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1 A m ultory distnce (cm ) % C enter distnce C e n te r tim e (s ) A m ultory distnce (cm ) % C enter distnce C e n te r tim e (s ) N o v e l c g e % h o m e c g e c o n s u m p tio n % M ic e fe e d in g (cum ultive frequency) % Increse in ody w eight over 1 dys SUPPLEMENTARY FIGURES 1 d s tre s s / lo w lig h t te s tin g Feeding ltency (s ) c n o s tre s s d 5 d s tre s s Supplementry Figure 1. 2-AG ugmenttion does not ffect coummtory or explortory drive. () Effect of cute JZL-184 on coumption nd ltency in less versive (low light) NIH novel-cge test 24h fter foot-shock stress. () Effect of 1 dys of JZL-184 tretment on ody weight in utressed mice. (c) Effects of cute JZL-184 on explortion in novel open field under control conditio nd (d) fter 5 dys of stress. P- vlues for unpired two-tiled t-test reported in ech pnel. Dt re presented s men ± SEM. 1
2 Corticosterone (ng/ml) Corticosterone (ng/ml) Corticosterone (ng/ml) Threshold to elicit ehviorl respoe (ma) Behviorl respoivity score % Component freezing Ltency to open rm (s) Open-rm entries Open rm time (s) Center time (s) Distnce trveled (cm) Susceptile EPM (n=23, n=17) Susceptile Susceptile OFT (n=23, n=17) Susceptile c d e Susceptile (n=19) Susceptile (n=21) 2.2. Flinch Run Susceptile Jump Voclize Behviorl Respoe 2 2. (n=19) Susceptile (n=21) week 1 week 2 Foot-shock # f g h 2 2 BL T1 T6 BL T1 T6 week 1 week 2 Component Susceptile 5 r 2 =.3658 p = Susceptiility (FS- ltency - ltency) 5 r 2 =.9828 p = Averge foot-shock respoivity score Supplementry Figure 2. Bseline nxiety, foot-shock respoivity, nd stress-induced corticosterone relese do not differ etween susceptile nd resilient groups. () nd susceptile pre-stress elevted plus mze (EPM) ltency to open rm, open rm entries, nd open rm time. () nd susceptile open field test (OFT) center time nd distnce trveled. (c) nd susceptile group verge foot-shock current thresholds to elicit specified ehviorl respoes. Although group sizes were n=19 nd n=21 for resilient nd susceptile groups, not every individul exhiited every ehviorl respoe. (d) nd susceptile verge ehviorl respoe scores (1-flinch/wlk, 2-run, 3-jump) to the first nd 6 th /lst 2 second.7ma shocks for 2 stress exposures 1 week prt. (e) nd susceptile percent freezing during seline (BL), nd the first tone (T1) nd lst tone (T6) of 2 foot-shock stress exposures 1 week prt. (f) nd susceptile plsm corticosterone mesurements 2 minutes fter the initition of footshock stress. (g) Correltion etween plsm corticosterone levels from pnel f nd susceptiility s defined y the difference in ltency etween NIH foot-shock novel cge test (FS-) nd seline novel cge test (). (h) Correltion etween plsm corticosterone levels in pnel f nd verge foot-shock respoivity scores cross ll 6 shocks during week 2 (immeditely prior to collection of plsm). Unpired two-tiled t-test conducted on pnels -, nd f. Two-wy ANOVA performed on pnels c-e. R 2 nd P vlue for liner regression reported in pnels g nd h. Dt re presented s men ± SEM. 2
