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1 doi:1.138/nture1194 Mterils nd methods: Experimentl subjects nd stereotxic surgery. Adult (25-3g) mle C57BL/6J mice (Jckson Lbortory, Br Hrbor, ME) were group-housed until surgery. Mice were mintined on 12:12 light cycle (lights on t 7:). Once the nimls were cclimted to the niml fcility for ~1 week, they were nesthetized with 15 mg/kg ketmine 5 mg/kg xylzine nd plced in stereotxic frme (Kopf Instruments). Microinjection needles were then inserted bilterlly directly bove the BLA (coordintes from Bregm: -1.6 AP, ±3.1 ML, -4.9 DV). Microinjections were performed using custom-mde injection needles (26 guge) connected to 2 µl Hmilton syringe. Ech BLA ws injected with.3.5 µl of purified nd concentrted AAV (~1 12 infectious units/ml) coding ChR2-EYFP, NpHR3.-EYFP, or EYFP under control of the CMKIIα promoter over 1 minutes followed by n dditionl 1 minutes to llow diffusion of virl prticles wy from the injection site. For opticl self-stimultion experiments mice were first injected unilterlly in the BLA with virus nd then guide cnnul ws implnted directly over the ipsilterl NAc (+1.3 AP, ±1. ML, -4. DV) to llow for insertion of the fiber optic cble during the experiment, which ws secured to the skull using Geristore ( dentl cement. Mice were then returned to their home cge. Body weight nd signs of illness were monitored until recovery from surgery (pprox. 2 weeks). All procedures were conducted in ccordnce with the Guide for the Cre nd Use of Lbortory Animls, s dopted by NIH, nd with pprovl of the UNC nd UCSF Institutionl Animl Cre nd Use Committees. Construct nd AAV preprtion. DNA plsmids coding paav-cmkiiα-chr2-eyfp (H134R), paav-cmkiiα-nphr3.-eyfp, or paav-cmkiiα-eyfp were obtined from the lbortory of Krl Deisseroth (see for dditionl detils). Plsmid DNA 1

2 RESEARCH ws mplified, purified, nd collected using stndrd plsmid mxiprep kit (Qigen). Following plsmid purifiction, restriction digest, nd sequencing to ssure DNA fidelity, purified recombinnt AAV vectors were serotyped with AAV5 cot proteins nd pckged using clcium phosphte precipittion methods by the UNC Vector Core fcilities (University of North Crolin, Chpel Hill). The finl virl concentrtion ws 1-2 x 1 12 genome copies/ml. Slice preprtion for ptch-clmp electrophysiology. Mice were nesthetized with pentobrbitl nd perfused trnscrdilly with modified CSF contining (in mm): 225 sucrose, 119 NCl, 2.5 KCl, 1. NH 2 PO 4, 4.9 MgCl 2,.1 CCl 2, 26.2 NHCO 3, 1.25 glucose. The brin ws removed rpidly from the skull nd plced in the sme solution used for perfusion t ~ C. Coronl sections of the NAc or BLA (2 µm) were then cut on vibrtome (VT-12, Leic Microsysytems). Slices were then plced in holding chmber nd llowed to recover for t lest 3 min before being plced in the recording chmber nd superfused with bicrbontebuffered solution sturted with 95% O2 nd 5% CO 2 nd contining (in mm): 119 NCl, 2.5 KCl, 1. NH 2 PO 4, 1.3 MgCl 2, 2.5 CCl 2, 26.2 NHCO 3, nd 11 glucose (t ~32 C). Ptch-clmp electrophysiology. Cells were visulized using infrred differentil interference contrst nd fluoresce microscopy. Whole-cell voltge-clmp or current clmp recordings of BLA nd NAc neurons were mde using n Axoptch 2A or B mplifier. Ptch electrodes ( MΩ) were bckfilled with internl solution contining 13 mm KOH, 15 mm methnesulfonic cid, 17 mm hydrochloric cid, 2 mm HEPES,.2 mm EGTA, 2.8 mm NCl, 2.5 mg/ml MgATP, nd.25 mg/ml GTP (ph 7.35, mosm). Series resistnce (15-25 MΩ) nd/or input resistnce were monitored online with 4 mv hyperpolrizing step (5 ms) given between stimultion sweeps. All dt ws filtered t 2 khz, digitized, nd collected using 2

