{"paper_id":"f842c8e7-18b4-46a4-9ad0-d35b718faa87","body_text":"Unravelling the neurocognitive mechanisms underlying 1 \ncounterconditioning in humans 2 \n 3 \nLisa Wirz 1,2‡*, Maxime C. Houtekamer 1‡, Jette de Vos 3, Joseph E. Dunsmoor 4, Judith R. Homberg 1‡, 4 \nMarloes J.A.G. Henckens1‡, Erno J. Hermans1 5 \n1 Donders Institute for Brain, Cognition, and Behaviour, Radboud University Nijmegen Medical Center, 6 \nNijmegen, The Netherlands 7 \n2 Cognitive Psychology, Ruhr University Bochum, Germany 8 \n3 Department of Psychiatry and Neuropsychology, MHeNs, Maastricht University, The Netherlands 9 \n4 Department of Psychiatry and Behavioral Sciences, University of Texas at Austin, United States 10 \n‡ These authors contributed equally to this work 11 \n* Corresponding author: 12 \nLisa Wirz 13 \nEmail: lisa.wirz@donders.ru.nl 14 \nPhone: (+31) 024 361 1301 15 \nKapittelweg 29, 6525 EN Nijmegen, The Netherlands 16 \n 17 \n 18 \nShort title: Neurocognitive mechanisms of counterconditioning 19 \nKeywords: Fear conditioning, counterconditioning, reward, ventral striatum, ventromedial prefrontal 20 \ncortex 21 \nAbstract: 200 words 22 \nMain text (excl. abstract, methods, references, figure legends): 5517 23 \nN Figures: 6 24 \nN Tables: 1 25 \nSupplementary Information 26 \n  27 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n2 \n \nAbstract 28 \nCounterconditioning (CC) aims to enhance extinction of threat memories by establishing new 29 \nassociations of opposite valence. While its underlying neurocognitive mechanisms remain largely 30 \nunexplored, previous studies suggest qualitatively different mechanisms from regular extinction.  In 31 \nthis functional MRI study, participants underwent categorical threat conditioning  (CS+/CS-: images of 32 \nanimals/tools), followed by either CC ( CS+ images  reinforced with monetary rewards , n=24) or 33 \nregular extinction (n=24). The following day, we assessed spontaneous recovery of threat responses 34 \nand episodic memory for CS+ and CS - category exemplars. While the ventromedial prefrontal cortex 35 \n(vmPFC) was activated during regular extinction, participants undergoing CC showed persistent CS+ -36 \nspecific deactivation of the vmPFC and hippocampus,  and CS+ -specific activation of the nucleus 37 \naccumbens (NAcc).  The following day, p hysiological threat responses returned in the  regular 38 \nextinction group, but not in the CC group. Counterconditioning furthermore strengthened episodic 39 \nmemory for CS+ exemplars presented during CC , and retroactively also for CS+ exemplars presented 40 \nduring the threat conditioning phase. Our findings confirm that CC leads to more persistent  41 \nextinction of threat  memories, as well as a ltered consolidation of the threat conditioning episode. 42 \nCrucially, we show a q ualitatively different activation pattern during CC versus regular extinction, 43 \nwith a shift away from the vmPFC and towards the NAcc. 44 \n  45 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n3 \n \nIntroduction 46 \nTrauma-related disorders are prevalent and highly detrimental to the individual’s quality of life 1. To 47 \ntreat these disorders, patients undergo exposure therapy in a safe therapeutic environment , causing 48 \nthreat responses to fade away 2. Although exposure therapy may be successful initially, relapse often 49 \noccurs and is the most pr evalent remaining challenge in optimizing treatment efficacy . Research 50 \nsuggests that exposure therapy creates a safety memory that competes for expression with the 51 \noriginal threat memory 3,4, suggesting that relapse may occur because of relatively weak learning and 52 \nretention of the safety memory . Therefore, identifying mec hanisms that can be used to strengthen 53 \nsafety learning is a key step in advancing treatment for trauma -related disorders. A promising 54 \napproach to strengthen safety learning is to create a new, positive association with the event that 55 \nwas previously linked to an aversive outcome. However, while there are indications that establishing 56 \npositive associations can prevent relapse, the underlying mechanism s are poorly understood (for a 57 \nreview, see 5). 58 \nTo study threat responses in a controlled setting, aversive Pavlovian conditioning is typically used. A 59 \nneutral stimulus (conditioned stimulus, CS; e.g., a picture) is coupled with a biologically aversive 60 \nunconditioned stimulus (US; e.g., an electrical shock), after which the CS alone also elicits a 61 \nconditioned threat response. Conditioned threat responses to the C S can be attenuated using 62 \nextinction, during which the CS is repeatedly presented in absence of the US . However, early theories 63 \nhave suggested that threat responses may more easily be inhibited by engaging appetitive systems 6,7. 64 \nIndeed, experiments provide evidence that coupling a CS to a p ositive US after threat conditioning, a 65 \nprocess known as aversive -to-appetitive counterconditioning (CC),  may be superior to regular 66 \nextinction. Specifically, CC compared to regular extinction was associated with a faster  attenuation of 67 \nlearned threat responses6,8, stronger decreases in threat expectan cy9,10, and more positive valence 68 \nratings of the CS11-13 immediately post-CC. 69 \nTests for spontaneous recovery, reinstatement, and renewal can subsequently be used to evaluate 70 \nthe return of threat  responses over time, after unsignaled presentation of the US, or in a novel 71 \ncontext, respectively 3,14. Thereby , one can investigate whether CC persistently attenuates threat 72 \nresponses. While early rodent studies showed that CC may be prone to the same relapse as 73 \nextinction15,16, recent neurobiological work in rodents showed that CC can enhance the activation of  74 \nan amygdala-striatal pathway, which is also recruited during extinction – albeit to a lesser degree –, 75 \nand that CC compared to regular extinction can reduce the return of  threat responses17. Recent 76 \nstudies suggest that CC may diminish the return of threat  responses in humans as well. Specifically, it 77 \nwas shown that CC compared to regular extinction reduced renewal of previously learned food -78 \nallergy associations when presented in a novel context one day later 18. Counterconditioning 79 \ncompared to regular extinction was also associated with reduced recovery of arousal and shock 80 \nexpectancy the following day9,19, as well as reduced reinstatement9.  81 \nExtinction learning appears to be mediated by activation of the ventromedial prefrontal cortex 82 \n(vmPFC), which inhibits the expression of threat responses  by suppressing amygdala activity 20-23. 83 \nWhen extinc tion is enhanced by replacing aversive with novel, neutral outcomes , the vmPFC was 84 \nfound to be engaged more effectively than  during standard extinction 24. When extinction is 85 \nenhanced by replacing aversive outcomes with a reward  (counterconditioning), evidence in rodents 86 \nsuggests stronger engagement of  the ventral striatum , a region known to be involved in the 87 \nanticipation and receipt of reward25. However, human studies only provide indirect evidence for such 88 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n4 \n \na mechanism since involvement of the ventral striatum could only be shown during spontaneous 89 \nrecovery19 or during reinstatement 26, but not during CC itself. Although it was observed that brain 90 \nareas of the fear network are reduced during CC versus regular extinction  in humans 19, it is unclear 91 \nhow this difference is achieved. Therefore, although evidence suggests that CC is more effective than 92 \nregular extinction in preventing the return of threat responses  the neural mechanisms are not well 93 \nunderstood yet. It remains unclear whether CC is a form of enhanced extinction that is mediated by 94 \nenhanced engagement  of extinction networks , including the vmPFC , or whether it is driven by 95 \nengagement of reward networks. 96 \nTo investigate the qualitative differences between CC versus regular extinction further, category 97 \nconditioning can be used, a procedure in which conditioned threat responses are learnt by coupling a 98 \nUS to conceptually linked exemplars that together form a category (e.g. , pictures of animals )27. It 99 \nallows for the typical measures of threat condition ing, but also provides the opportunity to probe 100 \nepisodic memory for the CS category exemplars 28. When episodic memory was probed 24h after CC 101 \nand extinction, it was shown that memory for CS+ stimuli that had undergone CC was stronger than 102 \nmemory for CS+ stimuli that had undergone regular extinction 29. This suggests that compared to 103 \nregular extinction, CC can enhance episodic memory consolidation and potentially provide stronger 104 \nretrieval competition against a threat memory. 105 \nTo investigate the neural mechanisms that distinguish CC from regular extinction and to establish 106 \nwhether CC is indeed associated with a memory that is qualitatively different from the safety 107 \nmemory established during regular extinction , we  performed a two -day fMRI study comparing CC 108 \nversus regular extinction in a between -subjects design (Figure 1A). Participants underwent category 109 \nconditioning and subsequently underwent either aversive-to-appetitive CC (CC group) or  regular 110 \nextinction (Ext group ; Figure 1 B-C). During the CC task, participants in the CC group obtain ed 111 \nmonetary rewards depending on how quickly they  responded to a cue superimposed on novel 112 \ncategory exemplars from t he CS+ category, a procedure similar to the monetary incentive delay  113 \n(MID) task30. To maximize task similarity between tasks and groups, the cued-response element was 114 \nkept consistent in all  tasks (acquisition, CC/extinction, spontaneous recovery, reinstatement),  but 115 \nresponse-time conting ent monetary rewards were  only present during the CC task  (Figure 1F ). To 116 \nassess the potential  of CC v ersus regular extinction in persistently attenuating the expression of 117 \nthreat response , we tested retrieval of the threat memory and reinstatement of threat responses 118 \none day later (Figure 1D). Episodic memory for exemplars of the CS categories that were presented 119 \nduring threat conditioning and CC/extinction was assessed by means of a surprise memory test. To 120 \ncharacterize pupil dilation responses  (PDRs) and skin conductance responses ( SCRs) during the 121 \nanticipation of shock - and reward-reinforcement independently from prior conditioning , a separate 122 \nvalence-specific response characterization task was included at the end of the experiment ( Figure 123 \n1E). 124 \nIn line with previous results 9,19, w e hypothesized that CC compared to extinction would lead to a 125 \nmore persistent attenuation of threat responses . As indicated above, t his could be mediated by  two 126 \npossible neural mechanisms: either through enhanced engagement of extinction networks, reflected 127 \nby increased engagement of the vmPFC , or through a shift towards reward networks, reflected by  128 \nactivation of the ventral  striatum. Based on previous results 29, we expect ed stronger episodic 129 \nmemory for CS exemplars presented during CC, whereas regular extinction would not  show such a 130 \nstrengthening effect. 