3 Ltency to Coume (s) Ltency to Coume (s) Interction F (2, 74) = 2.63 P <.1 **** (n=23) Susceptile (n=16) 18 p=.157 * p <.1**** Interction F (2, 74) = 2.13 P <.1**** (n=28) Susceptile (n=11) 18 p=.416* p <.1**** No stress 1 3 No stress 1 3 Dely etween stress nd novel cge test (dys) No stress 1 14 No stress 1 14 Dely etween stress nd novel cge test (dys) Supplementry Figure 3. Anxiety-like ehvior elicited y cute stress is long-lsting. () nd susceptile novel cge test ltencies in the repeted novelty-induced hypophgi prdigm t seline (No stress), 1 dy fter stress, nd 3 dys or () 14 dys fter second foot-shock stress exposure. F nd P vlues for two-wy ANOVA shown ove individul pnels. P vlues for pirwise compriso derived from Holm-Sidk multiple compriso test fter ANOVA shown in pnels. Dt re presented s men ± SEM. 3
4 % Open rm distnce Open Arm Time (s) Outside hlf open rm time (s) VEH JZL Drug VEH JZL Drug VEH JZL Drug Supplementry Figure 4. JZL-184 does not ffect nxiety-like ehvior in the elevted plus mze in repeted prdigm. () Elevted plus mze % open rm distnce (left), open rm time (middle), nd time on the outside hlf of the open rm (right) for resilient (lck circles) nd susceptile (red circles) individuls 24h fter foot-shock stress t lest 1 week fter susceptiility ctegoriztion in the repeted NIH prdigm nd treted with either vehicle (VEH) or JZL-184 (JZL; lue r). Dt comined from 2 independent cohorts. Unpired two-tiled t-tests performed for ech mesure. Dt re presented s men ± SEM. 4
5 Stress-induced chnge in ltency (s) Feeding ltency (s) Feeding ltency (s) ****p <.1 9 All Femles (n=51) F (2.111, 15.6) = P <.1**** 9 Interction F (1, 49) = P <.1 **** 6 6 (39) Susceptile (12) 3 p <.1**** Trining -FS-JZL F (6.14, 3.7) = P <.1**** 3 2 Trining Interction F (1, 49) = P =.2 *** -FS-JZL (39) Susceptile (12) 1 ***p=.4 ****p <.1 1 **p=.45 **p=.99 *p=.152 Trining -FS-JZL Trining -FS-JZL c n=12 **p=.11 Susceptile n=1 d r 2 =.1227 *p =.117 n=39 n=5-2 Totl=51 VEH Totl=51 JZL Supplementry Figure 5. JZL-184 promotes resilience in femle mice. () Femle home cge (lue lines) nd novel cge () ltencies nd () coumption t seline () nd fter stress with vehicle () or JZL (-FS-JZL). (c) Proportion of susceptile nd resilient femles fter vehicle (VEH) nd JZL-184 tretment. (d) Correltion etween seline novel cge coumption nd stress-induced chnge in ltency from seline. Blue rrows indicte stress exposure. F nd P vlues for onewy (left) or two-wy (right) ANOVA shown ove individul pnels (-). P vlues for pirwise compriso derived from Holm-Sidk multiple compriso test fter ANOVA shown in pnels. R 2 nd P vlue for liner regression reported in pnel (d). P vlue from chi-squred test reported in pnel (c). Dt re presented s men ± SEM. 5
6 MAGL Protein (Norm to Ponc.) MAGL Protein (Norm to Ponc.) MAGL Protein (Norm to Ponc.) MAGL Protein (Norm to Ponc.) DAGL Protein (Norm to Ponc.) DAGL Protein (Norm to Ponc.) DAGL Protein (Norm to Ponc.) DAGL Protein (Norm to Ponc.) CB1 Protein (Norm to Ponc.) CB1 Protein (Norm to Ponc.) CB1 Protein (Norm to Ponc.) CB1 Protein (Norm to Ponc.) AMY PFC NAC vhip R S R S R S R S R S R S R S R S Susceptile Susceptile Susceptile Susceptile R S R S R S R S R S R S R S R S c Susceptile Susceptile Susceptile Susceptile R S R S R S R S R S R S R S R S Susceptile