3 RESEARCH pclmp1 softwre (Moleculr Devices). For current clmp experiments to chrcterize cell firing, 1 pulses t frequencies of 1, 5, 1, nd 2 Hz, respectively, were tested to determine spike fidelity (the percentge of light pulses tht led to ction potentils). For opticl stimultion of EPSCs, stimultion (pulses of 1-2 mw, 473 nm light delivery vi 2 µm opticl-fiber coupled to solid-stte lser) ws used to evoke presynptic glutmte relese from BLA projections to the NAc. NAc MSNs were voltge-clmped t -7 mv. For phrmcologicl chrcteriztion of glutmte currents, light-evoked EPSCs were recorded for 1 min followed by bth ppliction of 1 µm CNQ for n dditionl 1 min sweeps pre- nd post-drug were verged nd pek EPSCs mplitudes were then mesured. For EPSC pulse trin experiments, input-specific currents were evoked by 6 opticl pulses (2 Hz stimultion, 5 ms pulse durtion.). This ws repeted 12 times t.1 Hz. 4 µm SCH2339 or vehicle ws then bth pplied for 1 min nd the stimulus trin ws gin repeted. The verge EPSC trin from the 6 sweeps immeditely prior to drug ppliction nd those for the 6 sweeps immeditely fter drug ppliction were then compred. In vivo optrode recording. Approximtely, d fter bilterl AAV-CMKIIα-ChR2-EYFP injection into the BLA, mice were deeply nesthetized with ketmine/xylzine nd plced in stereotxic frme equipped with temperture controller to regulte body temperture. The skull ws then removed directly bove the NAc. Prylene coted tungsten electrodes (1 MΩ), epoxied to n opticl fiber 2 µm core dimeter,.37 N.A.) coupled to 473 nm lser were then lowered into the NAc to record unit ctivity of postsynptic MSNs following trins of light pulses used to evoke BLA-to-NAc-specific glutmte relese. Ten pulses of light (1-2 mw, 5 ms) t frequencies of 1, 5, 1, nd 2 Hz, respectively, were used to determine spike fidelity in vivo nlogous to wht ws with performed during whole-cell recording. Unit ctivity ws 3

4 RESEARCH mplified with n extrcellulr mplifier (A-M systems), bnd-pss filtered t 3Hz low/5 khz, nd digitized using pclmp1 softwre. Freely moving opticl self-stimultion d following injection of paav-cmkiiα- ChR2-EYFP or control virus into the BLA, mice with cnnul plced bove the NAc were prepred for nosepoke trining. Mice were mildly food restricted to four grms of food per dy to stbilize body weight nd fcilitte behviorl responding. Body weight ws monitored throughout the experiment nd did not fll below ~9% of their free feeding weight. Immeditely before plcing mice in the opernt chmbers, stylets were removed from the cnnul, nd flt cut 125 µm dimeter fiber optic cble, coupled to solid stte 473 nm lser outside of the opernt chmber, ws inserted through the guide cnnul nd plced directly bove the NAc. Immeditely prior to insertion, through the guide cnnul, light output through the opticl fibers ws djusted to 1-2 mw. The opticl fiber ws then secured into plce vi custom-mde locking mechnism to ensure no movement of the fiber occurred during the experiment. Mice were then plced in stndrd Med-Assocites opernt chmbers equipped with n ctive nd inctive nosepoke operndum directly below two cue lights. The chmbers were lso equipped with house lights, udio stimulus genertors, nd video cmers coupled to DVD recorders. A one hour opticl self-stimultion session begn with the onset of the cue light bove the ctive nosepoke operndum. Ech ctive nosepoke performed by the niml resulted in n opticl stimultion of BLA-to-NAc fibers (6 pulses, 2 Hz, 5 ms pulse durtion). Both ctive nd inctive nosepoke timestmp dt ws recorded using Med-PC softwre nd nlyzed using Neuroexplorer nd Microsoft Excel softwre. 4