131 \n 132 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n5 \n \n 133 \n 134 \nFigure 1. Overview of the experimental design . (A). Participants were assigned to the counterconditioning (CC) or 135 \nextinction (Ext) group. On day 1, participants performed two blocks of acquisition of category -conditioned threat responses 136 \nseparated by a 30 second  break, followed by  CC or extinction. Day 2 consisted of  a spontaneous recovery test,  a 137 \nreinstatement procedure and test, an item memory test and a valence -specific response characterization. Valence and 138 \narousal ratings for the different categories were taken before or after  the tasks as indicated by ‘V+A Rating’. All tasks were 139 \nperformed in an MRI scanner.  (B) During acquisition, participants viewed trial -unique exemplars of objects and animals. 140 \nExemplars of one category (CS+ animals or objects counterbalanced) were paired with a shock in 50% of trials . CS- trials 141 \nwere not reinforced. (C) Participants in the CC group could earn a monetary reward if they responded quickly enough to 142 \nexemplars in the CS+ category. (D) Participants in the Ext group underwent extinction. During the ex tinction task, recovery 143 \ntest and reinstatement test, neither CS+ nor CS - exemplars were paired with a shock . (E) In the valence -specific response 144 \ncharacterization task, participants viewed three different coloured squares. One colour was associated with sh ock (CS+S), 145 \none colour with reward (CS+R) and one colour served as CS -. The trial structure was otherwise identical to the acquisition 146 \nand CC tasks. (F) In all Pavlovian tasks, trial onset was marked by presentation of a unique category exemplar. After a 147 \nvariable interval, a ring appeared, to which participants were instructed to respond as quickly as possible. Upon response, 148 \nthe ring shifted in colour as response confirmation . In the acquisition task, shocks could occur  0.5-1.5-seconds after the 149 \nresponse w indow had elapsed (indicated as ‘pre -shock). The category exemplar and cue remained visible 1 second after 150 \npotential shock administration (indicated as ‘post -shock’). During CC, participants received visual feedback for 2 seconds 151 \n(+€0.50 approximately the fastest 70% of trials, +€0.00 on other trials).  During the other tasks, participants viewed neutral 152 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n6 \n \nfeedback (three dots). Trials were separated by an 8-10 s intertrial interval, during which a fixation cross is displayed in the 153 \ncentre of the screen. 154 \nResults 155 \nIn the valence-specific response characterization task, we observed that both threat and reward-156 \nanticipation induced strong arousal -related PDRs and SCRs (see Supplementary Information ). 157 \nHowever, PDRs allowed for a better differentiation of the two compared to the CS- (Supplementary 158 \nFigure 1A). Therefore, we focus ed on PDRs in all analyses and refer to the Supplementary 159 \nInformation for details on the analysis of SCRs. During the acquisition task, both groups showed 160 \ncomparable and successful acquisition of differential conditioned threat responses ( PDR means ± SD: 161 \nCC CS+=1.085±.030, CC CS -=1.054±.033, Ext CS+ =1.084±.050, Ext CS -=1.050±.035; for PDR, SCR and 162 \nfMRI results see Supplementary Information). 163 \nExtinction and aversive-to-appetitive counterconditioning  164 \nAfter threat acquisition, participants in the CC group underwent CC, while participants in the Ext 165 \ngroup underwent regular extinction. Across both groups and pha ses (early vs. late),  we observed 166 \nretention of conditioned differential PDRs (CS-type (CS+, CS -) x Phase (Early, Late) x Group (CC, Ext) 167 \nrmANOVA, main effect CS-type: F(1,34)=15.393, p<0.001, η²=0.312, Figure 2A), as well as a decrease in 168 \nPDRs over the course of the task (main effect phase: F( 1,34)=10.121, p=0.003, η²=0.229). These 169 \nfindings are in contrast to our expectation of a  CS-type x Phase x Group interaction. Specifically, we 170 \nexpected differential PDRs to become extinguished in the Ext group , while being sustained in the CC 171 \ngroup, potentially due to increased reward anticipation. Extinction in the Ext group however already 172 \noccurred during the early phase (paired t -test, early CS+ vs. CS -, p=0.233), and differential responses 173 \ndid not change to wards the late phase (p=0.979) . As a result, we found distinct differential 174 \nconditioned PDRs  throughout the CC/extinction task between groups ( CS-type x Group interaction : 175 \nF(1,34)=6.053, p=0.019, η²=0.151) , with participants undergoing CC showing stronger PDRs to CS+ vs. 176 \nCS- category exemplars (paired t-test average CS+ vs. CS -, t(20)=3.602, p=0.002, CS+: 1.07 ±0.04, CS-: 177 \n1.04±0.04), whereas differential PDRs were extinguished in participants undergoing extinction 178 \n(paired t-test average CS+ vs. CS -, p=0.246, CS+: 1.05±0.04, CS-: 1.04±0.04). Results of t he valence-179 \nspecific response characterization task showed that differential PDRs can also be  indicative of 180 \nanticipation of reward (Supplementary Figure 1A). Thus, while PDRs in the Ext group indicated that 181 \ndifferential conditioned threat responses were successfully extinguished, differential PDRs persist ed 182 \nin the CC group, likely reflecting reward anticipation. Differential SCRs persisted during the late phase 183 \nFigure 2. Differential PDRs during CC/extinction and \nexplicit ratings of arousal and valence provided after the \ncounterconditioning or extinction phase. (A) Differential \nPDRs for the early (light red) and late (dark red) phase of \ncounterconditioning (CC, solid bars) or extinction (EXT, \nopen bars). Participants undergoing CC showed increased \ndifferential PDRs as compared to participants undergoing \nextinction. (B) Arousal and (C) valence ratings displayed \nseparately for part icipants assigned to the \ncounterconditioning (CC, solid bars) and extinction (EXT, \nopen bars) groups.  Participants that had undergone CC \ngave stronger differential arousal scorings than \nparticipants that had undergone extinction. In addition, \nparticipants that underwent CC showed flipped \ndifferential valence ratings: while valence differential \nvalence ratings were negative after extinction, the \ndirection reversed to positive differential ratings after CC. \nError bars represent ± standard error of the mean . \n*=p<0.05, **=p<0.01, ***=p<0.001, ≠ indicates that the \nbar is significantly different from 0. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n7 \n \nof both CC and extinction but were no longer detectable in the last two trials and were comparable 184 \nbetween groups (see Supplementary Information). 185 \nValence and arousal ratings provide further support for the extinction of differential responses in the 186 \nExt group and positive, reward -induced arousal for CS+ items in the CC group (Figure 2B-C). 187 \nDifferential valence ratings for the CS+ and CS - differed between groups after the CC/extinction task 188 \n(CS-type (CS+, CS-) x Group (CC, Ext) rmANOVA, CS-type x Group interaction: F(1,44)=12.054, p=0.001, 189 \nη²=0.215). Participants in the CC group rated CS+ stimuli more positive than CS - stimuli (t(21)=3.469, 190 \np=0.002, CS+: 7.5 ±0.30, CS -: 5.41±0.38) , while participants in the Ext group gave both categories 191 \nsimilar valence ratings (p=0.245, CS+: 5.63±0.32, CS -: 6.21±0.28). Differential arousal ratings for the 192 \nCS+ and CS- also differed between groups (CS-type (CS+, CS-) x Group (CC, Ext) rmANOVA, CS-type x 193 \ngroup interaction : (F( 1,44)=20.862, p<0.001, η²=0.322).  Participants in the CC group reported higher 194 \narousal levels for the CS+ category than for the CS - category (t(21)=6.370, p<0.001, CS+: 6.64 ±0.20, 195 \nCS-: 3.45±0.38) while participants in the Ext group gave similar arousal ratings for the CS+ and CS - 196 \ncategories (p=0.290, CS+: 4.21±0.43, CS -: 3.80±0.40).  Taken together, more positive valence and 197 \nhigher arousal ratings for the CS+ in the CC group as compared to the Ext group further support the 198 \ninterpretation of increased differential PDRs reflecting arousal induced by reward anticipation. 199 \nCC prevents differential spontaneous recovery 200 \nTo investigate whether CC prevented the spontaneous recovery of differential conditioned threat 201 \nresponses, we compared PDRs in the last two trials of the CC/extinction phase and the first two trials 202 \nof the spontaneous recovery test  in a CS -type (CS+, CS -) x Group (CC, Ext) x Phase (last two trials of 203 \nCC/extinction, first two trials of the spontaneous recovery test)  rmANOVA. We exp ected the E xt 204 \ngroup to show an increase in PDRs from the extinction task to the spontaneous recovery task, while 205 \nwe expected PDRs for the CC group  to remain stable or decrease . Critically, differential spontaneous 206 \nrecovery of PDRs differed between groups ( Group x CS-type x Phase interaction : F( 1,28)=6.329, 207 \np<0.018, η²=0.184,  Figure 3). While the CC group showed a decrease in differential PDRs from CC to 208 \nspontaneous recovery (t(14)= -1.807, p=0.046, one -tailed, CC: 0.34 ±0.2, spon taneous recovery : -209 \n0.01±0.18), the Ext group show ed an increase in differential PDRs (t(14)=1.850, p=0.043, one -tailed 210 \nsignificance, extinction: 0.11±0.01, spon taneous recovery : 0.04±0.02). To conclude , while we 211 \nobserved differential spontaneous recovery in the Ext group, we did not find evidence for differential 212 \nspontaneous recovery in the CC group , suggesting that CC attenuated the recovery of threat-213 \nresponses compared to regular extinction. 214 \nHowever, since participants undergoing CC showed persistent differential PDRs during the last two 215 \ntrials of the CC phase, while participants undergoi ng extinction did  not, we additionally explored 216 \nwhether there was differential responding during the first two trials of the spontaneous recovery  217 \ntest. During the first two trials of the spontaneous recovery test, participants in the CC group showed 218 \ndecreased differential PDRs as compared to the Ext group (CS-type (CS+, CS -) x Group (CC, Ext) 219 \nrmANOVA, CS-type x Group interaction:  F(1,29)=3.901, p=0.029, one -tailed, η²=0.119). Further 220 \nexploration within the groups confirmed that participants in the CC group did not show retention of 221 \ndifferential responses ( paired t-test, CS+ and CS - responses during the first two trials of the 222 \nspontaneous recovery test, p=0.219, one-tailed), while the Ext group did show increased responses 223 \nto the CS+ as compared to the CS - (t(14)=1.958, p=0. 035, one -tailed). Thus, both the differential 224 \nspontaneous recovery of PDRs between sessions, and differential responding within the firs t two 225 \ntrials of the spontaneous recovery test suggest ed that CC prevented spontaneous recovery of 226 \ndifferential responses compared to extinction.  SCRs did not show differential recovery and were 227 \ncomparable between groups (see Supplementary Information). 228 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n8 \n \n 229 \nFigure 3. Differential PDRs during the last two trials of extinction (grey) and the first two trials of the spontaneous 230 \nrecovery test (dark red). Differential PDRs show selective spontaneous recovery after extinction (Ext group , open bars) but 231 \nnot after CC (CC group , solid bars ). During the first two trials of the spontaneous recovery test , differential PDRs are 232 \nincreased in the Ext group as compared to the CC group. Insets show PDRs to the CS+  (red) and CS- (blue) during the last 233 \ntwo trials of extinction an d the first two trials of the spontaneous recovery test. While the Ext group shows differential 234 \nresponding during the spontaneous recovery test, the CC group does not. Error bars represent ± standard error of the 235 \nmean. *=p<0.05, #=p<0.05 one-tailed significance. 