SUPPLEMENTARY INFORMATION. Observational fear learning involves affective pain system and Ca v. 1.2 Ca 2+ channels in ACC

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1 SUPPLEMENTARY INFORMATION Oservational fear learning involves affetive pain system an Ca v 1.2 Ca 2+ hannels in ACC Daejong Jeon, Sangwoo Kim, Mattu Chetana, Daewoong Jo, H. Earl Ruley, Shih-Yao Lin, Dania Raah, Jean-Pierre Kinet & Hee-Sup Shin Supplementary Disussion To optimize the onitioning we hose the 1-s interval for 2-s foot shok (1 ma) for 4-min training perio from our pilot experiments with ifferent protools. When emonstrators reeive.7-ma foot shoks whih are use in a typial fear onitioning, they showe heterogeneous ehavioral responses: running, voalization an/or jumping. However, foot-shok intensity with 1 ma always mae them isplay a homogeneous response inluing all three ehaviors, runnig, voalization an jumping. Thus oservers an always get similar inputs from emonstrators uring the training. When the protools with the 2-s interval for 2-s foot shok (1 ma) for 4-min training perio (Supplementary Fig. 1a,) an the 1-s interval for 2-s foot shok (1 ma) for 2-min training perio (Supplementary Fig. 1,) were use, oservers isplaye low freezing levels uring the training or in the 24-hr ontextual memory test. However, high an stale freezing levels in the oservers were inue y the protool with the 1-s interval for 2-s foot shok (1 ma) for 4- min training perio. With this protool we performe some ontrol experiments an otaine the ehavior profile of oserver mie: 1) when there were no emonstrator mie, with or without foot shoks (Supplementary Fig. 1e), 2) simply in the presene of emonstrator mie for 9 min without foot shoks (Supplementary Fig. 1f,g), an 3) in a novel ontext (a ifferent hamer from the one for training) 24 h after training (Supplementary Fig. 1h). The novel hamer was a lak-olore plasti yliner (iameter 2 m an height 3 m) an a small amount of mouse eing was plae on the floor of the hamer. In experiments 1) an 2) the oservers i not show freezing ehavior, iniating that freezing response of the oservers totally ame from oservation of freezing ehaviors of the emonstrators. In experiment 3), the oservers also i not show freezing ehavior, iniating that the freezing response of the oservers ame from a speifi ontextual fear memory, rather than just an inrease in generalize fear. We also analyze the freezing response of ifferent emonstrators (silings or ouples vs. non-silings or non-ouples). There was no ifferene in the freezing levels among the emonstrators (Supplementary Fig. 9,). Therefore, we oul not examine the possiility of a orrelation etween the intensity of the freezing response of oservers an that of emonstrators, eause the extensive training sheule might have inue maximum freezing response in the emonstrators. To hek a possile non-speifi effet of p-cre on the animals, we miroinjete p-cre into the ACC of wil-type mie (C57BL/6J), an then performe several ehavioral experiments. The mie treate with p-cre showe similar levels of ehavioral responses to those of non-treate mie in oservational fear onitioning (Supplementary Fig. 11a,), elevate plus-maze test (Supplementary Fig. 11) an the lightark transition task (Supplementary Fig. 11), iniating a lak of non-speifi effets of p-cre proteins on those ehaviors. Nature Neurosiene: oi:1.138/nn.254

2 e g Only oserver Only oserver with foot-shok Couple (>1 weeks) Non-ouple f h Silings Non-silings Other ontext Supplementary Figure 1. Pilot experiments with ifferent protools an ontrol ehavioral profiles in oservational fear onitioning. (a,) When the protools with the 2-s interval for 2-s foot shok (1 ma) for 4-min training perio were use, oservers showe low freezing levels uring the training an in the 24- hr ontextual memory test. (,) When the protools with the 1-s interval for 2-s foot shok (1 ma) for 2- min training perio were use, oservers isplaye low freezing levels in the 24-h ontextual memory test. (e) Freezing ehavior of oserver mie, when there are no emonstrator mie, with or without foot shoks. (f,g) Freezing ehavior of oserver mie, simply in the presene of emonstrator mie for 9 min without foot shoks. (h) Context speifiity. Freezing ehavior in a ifferent hamer from the training hamer (a novel ontext, not the training ontext), 24 h after training. Nature Neurosiene: oi:1.138/nn.254