5 RESEARCH NAc microinjections prior to opticl self-stimultion. Stylets were removed from guide cnnul nd 26-guge injector needle connected to 1 µl Hmilton syringe ws inserted. All microinjections were delivered in.3 µl sterile sline t rte of.1 µl/min. Injector needles remined in plce for n dditionl 2 min before being removed nd replced immeditely with wither stylets or opticl fibers for self-stimultion sessions. Doses of drugs used for microinjections were: 6 ng/.3 µl for SCH2339; 1 ng/.3 µl nd 3 µg/.3 µl for rclopride, nd 1 ug/.3 µl for lidocine. Implntble opticl fibers for NpHR inhibition during behvior. For these experiments, mice were bilterlly injected into the BLA with virus coding for NpHR3.-EYFP or EYFP s described bove. Mice were lso implnted with bilterl opticl fibers trgeted directly bove ech NAc. Opticl fibers were constructed in-house by interfcing 7 1 mm piece or 2 µm,.37 NA opticl fiber with 1.25 mm zirconi ferrule (fiber extending 5 mm beyond the end of the ferrule.) Fibers were epoxied into ferrules, nd cut nd polished. After construction, ll fibers were clibrted to determine percentge of light trnsmission t the fiber tip tht would interfce with the brin. Prior to bilterl implnttion, fibers were mtched to ech other so tht ech fiber output equl mounts of light (within 1%). This ws done to ensure tht n equl mount of light ws delivered to ech hemisphere. Following surgery, protective plstic cps were plced on the implnted opticl fibers to protect them from dust nd debris. Four to five weeks fter implnttion surgery nd three dys prior to the experiment, mice were connected to dummy opticl ptch cbles ech dy for 3 6 min to hbitute them to the tethering procedure in their home cge. On experiment dys, protective cps were removed from the implnted fibers. Fibers were then connected to custom-mde opticl ptch cbles (62.5 µm core dimeter) tht were covered with furction tubing to protect the cbles nd 5

6 RESEARCH prevent light from the lser from illuminting the opernt chmber. Bilterl fibers were connected to fiber splitter (5:5 split rtio) tht interfced with fiber-coupled 532 nm DPSS lser (2 mw). Bsed on ech pir of fibers clibrtion fctor, light intensity ws set to 1 mw illumintion t ech fiber tip in the brin. Opticl inhibition of BLA-to-NAc fibers during sucrose responding. Mice with opticl fibers implnted bove the NAc nd expressing either NpHR3.-EYFP or EYFP in BLA-to-NAc fibers were trined to drink sucrose in response to n environmentl stimuli tht predicted sucrose delivery. The strt of the session ws signled by the onset of white noise in the opernt chmber. Ech session consisted of 5 cue-rewrd pirings with rndom inter-tril intervl of 12 s. During ech tril, TTL ws sent from the behviorl hrdwre to engge the lser 2 ms prior to the onset of 5 s rewrd-predictive stimulus (tone/house light compound stimulus). Delivery of 2 µl of 2% sucrose to receptcle occurred immeditely fter the termintion of the rewrd-predictive cue, nd the lser pulse ws terminted 2 ms fter cue offset. Strting nd extending the lser pulse 2 ms before nd fter the cue were done bsed on in vitro experiments where we observed tht ctivtion of NpHR led to mximl inhibition 2 ms fter the strt of the lser pulse. Cue presenttion, rewrd delivery, lick, nd lser timestmps were stored s seprte dt rrys nd nlyzed offline with Microsoft Excel nd Neuroexplorer. Timelocked licking behvior ws quntified for ll mice. Mice tht did not mke t lest 2 licks on t lest one of the four conditioning sessions were excluded from nlysis. This resulted in the removl of n = 2 NpHR nd n = 2 EYFP mice from nlysis. Timelocked lick histogrms with.5s time bins were then constructed from -1 3 s timelocked to cue onset (t = ). Lick rtes were normlized to bseline periods using z-score procedure (z = (x - µ)/ σ) 6