236 \nCC also appeared to have lasting beneficial effects on valence ratings compared to extinction. At the 237 \nstart of the second testing day, differential valence ratings continued to differ between groups (CS -238 \ntype (CS+, CS -) x Group (CC, Ext) rmANOVA, CS -type x Group interaction: F( 1,44)=5.160, p=0.028, 239 \nη²=0.105). While participants in the CC group gave similar valence ratings to both categories 240 \n(p=0.179, CS+: 6.3 ±0.34, CS -: 5.4±0.35), participants in the Ext group gave more negative  valence 241 \nratings t o the CS+ category than to the CS - category (t(23)= -1.964, p=0.031 one -tailed test, CS+: 242 \n5.5±0.30, CS-: 6.3±0.24), also illustrative of relapse of threat associations.  243 \nSurprisingly, while participants in the CC group showed heightened differential arousal ratings 244 \nimmediately after CC as compared to ratings from participants who had undergone extinction (Figure 245 \n2B), p articipants in both groups gave comparable differential arousal ratings  at the start of the 246 \nsecond day immediately before the spontaneous recovery test (CS-type (CS+, CS -) x Group (CC, Ext) 247 \nrmANOVA, main effect of CS -type: F( 1,44)=10.932, p=0.002, η²=0.022, CS+:  4.8±0.28, CS -: 3.9±0.24). 248 \nLikewise, response times to the CS+ and CS - during the first two trials of the spontaneous recovery 249 \ntask were similar across both groups (all p’s>0.2). These findings may suggest that differential arousal 250 \nevoked by the categories was similar in both groups immediately before and  during the spontaneous 251 \nrecovery test. 252 \nThe spontaneous recovery test was followed by a reinstatement procedure, consisting of three 253 \nunsignaled shocks, and a reinstatement test. However, mean PDRs decreased from spontaneous 254 \nrecovery to reinstatement (t(30)=3.063, p=0.005, last two trials of spontaneous recovery: 1.04 ±0.01, 255 \nfirst two trials of reinstatement: 1.01±0.01). Given that we did not observe successful reinstatement 256 \nin either group, our reinstatement test was not informative on whether CC can lead to a more 257 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n9 \n \npersistent attenuation of threat responses as compared to regular extinction. A full description of 258 \nPDR and SCR results of the reinstatement test can be found in the Supplementary Information. 259 \nDistinct CS-type specific activation for extinction and appetitive counterconditioning 260 \nAfter acquisition, the CC group underwent appetitive CC, while the Ext group underwent regular 261 \nextinction. Whole brain analysis revealed that over the course of this task, CS-type specific activation 262 \nchanged differe ntially between the two groups in  a large cluster encompassing multiple regions in  263 \nthe medial temporal lobe  (Group x CS -type x Phase interaction, cluster size = 1760 mm3, p=0.034, 264 \nwhole-brain FWE-corrected, Figure 4B and Table 1). We further investigated the anatomical location 265 \nof the cluster using our ROIs to probe for activity and fou nd that the effect encompassed the 266 \namygdala.  To further investigate the interaction effect in the amygdala, we extracted parameter 267 \nestimates from the complete bilateral amygdalae (Automated Anatomic Labelling, AAL, atlas in the 268 \nWFU PickAtlas toolbox in MN152 space) and performed post -hoc comparisons. In the early phase, 269 \nCS-type specific responses differed between the groups (t(1,44)=2.173, p=0.035, CC: 0.18±0.08, Ext: -270 \n0.073±0.08). Specifically, the CC group showed increased amygdala activation to the CS + as 271 \ncompared to the CS- (t(23)=2.210, p=0.037) while that was not the case in the Ext group (p=0.39 0). In 272 \nthe late phase, differential responses were comparable between the groups (p=0.503). 273 \nWhole-brain analysis further revealed a number of clusters showing distinct CS-specific activations 274 \nbetween groups throughout the task, including the anterior cingulate, cuneus, nucleus accumbens, 275 \ncaudate, thalamus and inferior frontal gyrus ( Figure 4A, Table 1 ). The group and stimulus -specific 276 \nactivation of the NAcc w as in line with a priori expectations for the CC phase  (Figure 4C). To further 277 \nexplore this effect, averaged parameter estimates from the bilateral NAcc ROI (mask acquired from 278 \nthe IBASPM 71 atlas in the WFU PickAtlas toolbox in MNI152 space) w ere extracted. Across the 279 \nbilateral NAcc, differential activation was increased in the CC as compared to the Ext group 280 \n(t(44)=2.731, p=0.009, CC: 0.37±0.10, Ext: 0.04±0.06) , with the CC showing increased NAcc activation 281 \nto the CS+ compared to the CS - (t(23)=6.194, p<0.001, CS+: 0.59±1.12, CS -: 0.16±0.09) wh ereas the 282 \nExt group did not (p=0.574).  283 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n10 \n \n 284 \nFigure 4. Stimulus-type specific activation differs between participants undergoing CC versus extinction.  A. Whole-brain 285 \nGroup x CS-type interaction effects revealed distinc t stimulus-specific activation of regions including the anterior cingulate, 286 \ncuneus, nucleus accumbens, caudate, thalamus and inferior frontal gyrus during the counterconditioning vs. extinction 287 \nphase. Panel A displays g roup F -images (see Table 1  for directions)  FWE-corrected at p<0.05, cluster -forming threshold 288 \np=0.001. B. The right amygdala showed a Group x CS-type x Phase interaction during the CC/extinction task, indicating that 289 \nCC compared to extinction is associated with decreased activation of the amygdala. C. The bilateral NAcc showed a Group x 290 \nCS-type interaction during the CC/extinction task, revealing increased NAcc activation in response to the CS+ compared to 291 \nthe CS - in the CC but not in the Ext group. Panel B and C display g roup F -images FWE -SVC at p<0.05, cluster -forming 292 \nthreshold p=0.001, along with post-hoc tests on mean parameter estimates from the complete ROI included in the analyses. 293 \n** p<0.01, * p<0.05, ≠ indicates that the value is significantly different from 0. 294 \n  295 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n11 \n \nTable 1. Whole-brain main effects of group (CC, Ext), CS type (CS+, CS-) and phase (early, late) and interactions, during 296 \nthe counterconditioning/extinction task. Cluster-forming threshold p=0.001, FWE-corrected at p<0.05, clusters were 297 \nlabelled using the Talairach Daemon atlas and the AAL atlas for ROIs. For each cluster, the peak voxel coordinates (MNI 298 \nspace) and regions are reported, and additional regions contained within the cluster are added in italics . See 299 \nSupplementary Table 1 for main effects of CS-type. 300 \nPeak MNI coordinate \nRegion Cluster x y z Size \n(mm3) \npFWE \n(cluster) \nPeak F-\nvalue \nDirection \n         \nGroup x CS-type x phase         \nParahippocampal Gyrus BA34R \nParahippocampal Gyrus Amygdala, \nUncus BA34R \n1 18 -8 -20 1760 0.034 23.40 CS+>CS- difference increases \nfrom early to late phase for \nCC, not for Ext \n         \nGroup x CS-type         \nLateral Geniculum Body LR, \nCaudate Head LR, Thalamus LR, \nLentiform Nucleus LR \n1 2 -26 -18 29920 <0.001 73.15 \n(CC CS+>CS-) > (Ext CS+>CS-) \n \nCuneus L \nLingual Gyrus BA17/BA18 LR, \nPosterior Cingulate LR, Cuneus \nBA18R, Cuneus BA30L Declive R  \n2 -6 -96 2 23272 <0.001 43.50 \nInferior Frontal Gyrus BA47L \nInsula BA13 L \n3 -36 18 -6 4504 0.009 30.62 \nExtra-Nucleus R 4 30 26 2 3136 0.016 37.67 \nSuperior Temporal Gyrus L \nSuperior Temporal Gyrus BA41 L, \nTransverse Temporal Gyrus L \n5 -60 -44 14 9088 0.002 43.56 \nTransverse Temporal Gyrus BA41 R \nSuperior Temporal Gyrus R, \nSuperior Temporal Gyrus \nBA42/BA22R \n6 44 -22 12 7784 0.003 42.17 \nAnterior Cingulate BA32R \nAnterior Cingulate BA32L, Cingulate \nGyrus R \n7 6 30 26 8880 0.002 27.90 \nPrecentral Gyrus L \nInferior Frontal Gyrus L \n8 -36 0 30 3624 0.014 30.10 \nPrecentral Gyrus R \nSub-Gyral R \n9 40 2 32 4056 0.011 40.64 \nPrecentral Gyrus BA6L \nMiddle Frontal Gyrus BA6L \n10 -44 -6 52 2184 0.028 24.34 (CC CS+>CS-) > (Ext CS+>CS-) \nAngular Gyrus R \nSupramarginal Gyrus R \n11 54 -60 36 1944 0.032 24.18 \n         \nGroup x Phase         \nNo significant clusters         \n         \nCS-type x Phase          \nNo significant clusters         \n         \nGroup          \nNo significant clusters         \n         \nPhase         \nInferior Frontal Gyrus R \nInferior Frontal Gyrus BA45 R \n1 30 26 8 4848 0.006 40.27 \nEarly Phase > Late Phase Insula L \nSuperior Temporal Gyrus BA22, \nPrecentral Gyrus L \n2 -28 26 0 4368 0.007 38.41 \nPostcentral Gyrus L 3 -54 -24 22 1768 0.031 23.75 \n 301 \nContrast estimates in further a priori defined ROIs during the CC/Ext task were submitted to a Group 302 \n(CC, Ext) x CS-type (CS+, CS-) x Phase (early, late) rmANOVA (Figure 5). The bilateral hippocampi (right 303 \nhippocampus cluster size: 664 mm 3, p=0. 001, FWE -SVC, left hippocampus cluster size: 112 mm 3, 304 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n12 \n \np=0.024, FWE-SVC) and the left vmPFC (mask defined as bilateral gyrus rectus and medial orbital gyri, 305 \ncluster size = 160 mm3, p=0.01 3, FWE-SVC) showed differentially changing CS -type-specific 306 \nactivations between the groups (Group x CS-type x Phase interaction). While CS+-specific suppression 307 \nof these regions appeared to increase during the CC task, this was not the case during the extinction 308 \ntask. Post-hoc comparisons on averaged  parameter estimates in the bilateral hippocampi confirmed 309 \nthat stimulus -specific suppression increased during  the course of the task in the CC group 310 \n(t(23)=3.280, p=0.003, early CS+ -CS-: 0.054 ±0.07, late: -0.150±0.07), but not in the Ext group 311 \n(p=0.266). Post -hoc comparisons across the vmPFC ROI also revealed increased CS+ -specific 312 \nsuppression in the CC group compared to the Ext group (t(44)=2. 221, p=0.032, CC: -0.189±0.06, Ext: -313 \n0.070±0.10). While the extinction group showed increased CS+ -specific activation from the early to 314 \nthe late phase of the extinction task (t(21)=2.235, p=0.036, early CS+: -0.149±0.08, late CS+: 315 \n0.040±0.09), the CC group did not (p=0.120). During the late phase , the CC group showed increased 316 \nvmPFC deactivation to the CS+ compared to the CS- (t(23)=3.174, p=0.004, late CS+: -0.284±0.06, late 317 \nCS-: -0.095±0.05), while the Ext group did not (p=0.503). Thus, across both the hippocampus and the 318 \nvmPFC, CC induced increased stimulus-specific suppression. 319 \n 320 \nFigure 5. ROI analyses during the CC/extinction task reveal distinct activity in the hippocampus and left vmPFC. During 321 \nthe CC/extinction task, stimulus-specific activation of the hippocampus (C) and left vmPFC ( D) changes differently between 322 \ngroups. ** p<0.01, * p<0.05, ≠ indicates that the bar is significantly different from 0. Group F-images FWE-SVC at p<0.05, 323 \ncluster-forming threshold p=0.001, along with post-hoc tests on mean parameter estimates from complete ROI included in 324 \nthe analyses. 325 \nDuring the spontaneous recovery task, a priori defined regions of interest did not reveal any effects 326 \n(see Supplementary Information). 