Susceptile Susceptile Susceptile Supplementry Figure 6. CB1 receptor, DAGLα, nd MAGL protein levels do not differ etween resilient nd susceptile groups. () CB1 receptor protein levels s mesured y western lot in the mygdl (AMY), prefrontl cortex (PFC), nucleus ccume (NAC), nd ventrl hippocmpus (vhip) for resilient (R) nd susceptile (S) mice. () DAGLα nd (c) MAGL protein levels in ech rin region s mesured y western lot. Representtive lots shown ove ech r grph. Unpired two-tiled t-tests were performed for ech mesure. Dt re presented s men ± SEM. 6
7 2-AG (pmol/mg tissue) AMY e PFC i NAC m 6 4 vhip Susceptile f 4 Susceptile j 4 Susceptile n 5 Susceptile c d R 2 =.4114 p= Stress-induced chnge in ltency R 2 =.2216 p= Susceptile R 2 =.97 p= Stress-induced chnge in ltency 15 Stress-induced chnge in ltency g h R 2 =.179 p= Stress-induced chnge in ltency R 2 =.219 p= Stress-induced chnge in ltency Susceptile 5 R 2 =.6778 p= Stress-induced chnge in ltency k l R 2 =.497 p= Stress-induced chnge in ltency R 2 =.2427 p= Susceptile R 2 =.1124 p= Stress-induced chnge in ltency 6 8 Stress-induced chnge in ltency o p R 2 =.1157 p= Stress-induced chnge in ltency 6 R 2 =.1282 p= Susceptile R 2 =.7669 p= Stress-induced chnge in ltency 6 8 Stress-induced chnge in ltency Supplementry Figure 7. 2-AG levels do not correlte with resilience. () Amygdl (AMY) 2-AG levels for resilient nd susceptile groups. () Correltion etween mygdl 2-AG nd susceptiility s mesured y stress-induced chnge in ltency for the whole popultion nd (c) resilient nd (d) susceptile supopultio. (e) Prefrontl cortex (PFC) 2-AG levels for resilient nd susceptile groups. (f) Correltion etween PFC 2-AG nd susceptiility s mesured y stress-induced chnge in ltency for the whole popultion nd (g) resilient nd (h) susceptile supopultio. (i) Nucleus ccume (NAC) 2-AG levels for resilient nd susceptile groups. (j) Correltion etween NAC 2-AG nd susceptiility s mesured y stress-induced chnge in ltency for the whole popultion nd (k) resilient nd (l) susceptile supopultio. (m) Ventrl hippocmpus (vhip) 2-AG levels for resilient nd susceptile groups. (n) Correltion etween vhip 2-AG nd susceptiility s mesured y stress-induced chnge in ltency for the whole popultion nd (o) resilient nd (p) susceptile supopultio. Unpired two-tiled t-tests were performed for pnels, e, i, nd m. R 2 nd P vlue for liner regressio reported in ll other pnels. Dt re presented s men ± SEM. 7
8 Feeding ltency (s) Feeding ltency (s) (n=35) Susceptile (n=5) 12 Resilent (n=35) F (2, 12) = 4.74 P =.199 * 12 Susceptile (n=5) F (2, 12) = 1.65 P =.22 ** *p= *p=.114 **p= Trining Resilent (n=35) F (2, 14) =.2536 P = FS-THC 3 Trining Susceptile (n=5) F (2, 12) =.1187 P = FS-THC Trining -FS-THC Trining -FS-THC Supplementry Figure 8. THC promotes resilience similrly to JZL-184. () Mle resilient nd susceptile home cge (lue lines) nd novel cge () ltencies nd () coumption t seline () nd fter stress with vehicle (FS-V) nd delt-9-tetrhydrocnninol (FS-THC). Blue rrows indicte stress exposure. F nd P vlues for one-wy ANOVA shown ove individul pnels. P vlues for pirwise compriso derived from Sidk multiple compriso test fter ANOVA shown in pnels. Dt re presented s men ± SEM. 8