7 RESEARCH where the µ ws the verge lick rte nd σ ws the stndrd devition in the 1 s preceding the cue onset. Opticl inhibition of BLA-to-NAc fibers nd conditioned plce preference/version. The sme mice used in the cue-evoke sucrose intke experiment were tested 1 2 weeks lter in modified conditioned plce procedure to ssy whether opticl inhibition of BLA-to-NAc fibers resulted in plce preference or version. On dy 1, ll NpHR nd EYFP mice underwent 25 min pre-test where mice were llowed to explore two contextully distinct comprtments to determine whether there ws n innte preference to one side of the chmber. Mice then underwent two 3 min conditioning sessions over the next two dys where one distinct environment ws pired with either no stimultion (but still tethered to opticl fibers) or the other environment where mice received 5 s lser pulses delivered to the NAc every 9 s. Stimultion nd no stimultion were counter-blnced cross mice. Following conditioning sessions, mice underwent finl preference test (25 min) where they gin hd ccess to both sides of the chmber. To exmine differences in preference or version in NpHR vs. EYFP mice, the mount of time spent on the side where mice received lser pulses ws normlized to the mount of time they spent on tht side during the pre-test session. Percentge of time spent on the stimultion-pired side ws then compred between NpHR nd EYFP groups. Dt nlysis. Sttisticl significnce ws ssessed using t-tests or nlysis of vrince (ANOVA) followed by post-hoc tests when pplicble using n α =.5. Dt ws nlyzed using Microsoft Excel with the Sttplus plugin nd Prism (GrphPd Softwre). 7

8 RESEARCH Virus expression nd histology. Following behviorl experiments, mice were deeply nesthetized with pentobrbitl nd perfused trnscrdilly with phosphte buffered sline (PBS) followed by 4% prformldehyde (PFA) dissolved in PBS. Brins were removed crefully nd post-fixed in 4% PFA for n dditionl hrs. Brins were trnsferred to 3% sucrose for hrs before slicing 5 µm sections of the BLA or NAc on freezing stge microtome or cryostt. Slices were then triple wshed in PBS for 5 min. Slices were then stined with 2% Neurotrce fluorescent Nissl stin (Invitrogen; excittion 53 nm, emission 615 nm) diluted in PBS with.1% Triton -1, for 1 hr. Slices were then wshed nd mounted on geltin-coted slides, treted with fluorescent-mounting medi, nd coverslipped. Expression of ChR2-EYFP, NpHR3.-EYFP, or EYFP ws then exmined for ll mice using either Nikon inverted fluorescent microscope with 4, 1, or 2 objective or Zeiss lser-scnning confocl microscope t 25 nd 63. Following injection of virus into the BLA, robust expression of ChR2-EYFP ws observed in BLA projection trgets including the NAc, mpfc, hippocmpus, insulr cortex, nd to lesser extent, the dorsl medil stritum. Mice showing no EYFP expression in the NAc due to fulty microinjections or mice showing cnnul or fiber plcements outside of the NAc were excluded from nlysis. Reconstruction of opticl stimultion or inhibition sites in the NAc. To determine opticl stimultion sites in experiments where guide cnnuls were used to introduce opticl fibers into brin tissue (BLA-to-NAc nd mpfc-to-nac opticl self-stimultion experiments, see Supplementry Figs. 3,14 for loction of opticl stimultion sites), fixed nd stined coronl brin sections (see bove) contining the NAc nd cnnul trcks were exmined on n upright conventionl fluorescent microscope. Cnnul trcks were locted in the slices nd opticl stimultion sites were determined by locting the site 1 mm ventrl to the end of the cnnul tip. A 1 mm distnce ws used in these experiments becuse the opticl fibers extended.5 mm 8