3 Fees # Silings Non-silings * Fees # Couple Non-ouple * Fees # Cav1.2Floxe/Floxe v Cav1.2ACC/PBS v * * Supplementary Figure 2. The numer of feal roppings (fees #) uring oservational fear learning. (a,) When silings or female mating partners were use as emonstrators, oservers she more feal roppings uring training than was the ase when unrelate mie were emonstrators. (a) The numer of roppings from sujets in silings (n = 2) an non-silings (n = 17) experiments (F 1,35 = 9.32, P =.43, one-way ANOVA). *P <.1. () The numer of roppings from sujets from in ouples (n = 15) an non-ouples (n = 14) experiments (F 1,27 = 4.76, P =.38, one-way ANOVA). *P <.5. Similar numer of roppings was otaine in tests with mating partners o-house for 1 to 15 weeks or 2 to 36 weeks, an therefore the results were poole for analysis. () Ca v 1.2 ACC/Cre mie (n = 22) she fewer feal roppings uring training than the other groups (Ca v 1.2 ACC/PBS, n = 22; Ca v 1.2 Floxe/Floxe, n = 13) *P <.1, one-way ANOVA. Nature Neurosiene: oi:1.138/nn.254

4 ACC-saline ACC-lioaine ACC-saline ACC-lioaine ACC-saline ACC-lioaine Supplementary Figure 3. Classial fear onitioning. (a) Aministration of lioaine into the ACC efore training ha no influene on the aquisition of fear (lioaine, n = 1; saline, n = 8) (F 1, 16 = 3.21, P =.92 two-way repeate ANOVA). The horizontal line iniates the uration of a tone (28 s) an the vertial arrow iniates the time of foot shoks (2 s). () There was no ifferene etween groups in 24-h ontextual memory (F 1, 16 =.16, P =.69, two-way repeate ANOVA). () No ifferene was also foun etween groups in 24-h ue memory. The horizontal line iniates the uration of a tone (3 min). Nature Neurosiene: oi:1.138/nn.254

5 14 15 Neo Frt site LoxP site Floxe Targete Null e Wil type 4 p 3 p Floxe Wil type Nature Neurosiene: oi:1.138/nn.254

6 Supplementary Figure 4. Generation of onitional Ca v 1.2 mie an ACC-speifi gene eletion of the Ca v 1.2 y p-cre. (a) Targeting strategy of Ca v 1.2 lous. Blak ox, the targete exon (exon14 an 15); Neo, the NEO assette; Frt, the Flipase Reognition Target; LoxP, the lous of X-over P1. () Southern lot (upper) an PCR analysis (lower) analysis for the targeting an genotyping. The 8.4 K segment orrespons to the wil-type allele; the 14.5 K, the targete allele; the 12.6 K, the floxe allele (Neoelete); the 9.7 K, the null allele (Neo, Exon 14, 15 elete). The floxe allele versus wil-type allele was etete y PCR with the following pair of primers (with annealing temperature at 6C): The 5 primer was 5'-CCT CCC TGT GAG CTG TTC-3' an 3 primer was 5'-CCT TTG ATG TGC CAG AGG-3'. The 3 p an, the PCR prout of the wil-type allele; the 35 p an, the prout of the floxe allele (Neoelete). () Gel staine with oomassie lue shows purifie p-cre. The arrow iniates p-cre. () Immunohistologial signals (green) for the GFP from the oronal rain setion inluing ACC of ROSA- GFP reporter mouse after ilateral injetion of the p-cre, whih iniates that the p-cre is iologially ative. (e) Immunohistologial laeling for the Ca v 1.2 in the ACC of Ca v 1.2 Cre/LoxP onitional mouse. Animals were anesthetize an perfuse through the heart with 5 ml of ol saline an 5 ml of 4% paraformalehye in.1 M phosphate uffer. Brains were then remove an were post-fixe overnight. Coronal setions (3 μm) ontaining ACC were staine with anti-ca v 1.2 (1:25, Alomone Las, ACC-3) as a primary antioy. A iotinylate seonary antioy an the aviin/iotin system were use for antioy followe y a DAB reation. Strong immunoreativity for Ca v 1.2 was seen in the non-injete ACC area (mile, 2 or 4 magnifie from left re retangle in the upper), ut the immunoreativity for Ca v 1.2 was asent in the large part of the ACC that was injete with p-cre (own, 2 or 4 magnifie from right lue retangle in the upper). Yellow arrow iniates the ACC area injete with p-cre (upper, 4 ). Nature Neurosiene: oi:1.138/nn.254