9 Amultory distnce (m) Open-rm entries % Open-rm time Amultory distnce (m) Open-rm entries % Open-rm time Amultory distnce (cm) Time in light (s) Light zone entries Amultory distnce (cm) Time in light (s) Light zone entries Amultory distnce (cm) Center time (s) % Center distnce Amultory distnce (cm) Center time (s) % Center distnce PFC Open Field d NAC Open Field CRE CRE Light-Drk CRE e CRE CRE Light-Drk CRE c CRE CRE Elevted Plus Mze CRE f CRE CRE Elevted Plus Mze CRE CRE -CRE -CRE Supplementry Figure 9. Bsl nxiety is not significntly ffected y loss of PFC or NAc 2-AG. () Open field test () light-drk nd (c) elevted plus mze ehvior of prefrontl cortex (PFC) control versus - CRE injected DAGL fl/fl mice. (d) Open field test (e) light-drk nd (f) elevted plus mze ehvior of nucleus ccume (NAC) control versus -CRE injected DAGL fl/fl mice. Dt for oth rin regio ws comined from two independent cohorts. Unpired two-tiled t-tests were performed for ech mesure; no significnt differences were found etween groups for ny mesure. Dt re presented s men ± SEM. -CRE -CRE -CRE 9
10 Stress-induced chnge in ltency (sec) Feeding Ltency (s) Feeding Ltency (s) Feeding Ltency (s) Stress-induced chnge in ltency (sec) Ltency to Coume (s) Feeding Ltency (s) Feeding Ltency (s) PFC c Interction F (8, 32) = P = Cre F (1, 4) =.2716 P =.651 Dy F (8, 32) = 34.6 P <.1 **** 1 5 Dy: e NAc f AAV- g AAV--CRE h Interction Interction Interction F (8, 296) =.984 P = Cre F (1, 37) =.781 P =.7916 Dy F (8, 296) = **** 15 F (8, 136) = 1.8 P <.1 F (8, 144) = 4.45 P =.2 *** P <.1 **** Susceptile (n=5) Susceptile (n=6) (n=15) AAV- (n=19) 1 AAV--CRE (n=2) 1 (n=13) 1 **** **** ** Dy: Dy: AAV- (n=22) AAV--CRE (n=2) Trining Trining -1FS -5FS Interction F (8, 296) = P <.1 **** Cre 2.5 F (1, 37) = 2.46 P =.1294 Dy F (8, 296) = 62.2 P <.1 **** AAV- (n=19) AAV--CRE (n=2) Trining ** -1FS -5FS Interction F (8, 32) =.6184 P =.7623 Cre F (1, 4) =.8119 P = Dy F (8, 32) = 14.9 P <.1**** Dy: AAV- (n=22) AAV--CRE (n=2) Trining -1FS -5FS **** **** -1FS -5FS Dy: Dy: 5 Dy: Dy: AAV- Interction F (8, 16) = P =.3 ** Trining AAV- Interction 2.5 F (8, 136) =.3296 P =.9534 Trining Susceptile (n=14) (n=8) Trining Susceptile (n=14) (n=8) Trining Susceptile (n=6) (n=13) ** AAV- Interction F (8, 16) =.394 P = FS -5FS -1FS -5FS ** -1FS -5FS -1FS -5FS Dy: Dy: Dy: Dy: AAV--CRE Interction F (8, 144) = 2.55 P =.126 * Susceptile (n=12) (n=8) Trining AAV--CRE Interction F (8, 144) = 1.2 P =.424 Trining AAV--CRE Interction 2.5 F (8, 144) = 1.87 P =.3756 Trining Susceptile (n=12) (n=8) Trining Susceptile (n=5) (n=15) *** -1FS -5FS -1FS -5FS -1FS -5FS -1FS -5FS d n=6 AAV- Totl=22 AAV- Totl= n=8 n=13 5d stress 5d stress Susceptile AAV- AAV--CRE AAV--CRE n=5 Interction F (1, 35) =.163 P =.6889 AAV--CRE n=14 n= n=12 Totl=2 Interction F (1, 38) =.6429 P =.9365 AAV- AAV--CRE Susceptile Totl=2 n=15 Susceptile Supplementry Figure 1. Stress susceptiility is not significntly ffected y loss of PFC or NAC 2-AG. () Ltency (top) nd coumption (ottom) mesurements for repeted novelty-induced hypophgi (rnih) testing of prefrontl cortex (PFC) control versus -CRE injected DAGL fl/fl mice. () PFC