9 RESEARCH beyond the end of the cnnul (ech fiber ws cut to this length prior to insertion). Bsed on light output from these opticl fibers (477 mw*mm -1 t the tip nd clculting intensity tking into ccount geometric loss nd scttering through tissue 1 loss t.5 mm beyond the fiber tip led to n estimted 2.6% trnsmission or 12.4 mw*mm -1 t this distnce. At 1 mm from the tip of the opticl fiber, estimted trnsmission dropped to.56% or 2.67 mw*mm -1, which pproximtes the minimum intensity required to ctivte opsin proteins (1 mw*mm -1 ). For NpHR-medited inhibition experiments, opticl inhibition sites (Supplementry Fig. 11) were determined in similr fshion; with.5 mm used s the distnce from fiber tip to the digrmmed inhibition sites, s no guide cnnul ws present..5 mm beyond the opticl fiber tip represents the center loction where opticl stimultion or inhibition occurs, (.5 mm bove nd below). All clcultions were performed using equtions nd constnts listed in Arvnis et l., References: 21 Arvnis, A.M., et l. An opticl neurl interfce: in vivo control of rodent motor cortex with integrted fiberoptic nd optogenetic technology. J Neurl Eng 4, S (27). 9

10 RESEARCH b Pek Stedy-stte 2 ms 5 pa Current (pa) Suppplementry Figure 1: ChR2 medited currents recorded from BLA neurons., Voltge-clmped BLA neurons t -7 mv respond to 5-ms pulse of 473 nm. b, verge dt for ll recorded cells (pek current: -115 ± 255 pa; stedy-stte current: ± 181 pa; P =.16; n = 1 cells). light-evoked extrcellulr ction potentils vi BLA-to-NAc fiber stimultion 5 ms 25 μv 1 ms 25 μv Supplementry Figure 2: Opticl stimultion of BLA-to-NAc fibers results in postsynptic ction potentils in vivo., In vivo optrode recordings of ction potentils from NAc neurons following opticl stimultion of BLA-to-NAc fibers. Lower trce shows expnded extrcellulr wveforms from single neuron. 1

11 RESEARCH BLA-to-NAc opticl self-stimultion b. NAc BLA b Supplementry Figure 3: Opticl stimultion sites for BLA-to-NAc mice., An opticl fiber is inserted through guide cnnul into the NAc to ctivte BLA-to-NAc ChR2- expressing fibers. b, Digrm of opticl stimultion sites locted 1mm from the cnnul tip loctions for ChR2-EYFP nd EYFP mice used in BLA-to-NAc stimultion experib. BLA-to-NAc opticl stimultion sites +.98 mm +1.1 mm mpfc mm mm 11

12 RESEARCH Active nosepokes ChR2-EYFP EYFP Time (min) Supplementry Figure 4: Averge cumultive nosepokes of BLA-to-NAc mice., Averge cumultive records for ctive nosepokes of mice expressing ChR2-EYFP or EYFP in BLA-to-NAc fibers. 12

13 RESEARCH b Active nosepokes/6 min 2 15 mpfc 1 5 BLA cell body stim BLA-to-NAc terminl stim Active nosepokes/6 min1 1 2 Opticl ICSS session Supplementry Figure 5: Direct optogenetic ctivity of BLA cell bodies leds to vrible self-stimultion behvior., Mice injected with AAV-CMKII-ChR2-EYFP into the BLA nd implnted with guide cnnul directly bove the BLA were tested for opticl self-stimultion behvior. While ll mice showed robust ChR2-EYFP expression, only 4/9 mice tested showed self-stimultion behvior. This is shown in comprison to cohort of BLA-to-NAc stimultion mice. b, Averge ctive nosepokes mde by mice receiving BLA cell body stimultion cross two consecutive behviorl sessions. 13

14 RESEARCH b Active nosepokes Lser on Lser off Time (min) Nosepokes/ 5 min lser on lser off * * * * * * * * * * Time (min) ** Supplementry Figure 6: Behviorl responding during extinction nd renwel of BLA-to-NAc opticl self-stimultion. Exmple cumultive nosepokes from single mouse diminish in the bsence of lser stimultion. b, Averge ctive nosepokes re reduced in the bsence of lser stimultion (n = 11 mice; P <.1). 4 ns Active nosepokes ** ns sline SCH2339 (6 ng) Rclopride (1 ng) Rclopride (3 μg) Microinjection tretment Supplementry Figure 7: Intr-NAc D1R receptor ntgonism blocks BLA-to-NAc self-stimultion., Averge number of ctive nosepokes mde for BLA-to-NAc stimultion in mice treted with SCH2339 or rclopride. Both moderte nd high doses of rclopride were ineffective t blocking BLA-to-NAc self-stimultion behvior, nd therefore Rclopride microinjection dt t both doses were pooled. 14