7 Response sore Cav1.2Floxe/Floxe v Cav1.2ACC/PBS v Cav1.2Floxe/Floxe v Cav1.2ACC/PBS v Lateny (se) Cav1.2Floxe/Floxe v Cav1.2ACC/PBS v Cav1.2Floxe/Floxe v Cav1.2ACC/PBS v Fore (g) 2 5 IR Intensity Supplementary Figure 5. Oservational fear learning (with an opaque partition) an pain ehavioral responses in the Ca v 1.2 ACC/Cre mie. (a,) Oservational fear learning of the Ca v 1.2 ACC/Cre (n = 7), Ca v 1.2 ACC/PBS (n = 6) an Ca v 1.2 Floxe/Floxe (n = 7) mie with an opaque Plexiglas partition. (a) Freezing ehavior on the ay of training an () 24 h after training. (,) Normal responses in aute pain ehaviors of the Ca v 1.2 ACC/Cre mie. () Paw withrawal responses to the mehanial stimuli with von Frey filaments in the Ca v 1.2 ACC/Cre (n = 14), Ca v 1.2 ACC/PBS (n = 13) an Ca v 1.2 Floxe/Floxe (n = 9) mie. () Tail flik lateny to raiant heat stimuli in the Cav1.2 ACC/Cre (n = 1), Ca v 1.2 ACC/PBS (n = 9) an Ca v 1.2 Floxe/Floxe (n = 5) mie. Nature Neurosiene: oi:1.138/nn.254

8 Time (se) Time (se) Open Enlose Center First time to light Ca v 1.2 Floxe/Floxe Ca v 1.2 ACC/PBS Ca v 1.2 ACC/Cre Ca v 1.2 Floxe/Floxe Ca v 1.2 ACC/PBS Ca v 1.2 ACC/Cre Crossing # 2 1 e Distane (m) Light Dark Total numer of entries Time in enter (se) Cav1.2Floxe/Floxe v Cav1.2ACC/PBS v Ca v 1.2 Floxe/Floxe Ca v 1.2 ACC/PBS Ca v 1.2 ACC/Cre Open Ca v 1.2 Floxe/Floxe Ca v 1.2 ACC/PBS Ca v 1.2 ACC/Cre First 5 min Enlose Supplementary Figure 6. Ca v 1.2 ACC/Cre mie showe a similar level of anxiety an loomotor ativity with that of ontrol mie. (a,) Elevate plus maze test. (a) Total time spent in eah arm an enter was similar among the Ca v 1.2 ACC/Cre (n = 15), Ca v 1.2 ACC/PBS (n = 14) an Ca v 1.2 Floxe/Floxe (n = 17) mie. () Total numer of entries into eah arm. () Light-ark transition test. There were no ifferenes in the first time to entry into the illuminate, light ompartment an the amount of time spent in eah ompartment among the Ca v 1.2 ACC/Cre (n = 7), Ca v 1.2 ACC/PBS (n = 8) an Ca v 1.2 Floxe/Floxe (n = 12) mie. Inset, the total numer of entry into the light ompartment. (,e) Open-fiel test. () The Ca v 1.2 ACC/Cre (n = 1), Ca v 1.2 ACC/PBS (n = 11) an Ca v 1.2 Floxe/Floxe (n = 7) mie spent similar time in the enter an (e) showe a similar loomotor ativity in open-fiel ox. However, all groups (Ca v 1.2 Floxe/Floxe, Ca v 1.2 ACC/PBS, an Ca v 1.2 ACC/Cre ) spent a little less time in the open arm in the elevate plus-maze test an in the enter in the open-fiel ox. This might e ue to the mixe geneti akgroun in onitional Ca v 1.2 mie. Nevertheless, Ca v 1.2 ACC/Cre have a similar level of anxiety responses as the ontrol mie. Nature Neurosiene: oi:1.138/nn.254