AAV- nd (c) PFC AAV--CRE rnih dt from pnel () split into resilient nd susceptile supopultio using the ltency difference etween novel cge testing 24h fter 5 dys of stress exposure (-5FS) nd seline (). (d) Susceptiility rtios for PFC AAV- control versus AAV--CRE injected mice from pnels () nd (c). (e) Ltency (top) nd coumption (ottom) mesurements for repeted novelty-induced hypophgi (rnih) testing of nucleus ccume (NAC) control versus -CRE injected DAGL fl/fl mice. (f) NAC AAV- nd (g) NAC AAV--CRE rnih dt from pnel () split into resilient nd susceptile supopultio using the ltency difference etween novel cge testing 24h fter 5 dys of stress exposure (- 5FS) nd seline (). (h) Susceptiility rtios for PFC AAV- control versus AAV--CRE injected mice from pnels (f) nd (g). Blue rrows indicte stress exposure which occurred once per dy for 5 coecutive dys. Dt for ech rin region ws comined from two independent cohorts. F nd P vlues for two-wy ANOVA shown in ll pnels. Asterisks indicte significnce s determined y pirwise compriso from Holm-Sidk multiple compriso test fter ANOVA. Chi-squred tests of susceptiility rtios were not significnt (d,h). Dt re presented s men ± SEM. 1
11 % odse % odse % mx odse % odse 3-5 weeks Injection site / Recording site vhip / BLA PreL / BLA RIM (n=5,2) VEH (n=9,2) Time (s) 15 1 F (2, 27) = P <.1**** ** **** LEC / BLA VEH (n=15,5) Time (s) 15 vhip PreL LEC 1 5 VEH (n=7,1) Time (s) Supplementry Figure AG most strongly modultes ventrl hippocmpl inputs to the BLA. () Schemtic figure for fferent-specific electrophysiologicl recordings in BLA. () Left: Representtive imges of YFP leled injection nd recording sites. Middle: Corresponding grphs of depolriztion induced suppression of excittion (odse) of opticlly evoked currents from ventrl hippocmpl (vhip), prelimic prefrontl corticl (PreL), nd lterl entorhinl corticl (LEC) inputs onto BLA pyrmidl cells with vehicle (VEH) incution. Rimonnt incution verified tht the vhip-bla odse is CB1-dependent (RIM, grey trce, top middle). Right: comprison of the mximlly evoked odse of the seprte inputs. Numer of (cells, nimls) re shown for ech group. F nd P vlue for one-wy ANOVA shown (d). Asterisks indicte significnce of P vlue from Holm-Sidk multiple comprison test etween PreL or LEC nd vhip. Dt re presented s men ± SEM. 11
12 Normlized oepsc Res (n=13,5) Susc (n=11,4) mM CP Time (min) Supplementry Figure 12. CB1 receptor gonist-induced depression in the BLA does not differ etween resilient nd susceptile mice. () CP-5594 induced depression of opticlly evoked EPSCs (oepsc) from ventrl hippocmpl synpses in solterl mygdl (BLA) pyrmidl cells. Numer of (cells, nimls) re shown for ech group. Dt re presented s men ± SEM. 12
13 Supplementry Figure 13. Full un-cropped lots re shown corresponding to Supplementry Figure 6. Arrows re shown mrking smples tht were not included in nlysis due to western lot rtifct. For MGL deity mesurements, severl nds were present, presumly corresponding to multiple isoforms tht were present in the tissue. For coistency, the deity of the top two moleculr weight isoforms were selected for nlysis. 13
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