15 RESEARCH b c Port entries Nosepokes/min 6 Vehicle SCH Time (min) Vehicle SCH Time (min) d Inter-nosepoke intervl Intrburst nosepoke frequency Vehicle Vehicle SCH2339 SCH2339 Supplementry Figure 8: Unilterl intr-nac D1R ntgonism does not ffect indices of generl locomotor ctivity., Rtes of ctive nosepokes mde for BLA-to- NAc stimultion erly in the behviorl session following intr-nac vehicle or SCH2339. Rtes of ctive nosepokes were comprble for both groups in the first 8 min of the behviorl sessions (~ 12 min fter microinjections). b, Burst nlysis of ctive nosepokes mde for BLA-to-NAc stimultion following intr-nac vehicle or SCH2339. All nosepokes with intervls between nosepokes of < 6 s were clssified s belonging to burst. Intr-NAc SCH239 did not differ significntly in their intr-burst frequency of responding reltive to vehicle-treted mice, suggesting tht unilterl SCH2339 does not led to motor-relted deficits in performnce. c, Averge cumultive records for port entries, mesure of locomotor ctivity in mice tht received intr-nac injections of vehicle or SCH2339. d, Averge port entries were not significntly different between mice tht received intr-nac vehicle or SCH2339. Port entries 15

16 RESEARCH Pre SCH pa 5 ms Post SCH pa b EPSC mplitude (% predrug) Vehicle SCH Pulse number 5 ms Supplementry Figure 9: Opticlly stimulted BLA-to-NAc EPSCs re reduced by D1R ntgonism ex vivo., Trins of BLA-to-NAc EPSCs before nd fter bth ppliction of 4 μm SCH2339 evoked by opticl stimuli used in vivo. b, Averge EPSC mplitudes for ll EPSCs evoked by stimulus trin s function of bseline non-drugtreted vlues (F59,66 = 71.9; P <.1, n = 7-8 cells per group). b c BLA-to-NAc opticl stimultion NAc BLA intr-bla lidocine Active nosepokes BLA sline BLA lidocine Nosepokes BLA sline BLA lidocine Time (min) Active Inctive Supplementry Figure 1: Inctivtion of BLA does not lter the cquisition of BLA-to-NAc opticl self-stimultion., Ipsilterl BLA ws inctivted by lidocine injections prior to BLA-to-NAc opticl self-stimultion sessions. b, Averge cumultive ctive nosepoke records for ll mice tht received intr-bla sline or lidocine immeditely prior to their first opticl self-stimultion session. c, Averge ctive nd inctive nosepokes for mice tht received intr-bla sline or lidocine prior to their first selfstimultion session. Intr-BLA lidocine did not lter the cquisition of opticl selfstimultion (n = 8 per group; P =.87). 16

17 RESEARCH b c d 2 μm 4 μm BLA:NpHR3.-EYFP e BLA:NpHR3.-EYFP 4 μm 4 μm NAc:NpHR3.-EYFP NAc:NpHR3.-EYFP BLA-to-NAc opticl inhibition sites for NpHR3. experiments EYFP +.98 mm +1.1 mm NpHR mm mm Supplementry Figure 11: NpHR3.-EYFP expression in the BLA nd NAc nd opticl inhibition sites for BLA-to-NAc expressing NpHR mice., Representtive imge showing expression of NpHR3.-EYFP in BLA neurons (green). b, Higher mgnifiction of the BLA showing NpHR3.-EYFP expressing neurons (green) c, Expression of NpHR3.-EYFP positive fibers from the BLA innervting the NAc. d, Higher mgnifiction of the NAc showing NpHR3.-EYFP fibers (green) intermixed with postsynptic neurons (red) e, Digrm depicting opticl inhibition sites locted.5 mm from the fiber tip loctions for NpHR-EYFP nd EYFP used in BLA-to-NAc inhibition experiments. 17