9 Preferene (%) e Cav1.2ACC/PBS v Training PBS Ca v 1.2 ACC/PBS Cre Ca v 1.2 ACC/Cre Cav1.2ACC/PBS v Time Preferene (%) f Cav1.2ACC/PBS v 1 hr 24 hr Cav1.2ACC/PBS v Time Cav1.2ACC/PBS v ACC/Cre Time Supplementary Figure 7. Ca v 1.2 ACC/Cre mie showe normal ehavioral responses in novel ojet reognition, preator exposure, or lassial fear onitioning tasks. (a,) Novel ojet reognition memory task. (a) Mean exploratory preferene uring training in the Ca v 1.2 ACC/Cre (n = 16) an Ca v 1.2 ACC/PBS (n = 14). Ca v 1.2 ACC/Cre mie an Ca v 1.2 ACC/PBS explore the two ojets for equal time, whih iniate no preferene of the animals for either ojet. () Exploration to a novel ojet after eah retention time. At 1 hr or 24 hr retention interval, when one of the familiar ojets was replae y a novel one, oth the Ca v 1.2 ACC/Cre mie an the Ca v 1.2 ACC/PBS exhiite inrease preferene for the novel ojet to the familiar one, ut no ifferene was foun etween the Ca v 1.2 ACC/Cre an the Ca v 1.2 ACC/PBS mie. () Preator exposure test for monitoring innate fear. The Ca v 1.2 ACC/Cre (n = 6), Ca v 1.2 ACC/PBS (n = 5) mie showe similar freezing ehavior when a rat was loate in opposite hamer as the ontrol group. (-f) Classial fear onitioning. () Freezing ehavior on the ay of training in the Ca v 1.2 ACC/Cre (n = 8) an Ca v 1.2 ACC/PBS (n = 9). Soli line iniates the uration of a tone (28 s) an the arrows iniate foot shoks (2 s). (e) Contextual fear onitioning 24 hr after training. (f) Cue fear onitioning 24 hr after training. A tone presentation is iniate y the horizontal line an foot shok, y vertial arrows. Nature Neurosiene: oi:1.138/nn.254

10 Duration (se) PF-lioaine PF-saline Time PF-lioaine PF-saline Time VPL/VPM-saline VPL/VPM-lioaine Time e PF-lioaine PF-saline Time Supplementary Figure 8 An inativation of the PF thalami nulei i not affet lassial fear onitioning (a-), an an inativation of the VPL/VPM thalami nulei reue inflammatory pain responses to formalin (,e). (a) Aministration of lioaine into the PF efore training ha no influene on the aquisition of fear (lioaine, n = 8; saline, n = 6). The horizontal line iniates the uration of a tone (28 s) an the arrow iniates the time of foot shoks (2 s). () There was no ifferene etween groups in 24-hr ontextual memory. () No ifferene was also foun etween groups in 24-hr ue memory. The horizontal line iniates the uration of a tone (3 min). () Behavioral responses to a formalin injetion, plotte in 5 min intervals, in the mie injete with lioaine in the VPL/VPM thalami nulei (n = 8) ompare with the saline group (n = 3). The miroinjetion into was performe 3 times at 2-min interval. (e) Data from () groupe into five time intervals. (f) The average total freezing response among the oservers with lioaine injetion into ifferent rain areas (ACC, PF, MD, an LA) efore the training. *P <.5, **P <.1, Stuent s t-test. Duration (se) f Average total freezing (se) VPL/VPM-saline VPL/VPM-lioaine ** ACC PF MD LA * * ** ** ** Time Nature Neurosiene: oi:1.138/nn.254