327 \nCounterconditioning retroactively enhances item recognition for conditioned exemplars  328 \nFollowing the reinstatement test and re-extinction, participants completed a surprise item 329 \nrecognition test approximately 24 hours after acquisition an d the CC/extinction task. One outlier was 330 \nexcluded from this analysis (CS - false alarm rate = 0.91). Threat conditioning has previously been 331 \nshown to enhance 24-hour item recognition for category exemplars presented during the acquisition 332 \nphase27. However, th is enhancement for CS+ items did  not extend to items presented during an 333 \nextinction session separated from the acquisition phase by a short break 31. We therefore analysed 334 \nitem recognition for the CS+ and CS - during acquisition and the CC/extinction phase separately to 335 \nexamine whether the groups differed in recognition memory performance (Figure 6).  336 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n13 \n \n 337 \nFigure 6. Twenty-four hour recognition memory results.  During acquisition and extinction on the first day of the 338 \nexperiment, participants viewed trial-unique exemplars from two semantic categories (objects, animals) that served as CS+ 339 \nand CS-. The next day, participants completed a surprise memory test for these items, mixed with an equal number of novel 340 \nexemplars. Participants recognized relatively more items from the CS+ category, and participants that underwent CC 341 \nshowed improved item recognition comp ared to participants in the Ext group. Error bars represent ± standard error of the 342 \nmean. *=p<0.05. 343 \nCorrected recognition scores (hits probability-false alarms probability) were subjected to a task 344 \n(acquisition, CC/extinction task) x CS -type (CS+, CS -) x Group ( CC, Ext) rmANOVA , including CS+ -345 \ncategory (animals, tools) as covariate. Overall, participants showed better memory for items from 346 \nthe CS+  category (main effect of CS -type: (F( 1,42)=10.615, p=0.00 2, η²=0.2 02) and participants who 347 \nunderwent CC showed better memory as compared to participants who underwent extinction (main 348 \neffect of Group: (F( 1,42)=4.963, p=0.0 31, η²=0.1 06). Stimulus -type specific item recognition differed 349 \nbetween the CC and Ext groups (CS -type x Group interaction: F(1,42)=4.535, p=0.039, η²=0.094). While 350 \nparticipants in the CC group showed better recognition memory for the CS+ category compared to 351 \nthe CS- category (t(22)=2.531, p=0.019, means ± SD: CS+ 0.39±0.17, CS- 0.31±0.10), this was not  the 352 \ncase for participants in the Ext group ( t(23)=0.889, p=0.384, means ± SD: CS+ 0.30±0.13, CS - 353 \n0.28±0.11). Although the effect of stimulus -type was stronger for tools as CS+,  this was not different 354 \nbetween groups  (see Supplementary Information ). Thus, across the acquisition and CC/extinction 355 \nphase, participants who underwent CC showed a stronger enhancement of CS+ memory compared to 356 \nthe participants that underwent extinction. 357 \nTo further investigate to what extent CC retroactively affected memory for items presented during 358 \nthe acquisition task, we examined item recognition during acquisition and the CC/extinction tasks 359 \nseparately. While threat conditioning increased memory for CS+  items presented during the 360 \nacquisition task across both groups (main effect  CS-type: (F( 1,42)=18.147, p=<0.001, η²=0.30 2), 361 \nsubsequent CC enhanced this effect (Group x CS -type interaction: (F( 1,42)=5.112, p=0.029, η²=0.109). 362 \nPost-hoc tests reveal ed increased item memory for the CS+  category compared to the CS - category 363 \npresented during acquisition in the CC group (t(2 2)=2.341, p=0.029, means ± SD: CS+ 0. 40±0.21, CS- 364 \n0.31±0.12) but not in the Ext group ( t(23)=0.818, p=0. 422, means ± SD: CS+ 0.3 3±0.16, CS - 365 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n14 \n \n0.30±0.13). Again, although the effect of stimulus-type was stronger for tools as CS+, this was not 366 \ndifferent between groups (see Supplementary Information ). As the  acquisition task was identical 367 \nbetween groups, it appears that CC  in comparison to extinction  retroactively enhanced memory for 368 \nCS+ items. For items presented during the CC/extinction task, overall item recognition was better in 369 \nthe CC group compared to the Ext group (main effect group: F( 1,42)=8.706, p=0.005, η²=0.172, means 370 \n± SD: CC 0.35±0.12, Ext 0.26±0.09). Thus, compared to regular extinction, CC enhanced recognition of 371 \nitems presented during CC, but interestingly also strengthened the emotional memory enhancement 372 \nof CS+ exemplars presented during acquisition, suggesting that immediate CC may alter consolidation 373 \nof a prior threat learning episode. 374 \nFollowing previous work 29,31,32, we explored stimulus-type specific decreases in item recognition 375 \nbetween tasks, as well as within -phase differences between item recognition for the CS+ and CS - 376 \nwithin each group. As expected, a post-hoc paired samples t -test showed that participants in the Ext 377 \ngroup remember ed significantly more CS+ items from the acquisition phase as compared to the 378 \nextinction phase (t(2 3)=2.238, p=0.036, means ± SD: acquisition 0.33±0.16, extinction 0.27±0. 13). In 379 \ncontrast, p articipants who had undergone CC remembered CS+  items presented during acquisition 380 \nand CC equally well ( t(22)=0.390, p=0.701, means ± SD: acquisition 0.40±0.21, CC 0.38±0.16). Thus, 381 \nwhile recognition memory for items encoded during the extinction task was substantially weaker 382 \nthan memory for items from the acquisition task, this was not the cas e for items presented during  383 \nCC.  384 \nDiscussion 385 \nThis study aimed to test whether CC compared to regular extinction can lead to a more persistent 386 \nattenuation of threat responses, and to investigate whether this is mediated by neural mechanisms 387 \nreflecting extinction-related enhanced engagement of the vmPFC  or engagement of reward -focused 388 \nnetworks. We found that CC prevented differential spontaneous recovery of PDRs compared to 389 \nregular extinction, suggesting that CC reduces the recovery of threat responses.  Our fMRI results 390 \nsuggested that CC engages different neural mechanisms compared to extinction. Most notably,  while 391 \nthe extinction group showed an increase in CS+ -specific vmPFC activation during extinction, the CC 392 \ngroup showed CS+-specific deactivation of the vmPFC that persisted throughout the late phase of CC. 393 \nFurthermore, CC led to increased NAcc activation for the CS+ compared to the CS -, whereas this was 394 \nnot the case for  extinction. Lastly, phase - and stimulus -specific activation of the hippocampus and 395 \nthe amygdala differed between extinction and CC. Compared to extinction, CC led to increased  396 \nactivation of the amygdala in the early phase, and increasing stimulus -specific deactivation of the 397 \nhippocampus over the course of the early and late phases.  In addition, CC retroactively enhanced 398 \nitem recognition for conditioned exemplars presented during acquisition and strengthened memory 399 \nfor conditioned exemplars presented during CC compared to extinction. 400 \nThe mechanism underlying CC appears to be qualitatively different from the mechanism underlying 401 \nregular extinction. Regular extinction is associated with activation of the vmPFC 23,33, which is thought 402 \nto inhibit the expression of threat responses by suppressing amygdala activity 20-23. In comparison to 403 \nregular extinction, n ovelty facilitated extinction, a  form of enhanced extinction , in which  aversive 404 \nevents are replaced  with novel  neutral outcomes, has shown stronger CS+ -specific vmPFC 405 \nactivation24. If CC was similarly mediated by enhanced recruitment of extinction networks, we would 406 \nhave expected increased activation of the vmPFC, yet we observed a CS+-specific deactivation of the 407 \nvmPFC during CC, disproving this hypothesis. Interestingly, deactivation of the vmPFC during CC was 408 \nalso found in studies investigating a form of counterconditioning induced by means of real-time fMRI 409 \ndecoded neurofeedback34,35. During neurofeedback CC, participants implicitly learned to obtain 410 \nmonetary rewards by generating a representation of the target CS+ in the visua l cortex 34. After 411 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n15 \n \nneurofeedback CC, reductions in threat responses were stronger in participants showing stronger 412 \nvmPFC deactivation, suggesting that vmPFC disengagement may be associated with fear reductions34. 413 \nTaken together, both our findings and previous neurofeedback studies  suggest that in contrast to 414 \nenhanced extinction, CC disengages the vmPFC.  Given that we replicate this finding using a different 415 \napproach that includes direct exposure to the CS+, vmPFC disengagement may be a distinguishing 416 \ncharacteristic of CC. The observed pattern of  activity, including vmPFC deactivation  further bears 417 \nresemblance to activity patterns  observed during goal-directed eye movements in  an experimental 418 \nmodel of eye -movement desensitization and reprocessing (EMDR) , which has also been shown to 419 \nimprove extinction learning 36. A similar activity pattern and effect has also been found for working 420 \nmemory-like tasks, such as a game of Tetris 37-39. Given that the above-mentioned tasks associated 421 \nwith vmPFC deactivation  share their  strong engage ment of  working memory and/or endogenous 422 \nattention mechanisms, th ereby engaging the executive control -network, deactivation of the vmPFC 423 \nand hippocampus could be the result of a deactivated default mode network due to competition 424 \nbetween activation of large scale brain networks40-42. 425 \nThe CC procedure led to clear CS+-specific activation of the NAcc, which is in line with expectations 426 \nfor reward anticipation in tasks with a monetary incentive delay aspect 43. Activation of the ventral 427 \nstriatum has also been reported for active avoidance, and may be generally associated with 428 \ninstrumental actions as opposed to passive delivery of an outcome 44,45. In line with studies on active 429 \navoidance, delivery of a reward contingent on instrumental action s has been shown to yield CC  that 430 \nis more resistant to renewal 46. CS+-specific activation of the NAcc was not seen in participants 431 \nundergoing extinction, suggesting that this activation is specific to CC. However, p revious work in 432 \nrodents revealed a n amygdala-ventral striatum (NAcc) pathway that  is activated during extinction 433 \ntraining17. The recruitment of this pathway was shown to be enhanced during CC, and reduced the 434 \nreturn of fear 17, suggesting that CC may in fact enhance activation of reward-related networks that 435 \nare weakly activated by extinction. Indeed, fMRI studies in humans that modelled prediction error for 436 \nomitted aversive outcomes during extinction training (i.e. outcomes “better-than-expected”) showed 437 \ninvolvement of the NAcc 47-49. Possibly, activation of the NAcc during extinction is limited to early 438 \nextinction trials generat ing prediction error s. Nevertheless, b ased on our findings, it appears that 439 \nsustained CS+ -specific activation o f the NAcc is a distinct mechanism underlying CC but not 440 \nextinction, which is potentially associated with instrumental actions. 