18 RESEARCH Opticl inhibition pired with nosepoke response: 5 s 9-12 s inter-tril intervl cue presenttion lser on 5.4 s sucrose delivery Supplementry Figure 12: Timing digrm of illustrting lser pulse reltive to rewrd- predictive cues nd sucrose delivery., Following vrible inter-tril intervl, onset of 532 nm light delivered to the NAc occurred 2 ms prior to onset of the rewrd-predictive tone/houselight cue (on t t =, lsting 5 s). Immeditely following the offset of the cue, 2 µl of 2% sucrose ws delivered to receptcle. 2 ms fter cue offset, the lser ws turned off. 3 * Totl licks 2 1 NpHR lser on NpHR lser off EYFP Supplementry Figure 13: Averge totl licks during NpHR inhibition nd noninhibition. Totl number of licks were reduced in sessions where NpHR-expressing mice received opticl inhibition compred to sessions where the BLA-to-NAc pthwys ws unperturbed (P =.44, n = 7 mice per group). 18

19 RESEARCH mpfc-to-nac opticl stimultion sites +.98 mm +1.1 mm mm mm Supplementry Figure 14: Opticl stimultion sites for mpfc-to-nac mice., Digrm illustrting opticl stimultion sites locted 1mm from the cnnul tip loctions for ChR2-EYFP nd EYFP mice used in mpfc-to-nac stimultion experiments. 19

20 RESEARCH b ChR2 medited firing in mpfc 5 pa 2 mv 1 ms 2 ms Supplementry Figure 15: mpfc neurons trnsduced with AAV-CMKII-ChR2- EYFP re opticlly excitble., mpfc neurons voltge clmped t -7 mv showed prominent trnsient nd stedy-stte inwrd currents in response to 5 ms, 2 mw, 473 nm light ppliction. b, Opticlly-induced firing of mpfc neurons in current-clmp mode t 2 Hz. 2

21 RESEARCH b cnnul trck c c mpfc-to-nac EPSC Bseline CNQ EPSC mplitude (pa) pa -8 2 ms -1 Supplementry Figure 16: Schemtic of neurons ptched in the NAc in close proximity to guide cnnul nd mpfc-to-nac EPSCs., Imge of brin slice with cnnul trck expressing ChR2-EYFP t mpfc-to-nac fibers. Ptch-clmp experiments in MSNs while stimulting mpfc-to-nac synpses were conducted < 2 μm ventrl to the end of the guide cnnul. c: nterior commissure. b, Imge showing voltge-clmped MSN filled with Alex-red biocytin in close proximity to the end of the guide cnnul lso surrounded by ChR2-EYFP positive fibers nd terminls originting from the mpfc. c, Averge EPSC mplitudes evoked following stimultion of mpfc-to-nac pthwy. EPSCs were significntly reduced by bth ppliction of 1 μm (F(1,56) = 9.63; P =.3, n = 7 cells per group). ** 21

22 RESEARCH b c 5 pa Electriclly-evoked mpfc-to-nac EPSC 1 ms 1 pa Opticlly-evoked BLA-to-NAc EPSC 1 ms EPSC mpltude (pa) * AMPA-medited EPSC mpfc-to-nac electricl stim. BLA-to-NAc opticl stim. Supplementry Figure 17: Individul NAc MSNs receive both corticl nd BLA excittory inputs, EPSC evoked by electricl stimultion of corticl fferents to the NAc. The electricl stimulting electrode ws plced dorsl to the corpus cllosum bove the NAc. b, BLA-to-NAc opticlly-evoked EPSC from the sme neuron. c, Averge EPSC mplitudes in cells tht responded to both opticl stimultion of BLA-to-NAc fibers nd electricl stimultion of corticl fferents (n = 1). Similir to opticlly-evoked dt presented in Fig. 4, BLA-to-NAc evoked EPSCs were significntly lrger thn those evoked by stimultion of corticl fferents to the NAc (mpfc-to-nac electricl stim: 23.1 ± 58.8 pa; BLA-to-NAc opticl stim: ± pa; t(9) = 2.7; P =.3). 22