11 Silings Non-silings Silings 4 3 * * 2 * ** * * Non-silings Couple (>1 weeks) Non-ouple Couple (> 1 weeks) Non-ouple Supplementary Figure 9. The fear responses of the silings/ouples oservers in the presene of an opaque partition (a,), an the freezing response of eah emonstrator in a transparent partition uring the training (silings or ouples vs. non-silings or non-ouples) (,). Freezing ehavior (a) in silings (n = 15) vs. nonsilings (n = 2) experiments (F 1, 33 = 17.44, P =.2, two-way repeate ANOVA) an () in ouples (n = 11) vs. non-ouples (n = 7) experiments (F 1,16 = 1.93, P =.45, two-way repeate ANOVA) using an opaque partition. Note that oservers exhiite higher freezing levels with silings or ouples as emonstrators than with non-silings or non-ouples, respetively. *P <.5, Sheffe s post-ho test. () There was no ifferene in the freezing levels of the emonstrators etween silings an non-silings. () There was also no ifferene in the freezing levels of the emonstrators etween ouples an non-ouples. Nature Neurosiene: oi:1.138/nn.254

ACC PF LA MD VPM/VPL ACC PF LA MD VPM/VPL ACC LA Supplementary Figure 1.

loal elivery y ye into the target rain area. (a) Cannula position.

() Fluoresent ye (Vyrant DiI ell-laeling solution) was use to monitor that our

After the injetion of DiI into the target regions with the same onition (.7 μl,.

oronally setione (1 μm) y a viratome (VIBRATOME ).

properly elivere into the eah restrite rain area. () Reoring eletroe position.

12 ACC PF LA MD VPM/VPL ACC PF LA MD VPM/VPL ACC LA Supplementary Figure 1. Postmortem histology of the reoring regions, annula positions, an verifiation of loal elivery y ye into the target rain area. (a) Cannula position. Coronal rain slies show the loation implante with a annula. () Fluoresent ye (Vyrant DiI ell-laeling solution) was use to monitor that our injetion was appropriately one into the target anatomial strutures. After the injetion of DiI into the target regions with the same onition (.7 μl,.1 μl min-1), rain was remove an post-fixe with 4% paraformalehye, an then oronally setione (1 μm) y a viratome (VIBRATOME ). The re fluoresent signals y injetion of DiI ye solution iniate that rug was properly elivere into the eah restrite rain area. () Reoring eletroe position. Coronal rain slies show the positions of eletroes in the ACC (left) an the LA (right). Arrows iniate where the eletroes an annula tip were loate. Nature Neurosiene: oi:1.138/nn.254

13 (se e Bregma mm mm +.98 mm +.74 mm +.5 mm +/+ ACC/Cre +/+ +/+ ACC/Cre +/ Time (se) Time (se) ACC1 ACC2 ACC1 ACC2 ACC1 ACC2 ACC1 ACC2 ACC1 ACC f +/+ +/+ ACC/Cre Open Enlose Center +/+ First time to light Cav1.2-positive ells/mm /+ ACC/Cre Light Dark Contralateral Ipsilatral * Supplementary Figure 11. Normal ehavioral responses in wil-type (C57BL/6J) mie injete with p- Cre (a-), an Ca v 1.2-elete proportion of the ACC (e,f). (a,) Normal oservational fear onitioning in wil-type (C57BL/6J) mie injete with p-cre. No ovious ifferene etween wil-type (C57BL/6J) mie treate with p-cre an non-treate mie in (a) the training an () the 24-hr ontextual memory. (,) Normal anxiety in wil-type mie injete with p-cre. The mie having the ACC miroinjete with p- Cre ha a similar level of ehavioral responses in () elevate plus maze an () light-ark transition tasks as the ontrol mie, whih iniates that p-cre injetions i not ause a non-speifi anormal ativity. (e) Shemati representative rawings of mouse rain oronal setions showing the ounary areas of Ca v 1.2- negative nulei (grey regions). (f) Ca v 1.2-positive ells in the ACC. For the ounting of Ca v 1.2-positive ells, after staining with anti-ca v 1.2 as a primary antioy, five oronal setions aroun the injetion site were selete an analyze. Square sampling regions (retangular, 3 3 μm) in (e) were use for Ca v 1.2- positive ell ounting. Total ounts in these sampling regions were onverte into ell ensities for quantifiation. *P <.1, Stuent s t-test. Nature Neurosiene: oi:1.138/nn.254

Supplementary Materials for A universal data based method for reconstructing complex networks with binary-state dynamics

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