441 \nA recent neuroimaging study suggests that the neural differences between regular extinction and CC 442 \nmay be maintained over time 19. In their within-subject study, two CS+ categories (animals, objects) 443 \nwere used during threat conditioning. Subsequently one of the CS+ categories was used for regular 444 \nextinction, whereas the other was used for CC. During CC, CS+ exemplars were  paired with positively 445 \nvalenced picture s. During a spontaneous recovery task the following day, it was shown that 446 \ninvolvement of the vmPFC (amygdala -vmPFC functional connectivity) was stronger for regular 447 \nextinction compared to CC. In contrast, CS+-specific increases in functional connectivity between the 448 \namygdala and the ventral striatum (NAcc) were only observed in the CC condition during a 449 \nspontaneous recovery task . Both findings are in line with  the CC-associated vmPFC deactivation and 450 \nNAcc activation that we observed  and suggest that differences in the neural mechanisms of regular 451 \nextinction and CC may be maintained during threat retrieval.  452 \nCC compared to regular extinction also strengthened item memory for the conditioned category. 453 \nWhile both reward and threat conditioning can enhance item recognition for the CS+ category27,50, 454 \nrecognition of CS+ exemplars presented during extinction  was shown to drop compared to 455 \nacquisition31. In contrast to extinction, within-session CC was previously shown to enhance memory, 456 \nsuggesting that CC has a unique, strengthening effect on memory29. In the current study, we replicate 457 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n16 \n \nthis finding, showing strengthened memory after CC compared to extinction. While enhanced 458 \nrecognition of items presented during CC could be mediated by attentional prioritization 51, CC also 459 \nretrospectively strengthened memory for items presented during acquisition, suggesting that CC may 460 \nalter the consolidation of a prior threat conditioning episode. Retroactive enhancement of memory 461 \nconsolidation for related items has previously been shown for conceptually related neutral items 462 \npresented prior to threat conditioning 32 and reward conditioning 50. At a neurobiological level, these 463 \nfindings have been related to the synaptic tagging-and-capture hypothesis postulating that memories 464 \nfor neutral events can be strengthened if they are followed by salient events, due to an initially short-465 \nlived synaptic “tag” that allows later events to stabilize the memory 32,52,53. At a systems level , 466 \nretroactive memory strengthening has been linked to reverse replay 54. Specifically, animal research 467 \nindicates that reward s increase reverse replay 55-57, and reward-induced reverse replay occurs 468 \nconcurrently with firing of midbrain dopamine neurons 58. Interestingly, spontaneous replay is also 469 \ninvolved in regular extinction, in which unexpected omission of the US drives spontaneous 470 \nreactivation of activity patterns in the vmPFC . This spontaneous reactivatio n was shown to be 471 \npredictive of extinction recall and c ould be amplified through pharmacological enhancement of 472 \ndopaminergic activity 59. Yet while physiological dopaminergic modulation during extinction may be 473 \nlimited to prediction error signals during the early phase 47-49, dopaminergic modulation may be 474 \nsustained throughout the MID -based CC task applied in this study. While we did not measure 475 \ndopaminergic activity  directly, activation of the N Acc during reward anticipation is predictive of 476 \ndopamine release within the NAcc60-63. Given the increased stimulus-specific activation of the NAcc in 477 \nthe CC group, it is likely that dopaminergic activity was enhanced during CC compared to regular 478 \nextinction. The enhanced dopaminergic modulation could strengthen memories through replay 55,64, 479 \nor may increase synaptic plasticity directly, potentially explaining enhanced item recognition after CC 480 \ncompared to regular extinction 54,65,66. In line with these findings, research in humans shows that 481 \nreward systematically modulates memory for neutral objects in a retroactive manner, with objects 482 \nclosest to the reward being prioritized54. It could be that reward-conditioning during CC similarly 483 \ndrives reward -driven reverse replay, enhancing episodic memory for  conceptually related  items 484 \npresented during the preceding acquisition task. 485 \nSeveral limitations of the current study are worth considering. First, while the monetary incentive 486 \naspect during CC clearly induced positive valence, it also increased physiological arousal, making it 487 \ndifficult to isolate the individual effects of positiv e valence and reward-induced arousal. While the 488 \ncurrent results are in line with previous work in CC using low -arousal, positive-valence pictures29, we 489 \ncannot exclude the possibility that the current findings (in part) reflect differences in task 490 \nengagement between participants  due to active instead of passive reward delivery . However, it is 491 \nquestionable whether it is  meaningful to tease individual effects of valence and arousal apart since 492 \narousal may facilitate reward processing. Indeed, striatal responses to obtained monetary rewards 493 \nare dependent on salience and are increased when rewards are dependent on active re sponses 494 \ncompared to passive delivery 67. Second, although we included a reinstatement procedure in the 495 \nexperiment, neither the Ext nor the CC group showed differential reinstatement. It is worth noting 496 \nhowever that reinstatement paradigms in humans may not reliably produce differential 497 \nreinstatement after extinction 68. Third, it is important to note that CC/extinction was carried out 498 \nwithin minutes after  the acquisition phase, and the effects of CC and extinction may differ when 499 \ncarried out after the acqui sition memory has been consolidated  69-72. Fourth, whole-brain analysis of 500 \nthe CS -specific activation  during the spontaneous recovery test  in the Ext group did not yield any 501 \nclusters above threshold , while  physiological results indicated spontaneous recovery of differential 502 \nthreat responses. Given that recovered threat responses are often quick to extinguish  and fMRI 503 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n17 \n \nanalyses require averaging across multiple trials to achieve sufficient signal- to-noise ratio, threat-504 \nevoked neural activity may have been too brief to be detected.  505 \nIn conclusion, our findings show that appetitive CC improves the retention of safety memory over 506 \nstandard extinction. Strikingly, in cont rast to activation of the vmPFC during extinction, CC  was 507 \nassociated with stimulus-specific deactivation of the vmPFC. These findings may inform development 508 \nof future treatments for fear - and anxiety disorders. While  a large body of research focuses on 509 \nenhancing regular extinction,  this study i ndicates that  another promising and potentially longer -510 \nlasting approach may be to engage reward -circuits. Although further work is needed, a  major 511 \nadvantage of CC -based interventions over extinction -based interventions may be that CC could be 512 \nmore tolerable as it may shift attention away from the experience of fear. 513 \nMaterials and Methods 514 \nParticipants 515 \nForty-eight healthy right-handed volunteers (15 males, 33 females; age [22.71±0.44]) with no 516 \nneurological or psychiatric history, and with normal  hearing and normal or corrected -to-normal 517 \nvision completed the study. Exclusion criteria were pregnancy, disorders of the autonomic system, 518 \nheart conditions, recreational drug use and any contraindications for MRI. Participants provided 519 \nwritten informed consent and were paid 55 euros for their participation. Participants in the CC group 520 \nwere able to earn an additional 14 euros. This study was approved by the local ethical review board 521 \n(METC Oost -Nederland and CMO Radboudumc). Participants were excluded from  the threat 522 \nacquisition, CC /Extinction, spontaneous recovery, and reinstatement analyses if there was no 523 \nevidence for successful threat acquisition  (mean CS->CS+ or CS+=CS-). For SCRs this was the case for 524 \nthree participants, for PDR this was the case for two participants. Additional participants were 525 \nexcluded in case of missing data due to technical failure. 526 \nDesign and procedure 527 \nThis study was a two-day between-subjects experiment carried out in the fMRI scanner (see Figure 1 528 \nfor an overview of the design). Participants were assigned to either the CC or Ext group according to 529 \na predetermined allocation sequence. At the start of each session, two  Ag/AgCl electrodes were 530 \nattached to the medial phalanges of the second and third d igit of the left hand, a pulse oximeter was 531 \nattached to the first digit of the left hand to measure finger pulse  and a respiration belt was placed 532 \naround the abdomen to measure respiration. All measures were taken using a BrainAmp MR system 533 \nand recorded us ing the BrainVision Recorder software (Brain Products GmbH, Munich, Germany). 534 \nThe first day consisted of individual adjustment of the electrical shock followed by a single fMRI 535 \nsession that included the following tasks: an object localizer task ( 17 min), a  category threat 536 \nconditioning task ( 23 min) and a CC or extinction task ( 23 min). The second session took place the 537 \nfollowing day and consisted of three runs: the spontaneous recovery and reinstatement test (12 538 \nmin), item recognition test (29 min) and the valence-specific response characterization task (17 min). 539 \nPavlovian conditioning paradigm  540 \nNote that CC included an instrumental and not Pavlovian conditioning procedure. This was done 541 \nbecause of pragmatic constraints in studies with humans. For example, w e cannot food deprive 542 \nhumans to make an appetitive reward truly reinforcing and make participants anticipate the reward. 543 \nPrevious work 50,67 and our pilot studies indicated that to maximize reward anticipation and evoke 544 \nconditioned responses, the reward conditioning needed to be instrumental. 