztion, the setions were rinsed one nd wshed twie in 2 SSC for 15 min t 6 C, followed y tretment with 2 µg ml 1 RNse A in 2 SSC t 37 C for 3 min. They were further rinsed one nd wshed twie in.2 SSC for 3 min t 6 C, followed y three rounds of wsh in.1 M PBS ontining.5% Tween 2 (PBT) for 1 min t 25~28 C. The setions were loked with 1% norml sheep serum (vol/vol) in PBT for 1 h t 25~28 C nd inuted in the sme solution with sheep ntiody to digoxygenin ntiody (1:5, Rohe ) t C overnight. On the seond dy, the setions were wshed three times in PBT for 1 min nd inuted in the mplifition solution with ynine 3 tyrmide (1:1, PerkinElmer NEL7B1KT) for 2 min t 25~28 C, followed y three rounds of wshing in.1 M PBS for 1 min. For FIHC, the setions were further inuted with rit ntiody to -fos (1:5, or 1:1,; Cliohem PC38) for 2 h t 25~28 C nd for 6 h t C. In the se of short tretment intervl ( 3 h, in CEA, PVH nd MEA regions), we visulized the protein signl diretly vi the Alex Fluor 88 fter inding of the got ntiody to rit (1:1,, Invitrogen A113) to the primry ntiody. For the long tretment intervl ( h, in NA, LSv nd BST regions), n dditionl tyrmide mplifition step ws introdued for ll the experimentl nd ontrol groups: the setions were further inuted with iotinylted rit ntiody to got ntiody (1:, Vetor Ls BA5) overnight t C, followed y reting with horserdish peroxidse onjugted streptvidin (1:8; Millipore ) for 1.5 h t 25~28 C. To intivte the peroxidse used in the FISH, the setions were treted with 2% sodium zide (wt/vol) in.1 M PBS for 15 min t 25~28 C immeditely fter the end of the primry ntiody inution. Finlly, the protein signl ws mplified nd visulized using the sme method s the FISH, ut inuted with oumrin tyrmide (1:15, PerkinElmer NEL731KT). After three rounds of wshing in.1 M PBS, the setions were mounted nd stored t C until further nlysis. For lultion of the perentge of -fos + ells, NeuN stining ws performed on one seril of setions of the sme niml. The setions were inuted with the mouse ntiody to NeuN (1:1,, Millipore MAB377) overnight t C nd further reted with Alex Fluor 59 got ntiody to mouse (1:1,, Invitrogen A3162). Mirosopy nd imge nlysis. Fluoresent imges were quired using n Olympus Fluoview FV1 onfol mirosope with 1 ojetive lens (NA =.) under the ontrol of the Olympus Fluoview FV1 version 2.1 softwre. The thikness of setions overed in eh imge nd used for quntifition ws 8.9 µm. The -fos protein signl showed nuler stining pttern, wheres its mrna ppered mostly in the ytoplsm. Imges shown in the figures were from the sme region illustrted in the shemtis ording to speifi neurontomil mrkers. Cell quntifition ws performed in 3 3 µm squre re for the NA DMS, NA VMS, NA Core nd NA LS regions in Figure 5, nd in outlined res for other rin regions. Signls were ounted from one slie per mouse t the leled Bregm position s outlined in the shemtis of eh orresponding figure. The ontrol nd experimentl groups were proessed in prllel. Imges were oded nd ounting ws performed in lind fshion. Brightness nd ontrst djustments were pplied to the whole imge. Positive protein signls should e solid round- or ovl-shped nd hve dimeter of 6~1 µm. Positive mrna signls were typilly prtile lusters 6~1 µm in dimeter. The kground signl ws determined y the men gry vlue of n unleled rin region within the mirosopi field. Intensity of mrna nd protein signls ws t lest 8-fold greter thn tht of the kground vlue. Clultion of overlp rtio. For lultion of the overlp rtio in the rin regions of onvergent nd intermingled ptterns, we ounted numers of red (R), green (G) nd yellow (Y) neurons from the doule-leled slies nd the numer of the NeuN-positive neurons (N) in seprte set of slies t the sme lotion in designted regions. Note tht R inluded oth red nd yellow neurons nd G inluded oth green nd yellow neurons. We use Y/R to represent the mesured overlp rtio. Sine the hne for given neuron to e oth red nd green (therefore ppering yellow) is R/N G/N, the predited Y = R/N G/N N = R G/N. Therefore the predited hne level of overlp rtio = predited Y/R = R G/N/R = G/N. Behviorl proedures. phine onditioned ple preferene ws performed in three-hmer devie (Med Assoites). Briefly, it onsisted of three phses. (i) Preonditioning. Mie were pled into the entrl omprtment nd llowed to freely explore the three omprtments for 15 min on dys 1 nd 2. Between the left nd right omprtments, the less preferred one on dy 2 ws piked for morphine piring on the onditioning dy. The time spent in this omprtment ws used s the seline. (ii) Conditioning. On dy 3, mie were injeted with morphine (15 mg per kg; i.p.) or sline nd pled in the orresponding omprtment for 3 min. (iii) Post-onditioning. On dy, mie were hndled in the sme wy s on dy 1 nd reeived no drug or sline dministrtions. Time spent in the omprtment where mie reeived the morphine injetion ws used nture NEUROSCIENCE doi:1.138/nn.3813