545 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n18 \n \nThe acquisition, counterconditioning, extinction, spontaneous recovery and reinstatement tasks 546 \nconsisted of a categorical differential delay threat conditioning paradigm 27 with elements of the 547 \nmonetary incentive delay task 30. Participants viewed trial-unique exemplars of pictures from two 548 \ncategories (animals or objects, see Figure 1). In a counter -balanced manner, exemplars from one 549 \ncategory served as CS+ (reinforced) stimuli, while exemplars form the other category served as CS - 550 \n(unreinforced stimuli). Each trial start ed with the presentation of a stimulus. After a variable delay of 551 \n2.5-4s, a cue appeared to which participants were instructed to respond as quickly as possible with a 552 \nbutton press. After the button press, or when a 1s response window ha d elapsed, the colour of the 553 \ncue shift ed from black to blue. 0.5 -1.5s after the response window elapsed, CS+ items presented 554 \nduring the acquisition phase could be reinforced with a shock. During the acquisition phase, 50% of 555 \nthe CS+ pictures w ere followed by a shock. After 1s, the stimulus was replaced by neutral feedback 556 \nduring the acquisition, extinction, and recovery tasks. During the CC phase, neutral feedback was 557 \nreplaced by monetary feedback. During the CC phase, participants could obtain a €0.50 reward for 558 \ntheir quickest responses to the cues presented on top of CS+ stimuli. The response time target was 559 \ndynamically adjusted to achieve a reward reinforcement rate of approximately 70%. Reward was 560 \nwithheld during the first three CS+ trials during the CC phase to make the tran sition from the 561 \nacquisition to the CC phase more gradual. The inter-trial interval (ITI) varied randomly between 8 and 562 \n10s. Pictures were presented in a pseudorandom order with no more than  3 consecutive 563 \npresentations of items from the same category and CC blocks consisted of 40  CS+ and 40 CS - 564 \npresentations each. The spontaneous recovery block consisted of 15  CS+ and 15  CS+ presentations, 565 \nand the reinstatement test consisted of 5 CS+ and 5 CS- presentations. 566 \nItem recognition memory test 567 \nParticipants carried out a surprise recognition memory test compromised of 160 pictures (80 CS+, 80 568 \nCS-) shown during the acquisition and CC/extinction phases , as well as 160 category-matched new 569 \nitems (80 CS+, 80 CS-). Participants rated on a 6 -point scale whether the picture was ‘definitely old’, 570 \n‘probably old’, ‘maybe old’, ‘maybe new’, ‘probably new’, ‘definitely new’.  571 \nValence-specific response characterization 572 \nThe valence-specific response characterization task consist ed of an adapted version of the 573 \nconditioning paradigm used during the acquisition phase. Instead of category items, participants 574 \nwere presented with squares in three different colours. One of the stimuli was reinforced with 575 \nshocks (CS+ -shock, 50% reinforceme nt rate), one stimulus was reinforced with monetary rewards 576 \n(CS+-reward, approximately 70% reinforcement rate, response time target adjusted dynamically) and 577 \nthe last stimulus was not reinforced (CS-). Each stimulus was presented 40 times in a pseudorandom  578 \norder with no more than three repetition s of each stimulus. Colours and reinforcement (shocks vs. 579 \nrewards) were counterbalanced across participants. 580 \nPeripheral stimulation 581 \nElectrical shocks were deliver ed using two Ag/AgCl electrodes attached to the medial phalanges of 582 \nthe second and third digit of the right hand using a MAXTENS 2000 (Bio -Protech) device. Shock 583 \nintensity varied in 10 intensity steps between 0 to 40 V and 0 to 80 mA. Shock duration was 200 ms. 584 \nIn line with prior threat conditioning protocol s, s hock intensity was calibrated using an ascending 585 \nstaircase procedure starting with a low voltage setting near a perceptible threshold and increasing to 586 \na level deemed “maximally uncomfortable but not painful” by the participant32,73,74. 587 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n19 \n \nArousal and valence ratings 588 \nArousal and valence ratings were acquired using self-assessment manikin scales. The arousal scale 589 \nranged from 1 (=extremely calm) to 10 ( =extremely excited). The valence scale ranged from 1  590 \n(=extremely negative) to 10 (=extremely positive).  The valence and arousal ratings were collected for 591 \nthe two categories (animals and tools) after the acquisition phase, after the CC/extinction phase, at  592 \nthe start of day 2 immediately before the spontaneous recovery test and after the reinstatement 593 \ntest. For the stimuli used in the valence-specific response characterization task , valence and arousal 594 \nratings were collected immediately after the task.  595 \nSCR pre-processing and analysis 596 \nElectrodermal activity data were pre-processed using in-house software; radio frequency (RF) 597 \nartefacts were removed and a low-pass filter was applied75,76. Skin conductance responses (SCR) were 598 \nautomatically scored with additional, blinded, manual supervision using Autonomate 77. SCR 599 \namplitudes (measured in μSiem) were determined for each trial as the maximum response with an 600 \nonset between 0.5 and 7.5s after stimulus onset and maximum rise time of 14.5s. Shock- and reward- 601 \nreinforced trials were excluded from analysis. All response amplitudes  were square-root transformed 602 \nand normalized according to each participant’s mean UCS response prior to statistical analysis.  The 603 \naverage SCRs were computed per CS-type, task, phase (early, late), and participant. 604 \nPDR pre-processing and analysis 605 \nPupil dilation was measured with a MR-compatible eye-tracker from SensoMotoric Instrument (MEye 606 \nTrack-LR camera unit, SMI, SensoMotoric Instruments) and sampled at a rate of 50 Hz. Data were 607 \nanalysed using in -house software 78 implemented in Matlab R2018b (MathWorks), based on 608 \npreviously described methods 79. Eyeblink artifact s were identified and linearly interpolated 100 ms 609 \nbefore and 100 ms after each identified blink. Data from scan runs missing 50% time points or more 610 \nwere excluded. After interpolating missing values, time series were band-pass filtered at 0.05 to 5 Hz 611 \n(by subtracting the mean and dividing by the standard deviation) within each participant and run to 612 \naccount for between -subjects variance in overall pupil size. Event -related pupil diameter responses 613 \nwere calculated by averaging pupil diameter during 3.5 to 7  sec period after stimulus onset, divided 614 \nby the 1 sec pre-stimulus pupil diameter (-1 to 0 sec). The average PDR s were computed per CS-type, 615 \ntask, phase (early, late), and participant. 616 \nMRI data acquisition 617 \nMRI scans were acquired using a Siemens (Erlangen, Germany) 3T MAGNETOM PrismaFit MR scanner 618 \nequipped with 32 -channel transmit -receiver head coil. The manufacturer’s automatic 3D -shimming 619 \nprocedure was performed at the beginning of each experiment. Participants were placed in a light 620 \nhead restraint withi n the scanner to limit head movements during acquisition. Functional images 621 \nwere acquired with multi -band multi-echo gradient echo -planar (EPI) sequence [5 1 oblique 622 \ntransverse slices; slice thickness, 2.5mm; TR, 1.5s; flip angle, 75°; echo times, 13.4, 34.8, and 56.2 ms; 623 \nFOV, 210 x 210 mm2 ; matrix size 84x84x64, fat suppression ]. To account for regional variation in 624 \nsusceptibility-induced signal drop out, voxel -wise weighted sums of all echoes were calculated based 625 \non local contrast -to-noise ratio after which echo-series are integrated using PAID weighting 80. Field 626 \nmaps were acquired (51 oblique transverse slices; slice thickness, 2.5mm; TR, 0.49 s; TE, 4.92 ms and 627 \n7.48 ms; flip angle, 60°; FOV, 210 x 210 mm2; matrix size 84x84x64 ) at the start of each session to 628 \nallow for correction of distortions due to magnetic field in homogeneity. A high-resolution structural 629 \nimage (1mm isotropic) was acquired using a T1 -weighted 3D magnetizatio n-prepared rapid gradient 630 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n20 \n \necho sequence [MP-RAGE; TR, 2300 ms; TE, 3.03 ms; flip angle, 8°; 192 contiguous 1 mm slices; FOV = 631 \n256 x 256 mm2]. 632 \nfMRI analysis 633 \nAnatomical and functional data were pre-processed using fMRIPrep 20.0.6 81. The complete 634 \nboilerplate can be found in Supplementary Information 1. In brief, MRI data were pr e-processed in 635 \nstandard stereotactic (MNI152) space. Pulse and respiration data were processed offline using in -636 \nhouse software and visually inspected to remove artefacts and correct peak detection , and corrected 637 \npulse and respiration data were used for retrospective image -based correction (RETROICORplus) of 638 \nphysiological noise artefacts in BOLD -fMRI data 82. Identical transformations were applied to all 639 \nfunctional images, which were resliced into 2 mm isotropic voxels. After pre -processing in fMRIPrep, 640 \nfunctional images were smoothed with a 6 mm FWHM Gaussian kernel (using SPM12; 641 \nhttp://www.fil.ion.ucl.ac.uk/spm; Wellcome Department of Imaging Neuroscience, London, UK).  642 \nFor the acquisition, extinction/cc and spontaneous recovery phases, BOLD responses to CS+, and CS- 643 \nduring the early phase (first half of the trials) and late phase (second half of the trials) were modelled 644 \nin 4 separate regressors using box -car functions. Additionally, during all these phases, target 645 \npresentation, button pres s and shocks were modelled using stick functions, and feedback 646 \npresentation and breaks were modelled using box-car functions and included as nuisance regressors. 647 \nFor the category localizer, BOLD responses to animals, objects, and phase-scrambled blocks were 648 \nmodelled in 3 separate regressors using box functions.  All first-level models also included  six 649 \nmovement parameter regressors (3 translations, 3 rotations) derived from rigid -body motion 650 \ncorrection, 2 5 RETROICOR physiolog ical noise regressors , high-pass filtering (1/128 Hz cut -off), and 651 \nAR(1) serial correlations correction. First-level contrast s were calculated for early and late CS+ and 652 \nCS- separately for the acquisition, CC/extinction, and spontaneous recovery phases.  653 \nFor the acquisition and CC/extinction, first-level contrast were entered into a second-level Group 654 \n(extinction, cc) x CS -type ( CS+, CS -) x Phase (early, late) mixed factorial model using the Multilevel 655 \nand Repeated Measures (MRM) toolbox 83. For the spontaneous recovery test, BOLD-responses from 656 \nthe early phase were entered into a second -level Group (extinction, cc) x CS -type (CS+, CS -) mixed 657 \nfactorial model. Thresholding was achieved using nonparametric permutation testing (5,000 658 \niterations), with a cluster -setting threshold of p <.001 for whole -brain analysis and familywise error 659 \n(FWE) correction at p<0.05 at cluster -level for whole -brain analysis and voxel -level for ROI -analysis 660 \n(Amygdala, Hippocampus, vmPFC, NAcc). Activations are displayed on the single -subject high-661 \nresolution T1 volume provided by the Montreal Neurological Institute (MNI). 662 \nRegion of interest definition  663 \nBased on a priori hypotheses, results for the amygdala, NAcc, hippocampus and the ventromedial 664 \nprefrontal cortex are corrected for reduced search volumes using small  volume. Masks were created 665 \nusing the WFU PickAtlas toolbox84 in combination with the Automated Anatomical Labeling atlas 85 for 666 \nthe bilateral amygdala , bilateral hippocampus and vmPFC ( Frontal_Med_orb_L&R and Rectus L&R ). 667 \nThe IBASPM 71 anatomical atlas toolbox86 as used to create a mask for the bilateral NAcc.  668 \nStatistical testing 669 \nStatistical analyses of behavioural and physiological variables were performed in SPSS (IBM SPSS 670 \nStatistics Inc.). Dependent measures were submitted to repeated measure ANOVAs and statistics 671 \nwere Greenhouse -Geisser or Huyn -Feldt corrected for non -sphericity when appropriate. Significant 672 \nfindings from ANOVAs were followed -up by paired - and independent samples t -tests. We report 673 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n21 \n \npartial eta-square as measure of effect size. Means ± s.e.m are provided where relevant unless 674 \notherwise indicated. 675 \nDeviations from the pre-registration 676 \nThe preregistration for this project can be found on OSF (https://osf.io/fbz6n). We pre-registered to 677 \nsample SCRS in a 0.75 and 3.15 s window after stimulus onset. However, visual inspection of SCR 678 \nresponses during the acquisition phase indicated that response latencies shifted towards the late 679 \nphase of the trial. We therefore opted to use a longer window (0.5s to 7.5s for stimulus onset) and 680 \nexclude reinforced trials. The pre -registration erroneously stated that pupil -dilation data would be z -681 \nscored and later divided by the pre -stimulus average. PDR data were not z -scored but  were only 682 \nnormalized to a 1-sec pre-stimulus baseline. In line with the SCR data, response onset latencies were 683 \nlater than expected. Based on visual inspection of the data from the acquisition phase,  we decided to 684 \nuse a window around the expected shock onset: 3.5 -7s after stimulus onset. Reinforced trials were 685 \nexcluded. Results for SCR, retrospective reinforcement estimations and the reinstatement test can be 686 \nfound in the Supplementary Information. Due to an error in the scripts for the item recognition test, 687 \ntrial-by-trial data w ere not recorded for the first 12 participants. Therefore, analysis of the me mory 688 \ndata focused on averaged data for the early and late phase of acquisition and CC/extinction, leaving 689 \nout planned change point analyses on bins of 4 trials.  690 \nWhile we planned to extract a vmPFC mask for ROI analysis based on a [CS- > CS+ shock] contrast of 691 \nBOLD responses during the valence -specific response characterization task to identify “extinction 692 \nregions”, this did not yield ventromedial prefrontal clusters that survived correction. Instead, in line 693 \nwith our other ROIs, we opted to create a mask based on the AAL atlas. 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A. & Burdette, J. H. An automated method for 930 \nneuroanatomic and cytoarchitectonic atlas -based interrogation of fMRI data sets. 931 \nNeuroImage 19, 1233-1239, doi:10.1016/S1053-8119(03)00169-1 (2003). 932 \n85 Tzourio-Mazoyer, N.  et al.  Automated anatomical labeling of activations in SPM using a 933 \nmacroscopic anatom ical parcellation of the MNI MRI single -subject brain. NeuroImage 15, 934 \n273-289, doi:10.1006/nimg.2001.0978 (2002). 935 \n86 Alemán-Gómez, Y., Melie-Garcia, L. & Valdés-Hernández, P. A. in Proceedings of the 12th 936 \nAnnual Meeting of the Organization for Human Brain Mapping    (2006). 937 \n Acknowledgement 938 \nThis work was supported by the European Research Council (ERC-2015-CoG 682591). 939 \nAuthor contributions 940 \nM.C.H., J.E.D., J.H., M.J.A.G.H. and E.H. designed the study. M.C.H. and J.V. implemented and 941 \nconducted the experiment. M.C.H., L.W. and J.V. analysed the results with support of E.H.. L.W. and 942 \nM.C.H. wrote the paper with contributions from all authors. 943 \nCompeting interests 944 \nThe authors declare no competing interests. 945 \n  946 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n27 \n \nSupplementary Information 947 \nValence-specific response characterization 948 \nAt the end of the experiment, participants underwent a simplified version of the main experimental 949 \ntask, in which category exemplars were replaced by colored squares. This task was used to 950 \ninvestigate to what extent sin conductance  responses (SCRs) and pupil dilation responses (PDRs) can 951 \nbe used to disentangle anticipation of shock and reward. Participants viewed three different 952 \ncoloured squares and learned that one colour was associated with shocks (CS+S), one colour with 953 \nrewards ( CS+R) and one colour served as CS -. The trial structure was otherwise identical to 954 \ncomparable trials from the acquisition and CC phases. At the end of the task, participants were asked 955 \nto rate the three stimuli on valence and arousal self -assessment maniki n scales (Bradley & Lang, 956 \n1994). 957 \nDuring this valence-specific response characterization task, we observed habituation in SCRs over the 958 \ncourse of the task (CS -type (CS+ S, CS+ R, CS -) x Phase (early, late) x Group (CC, Ext) rmANOVA, main 959 \neffect phase: F( 1,38)=13.921, p=0.001, η²=0.268) and different SCR magnitudes for the three different 960 \nCS-types (main effect CS -type (CS+S, CS+R, CS -): F( 2,76)=78.460, p<0.001, η²=0.674). In addition, 961 \nhabituation depended on CS -type (CS -type x Phase interaction: F( 2,76)=6.825, p=0.002, η²=0.152). 962 \nDuring the early phase, SCRs in response to the CS+R and the CS - were not distinguishable 963 \n(t(40)=0.115, p=0.909, CS+R: 0.32 ±0.03, CS -:0.32 ±0.03), while during the late phase, SCRs to the 964 \nCS+R were larger than the CS - (t(40)=4.993, p< 0.001, CS+R: 0.29±0.03, CS -:0.19±0.02). SCRs to the 965 \nCS+S were consistently larger than SCRs to the CS+R (early: t(41)=9.345, CS+S: 0.62±0.04, p<0.001, 966 \nlate: t(40)=5.952, p<0.001, CS+S: 0.56±0.04) and the CS - (early: t(40)=10.020, p<0.001, late: 967 \nt(4)=10.122, p<0.001). Thus, anticipation of aversive reinforcement (CS+S) led to increased SCRs 968 \ncompared to anticipation of reward (CS+R) and CS - presentation throughout the task. Due to the fact 969 \nthat SCRs performed less well in differentiation between CS+R and CS -, we focused our analyses on 970 \nPDRs, but report SCR results here as well. 971 \nWe also observed CS-type dependent differences in PDRs (CS-type (CS+ S, CS+ R, CS-) x Phase (early, 972 \nlate) x Group (CC, Ext) rmANOVA, main effect CS -type (CS+S, CS+R, CS -): F( 2,68=19.783, p<0.001, 973 \nη²=0.368). In comparison to the neutral CS -, both the shock-reinforced CS+ (CS+S) and reward -974 \nreinforced CS+ (CS+R)  evoked larger PDRs (Supplementary Figure 1A, t(36)=7.071, p<0.001 and 975 \nt(26)=4.900, p<0.001 respectively, CS+S: 1.05 ±0.03, CS+R: 1.04 ±0.04, CS -: 1.01±0.02). However, 976 \nreward- and shock anticipation-induced PDRs did not differ statistically (t(36)=1.146, p=0.259). While 977 \nboth shock anticipation and reward anticipation led to similar increases in  PDRs as compared to the 978 \nneutral con dition, valence and arousal ratings indicated that participants experienced shock and 979 \nreward trials differently . Specifically, the CS+R was rated more positive than the CS - (t(47)=9.046, 980 \np<0.001, CS+R: 7.79±0.14, CS-: 5.96±0.16, Supplementary Supplementary Figure 1C), while the CS+S 981 \nwas rated less positive than the CS - (t(47)=-10.337, p<0.001, CS+S: 2.96±0.25). Participants reported 982 \nincreased arousal to both the CS+S and CS+R as compared to the CS - (t(47)=4.666, p<0.001 and 983 \nt(47)=8.897, p<0.001 respectively, CS+S: 5.42±0.35, CS+R: 6.31±0.21, CS -: 3.33±0.30, Supplementary 984 \nFigure 1B). While  it was not possible to distinguish  PDRs to the CS+S and CS+R, explicit ratings of 985 \narousal were marginally increased for the CS+R as compared to the CS+S (t(47)= -2.100, p=0.041). In 986 \nconclusion, the response characterization task showed that while anticipa tion of reward and shock 987 \nboth generate increased PDRs as compared to the CS -, distinct retrospective valence ratings show 988 \nthe expected directions. 989 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n28 \n \nThreat acquisition 990 \nPhysiological and behavioural evidence for acquisition of conditioned threat responses 991 \nParticipants pre-assigned to the CC and Ext groups underwent an identical threat acquisition 992 \nprocedure. To verify that participants pre -assigned to both groups acquired conditioned threat  993 \nresponses of comparable strength, we compared PDRs, explicit valence and arousal ratings , and 994 \nresponse times between groups. During the acquisition task, participants pre -assigned to both 995 \ngroups showed stable and comparab le differential conditioned threat responses as measured by 996 \nPDRs (Supplementary Supplementary Figure A, CS-type (CS+, CS-) x Phase (Early, Late) x Group (CC, 997 \nExt) rmANOVA, main effect CS -type: F( 1,37)=41.172, p<0.001, η²=0.533, other main effects and 998 \ninteractions: all p’s>0.2). Both groups also acquired comparable differential SCRs (main effect CS -999 \ntype: F( 1,42)=58.633, p<0.001, η²=0.583 ), although SCRs showed habit uation over the course of the 1000 \ntask (main effect phase: F(1,42)=66.907, p<0.001, η²=0.614, all other p’s>0.3 ). Thus, both SCRs and 1001 \nPDRs demonstrated comparable acquisition of conditioned threat responses between groups. 1002 \nSuccessful threat acquisition was further confirmed by valence and arousal ratings for the CS+ and 1003 \nCS- categories at the end of the acquisition task. Arousal ratings for the CS+ category exceeded 1004 \narousal ratings for the CS - category (Supplementary  Supplementary Figure B, CS-type (CS+, CS-) x 1005 \nGroup (CC, Ext) rmANOVA, main effect CS -type: F(1,44)=27.573, p<0.001, η²=0.385), and did not differ 1006 \nbetween groups (all p’s>0.2). Similarly, the CS+ category was given lower valence  (less positive)  1007 \nratings than the CS - category (Supplementary Supplementary Figure C, CS-type (CS+, CS-) x Group 1008 \n(CC, Ext) rmANOVA , main effect CS -type: F( 1,44)=12.626, p<0.001, η²=0.223). Although there was no 1009 \nmain effect of group on valence ratings (p>0.7), the effect of CS -category unexpectedly differed 1010 \nbetween the CC and Ext group (CS -type x Group interaction: F( 1,44)=4.512, p=0.039, η²=0.093), due to 1011 \nmore positive ratings to the CS - category in the Ext group (CC: 5.8±0.4, Ext: 6.9±0.3, t(44)=2.156, 1012 \np=0.037). Nevertheless, v alence ratings for the CS+ category were comparable between groups (CC: 1013 \n5.1±0.5, Ext: 4.2 ±0.4, p>0.1), suggesting that the s trength of the acquired threat responses is likely 1014 \nsimilar between groups. 1015 \nSupplementary Figure 1. Pupil dilation responses (PDRs), \nexplicit arousal and valence rating for the diffe rent CSs \npresented during the valence-specific response characterisation \ntask. (A) PDRs to the shock reinforced (CS+S), reward reinforced \n(CS+R) and CS - stimuli, averaged across the task and all \nparticipants. PDRs were increased for the CS+S and CS+R as \ncompared to the CS- (B) Explicit ratings of arousal and (C) valence \nprovided immediately after the task. Explicit ratings of arousal \nfor the CS+S exceeded ratings for the CS -, and the CS+R was \nrated higher in arousal than the CS+S. Valence ratings \n(1=extremely negative, 10=extremely positive) for the CS+R were \nmore positive than ratings for the CS -, while ratings for the CS+S \nwere more negative than for the CS - and CS+R. Error bars \nrepresent ± standard error of the mean *=p<0.05, ***=p<0.001 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n29 \n \n 1016 \nTo keep all experimental tasks similar between groups, participants in both groups were asked to 1017 \nrespond to targets that were superimposed on the stimuli as quickly as possible. T o verify that both 1018 \ngroups performed similarly on this task, we compared response times for the different stimuli 1019 \nbetween the groups. During the acquisition task, participants responded faster to targets in CS+ trials 1020 \ncompared to CS - trials ( CS-type (CS+, C S-) x Group  (CC, Ext) rmANOVA , main effect CS -type: 1021 \nF(1,45)=10.839, p=0.002, η²=0.194), with no differences between groups (all p’s>0.058). 1022 \nAfter the acquisition task, participants in both groups reported higher estimated reinforcement rates 1023 \nfor the CS+ ca tegory as compared to the CS - category (CS-type (CS+, CS -) x Group rmANOVA, main 1024 \neffect CS -type: F( 1,45)=82.176, p<0.001, η²=0.646). The reported reinforcement rates did not differ 1025 \nbetween groups (all p’s>0.3). 1026 \nBrain activation supports successful acquisition of conditioned threat responses 1027 \nThe acquisition of conditioned fear on the first day reliably activated networks associated with fear 1028 \nconditioning. Whole -brain analysis identified regions that were more responsive to the CS+ versus 1029 \nthe CS- category (Supplementary Figure 3, see Supplementary Table 1 for a complete overview of 1030 \nfindings). We observed differential BOLD responses in a large number of brain areas, including the 1031 \nbilateral insula, posterior and anterior cingulate, thalamus, precuneus ( undirected test, cluster size = 1032 \n425400 mm 3, p<0.001, whole-brain FWE-corrected) and the bilateral amygdala (right cluster size = 1033 \n1088 mm3, p<0.001, FWE-SVC, left cluster size = 736 mm3, p<0.001, FWE-SVC). 1034 \nSupplementary Figure 2. Differential PDRs during \nacquisition and explicit ratings of arousal and valence \nprovided after acquisition. (A) Differential PDRs for \nthe early (light red) and late (dark red) phase of the \nacquisition task, (B) arousal and (C) valence ratings, \ndisplayed separately for participants assigned to the \ncounterconditioning (CC, solid bars) and extinction \n(EXT, open bars) groups. Both groups showed \ncomparable di fferential PDRs and arousal ratings \nduring the acquisition task. Participants in both groups \nshowed negative differential valence ratings (stronger \nnegative valence for CS+ vs. CS -), although this effect \nwas stronger in the Ext group.  Error bars represent ± \nstandard error of the mean . *, p<0.05. ≠. Significantly \ndifferent from 0 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n30 \n \n  1035 \nSupplementary Figure 3. Differential threat responses during acquisition revealed CS-specific activation of clusters 1036 \nencompassing a range of regions including the bilateral insula, thalamus, precuneus, anterior cingulate and midbrain . 1037 \nGroup F -image of the effect of CS type, thresholded at cluster -level FWE-corrected p<0.05, cluster -forming threshold 1038 \np=0.001, displayed on the single-subject high-resolution T1 volume provided by the Montreal Neurological Institute (MNI).  1039 \nSupplementary Table 1. Whole-brain main effects of group (CC, Ext), CS type (CS+, CS-) and phase (early, late) and 1040 \ninteractions, during the acquisition task. Cluster-forming threshold p=0.001, FWE-corrected at p<0.05, clusters were 1041 \nlabelled using the Talairach Deamon atlas and the AAL atlas for ROIs. For each cluster, the peak voxel coordinates (MNI 1042 \nspace) and regions are reported, and additional regions contained within the cluster are added in italics. 1043 \n  Peak MNI coordinates     \nRegion Cluster x y z Size \n(mm3) \npFWE \n(cluster) \nPeak F-value Direction \n         \nCS-type x phase         \nParahippocampa Gyrus L \nInsula L, Parahippocamal Gyrus \nHippocampus L, Claustrum L, Lentiform \nNucleus Putamin L, Uncus L, Postcentral \nGyrus BA43 L \n1 -18 -10 -16 6656 0.005 25.86 \nEarly CS+ > Late CS+ \nParahippocampa Gyrus Amygdala R \nInferior Frontal Gyrus R, Subcallosal \nGyrus BA34R \n2 20 -4 -22 2116 0.033 25.34 \nCulmen L \nDeclive L, Lingual Gyrus L \n3 -8 -54 -16 1720 0.027 20.57 \nParahippocampal Gyrus L \nParahippocampal gyrus BA36L/BA30L, \nCulmen L \n4 -20 -42 -2 5236 0.006 31.65 \nMedial Frontal Gyrus  BA11 L \nAnterior Cingulate BA32L, Medial \nFrontal Gyrus BA10 R, BA11 R \n5 -4 38 -14 1104 0.046 17.23 \nSuperior Temporal Gyrus L \nMiddle Temporal Gyrus BA21/BA22 L \n6 -52 10 -14 3056 0.015 28.06 \nLingual Gyrus BA18/BA19 R 7 -6 -68 -2 2584 0.018 35.21 \nInsula R \nInferior Parietal Lobule R, Superior \nTemporal Gyrus BA22 R, Postcentral \ngyrus BA3 R, Superior Temporal Gyrus \nBA22 R, Precentral Gyrus BA4/BA6, \nInferior Parietal Lobule BA40, Middle \ntemporal gyrus, Superior temporal \ngyrus BA42 \n8 38 -6 18 24488 0.001 27.62 \nParahippocampal Gyrus R 9 24 -36 -4 1432 0.035 22.26 \nInferior Frontal Gyrus BA45 R 10 52 14 14 1392 0.036 21.37 \nPrecentral Gyrus L 11 -60 -8 32 5640 0.006 22.09 \nInferior Parietal Lobule BA40L \nPostcentral gyrus BA2L \n12 -56 -36 42 1104 0.046 20.42 \nPrecuneus L \nPostcentral gyrus L, cingulate gyrus L \n13 -14 -42 54 1496 0.034 19.85 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n31 \n \nPrecuneus R \nParacentral Lobule Ba7 R, Precuneus R, \nCingulate gyrus R, Superior Parietal \nLobule BA7 R \n14 20 -52 54 5528 0.006 24.86 \nMedial Frontal gyrus L (23) \nMedial frontal gyrus BA6LR, Paracentral \nLobule L \n15 -6 -20 64 1840 0.025 21.47 \n         \nCS-type         \nPostcentral Gyrus L \nInferior Parietal Lobule LR, Insula LR, \nPostcentral gyrus R, Cingulate Gyrus LR, \nThalamus LR, Caudate LR, Inferior- \nMiddle- and Superior Frontal Gyrus LR, \nPosterior Cingulate R, Precentral Gyrus \nLR, Precuneus L, Delice R, Culmen R, \nCuneus L, Superior Temporal Gyrus LR, \nAnterior Cingulate LR, Parahippocampal \nGyrus BA27 R, Lentiform nucleus LR \n1 -50 -20 16 425400 <0.001 195.37 \nCS+>CS- \nPosterior Cingulate BA31 L \nPrecuneus M \n2 -4 -56 24 2816 0.021 26.17 \nCS+<CS- \nCorpus Callosum M \nCorpus Callosum R \n3 0 0 22 1296 0.049 35.45 \nAngular Gyrus R \nAngular Gyrus BA39 R, Precuneus R \n4 56 -66 30 2432 0.024 31.42 \nAngular Gyrus BA39L 5 -54 -68 30 5584 0.010 36.02 \nSuperior Frontal Gyrus BA9L \nSuperior Frontal Gyrus BA8L, Middle \nfrontal gyrus BA6L \n6 -18 40 42 7200 0.007 33.18 \n         \nPhase         \nSuperior Temporal Gyrus LR, \nInferior Parietal Lobule R, Middle \nTemporal Gyrus LR, Inferior- Middle- \nand Superior Frontal Gyrus LR, Caudate \nLR, Middle Occipital Gyrus LR, Cingulate \nGyrus LR, Anterior Cingulate LR, Declive \nLR, Precuneus LR, Insula LR, Culmen LR, \nSuperior Temporal Gyrus LR, Lingual \nGyrus LR, Fusiform Gyrus LR, Angular \nGyrus R, Claustrum LR, Thalamus LR, \nParahippocampal Gyrus LR, Cuneus LR \n1 -64 -38 12 784632 <0.001 77.44 \nEarly>Late \n         \nCounterconditioning and extinction 1044 \nSkin conductance responses 1045 \nDifferential SCRs were still apparent during the CC/extinction task (CS-type (CS+, CS-) x Phase (Early, 1046 \nLate) x Group (CC, Ext) rmANOVA, main effect CS -type: F( 1,40)=17.609, p<0.001, η²=0.306). To verify 1047 \nthat successful extinction was reached by the end of  the task, we explored SCRs in the late phase 1048 \nseparately, but found that differential SCRs were still apparent in that phase (F( 1,41)=12.166, p=0.001, 1049 \nη²=0.229). Finally, we explored whether the last two trials of the extinction task showed evidence of 1050 \nresidual differential SCRs. In the last two trials of extinction, across both groups, there was no 1051 \nevidence for differential SCRs (all p’s>0.2). Thus, while differential SCRs persisted during the late 1052 \nphase of the extinction task, differential responses were no longer apparent in the last two trials. 1053 \nThroughout the CC/extinction, there was no evidence for different SCRs between groups, suggesting 1054 \nthat participants in both groups underwent comparable but slow extinction of differential SCRs. 1055 \nOverlapping stimulus-specific activation during counterconditioning and extinction 1056 \nA number of clusters showed comparable stimulus-specific activations during CC and extinction 1057 \n(Supplementary Table 2). 1058 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n32 \n \nSupplementary Table 2.  Whole-brain main effect of CS-type during the CC/extinction task . Cluster-forming threshold 1059 \np=0.001, FWE-corrected at p<0.05, clusters were labelled using the Talairach Daemon atlas and the AAL atlas for ROIs. For 1060 \neach cluster, the peak voxel coordinates (MNI space) and regions are reported, and additional regions contained within the 1061 \ncluster are added in italics. 1062 \nPeak MNI coordinate \nRegion Cluster x y z Size \n(mm3) \npFWE \n(cluster) \nPeak F-\nvalue \nDirection \n         \nCS-type         \nCaudate Head L \nThalamus LR, Caudate Head R, \nSubstantia Nigra LR \n1 -10 10 -2 25136 0.001 60.98 \nCS+>CS- \n \nInsula R \nInferior Frontal Gyrus R, Precentral \nGyrus BA44 R, Inferior Frontal Gyrus \nBA45 R \n3 28 26 0 22800 0.001 89.75 \nInferior Frontal Gyrus L \nInsula BA13 L \n4 -32 28 0 7808 0.004 52.01 \nLingual Gyrus L \nInferior Occipital Gyrus L, Cuneus L, \nMiddle Occipital Gyrus L \n5 -24 -80 -12 5696 0.006 32.81 \nSuperior Temporal Gyrus R \nTransverse Temporal Gyrus R \n6 50 -18 6 3744 0.012 30.65 \nLingual Gyrus R \nCuneus R \n7 8 -94 6 3688 0.012 27.81 \nSuperior Temporal Gyrus L \nTransverse temporal Gyrus L \n8 -44 -24 8 3864 0.011 44.50 \nAnterior Cingulate BA32 R \nMedial Frontal Gyrus BA8 R, \nAnterior Cingular LR, Cingulate \nGyrus BA32 R \n9 4 38 20 7064 0.004 26.61 \nSuperior Temporal Gyrus R \nSupramarginal Gyrus R, Inferior \nParietal Lobule BA40R \n10 64 -34 14 3720 0.012 25.88 \nSuperior Temporal Gyrus L 11 -60 -46 16 2504 0.023 36.62 \nCingulate Gyrus L \nPosterior Cingulate BA23R, \nPosterior Cingulate L \n13 -6 -20 30 3928 0.011 38.33 \nAngular Gyrus L \nMiddle Temporal Gyrus L, Angular \nGyrus BA39 L \n12 -44 -64 32 4104 0.010 23.58 \nCS->CS+ \nInferior Temporal Gyrus BA21 L, \nMiddle Temporal Gyrus BA21 L \n2 -64 -10 -22 1696 0.039 27.05 \nAngular Gyrus R \nSupramarginal Gyrus R \n14 44 -66 34 1392 0.050 18.35 \nPostcentral Gyrus BA40R \nPrecentral Gyrus Ba4/BA3 R \n15 34 -40 58 1704 0.038 19.84 \nMiddle Frontal Gyrus BA8/BA6 L 16 -24 16 48 4576 0.008 34.03 \n         \nSpontaneous recovery test 1063 \nSkin conductance responses 1064 \nTo investigate whether CC can prevent spontaneous recovery of differential SCRs, SCRs during the 1065 \nlast two trials of extinction and the first two trials of the spontaneous recovery test were submitted 1066 \nto a CS -type (CS+, CS -) x Phase (last 2 trials of the CC/extinction phase, first two trials of the 1067 \nspontaneous recovery test) x Group (CC , Ext) rmANOVA. SCRs showed a generalized increase from 1068 \nthe last two trials of extinction to the first two trials of the spontaneous recovery test (main effect 1069 \nphase: F(1,38)=32.392, p<0.001, η²=0.460)). There was evidence for differential SCRs across both  1070 \nphases (main effect CS -type: (F(1,38)=9.560, p=0.004, η²=0.201), as CS+ stimuli evoked higher SCRs 1071 \nthan CS - stimuli (t(43)=2.518,p=0.016, CS+:0.41 ±0.03, CS -:0.35±0.03), yet we did not find evidence 1072 \nfor CS+ -specific spontaneous recovery or effect of group (all p’s>0.4). Thus, although there was a 1073 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n33 \n \ngeneralized increase in responding from the end of extinction to the start of the spontaneous 1074 \nrecovery test, SCRs did not show differential recovery and were comparable between groups. 1075 \nBrain activation 1076 \nDuring the spontaneous recovery test, CS-specific activation differed between groups in the inferior 1077 \ntemporal gyrus (cluster size = 2008 mm3, p=0.020, FWE-corrected, Supplementary Figure 4A) and the 1078 \ninferior frontal gyrus (cluster size = 1920 mm 3, p=0.022, FWE-corrected, Supplementary Figure 4B). 1079 \nSeparate analysis of the spontaneous recovery phase within each group did not reveal any 1080 \nsuprathreshold clusters in the Ext group, while a number of clusters showed stimulus -specific 1081 \nactivation i n the CC grou p. Spe cifically, the CC group showed stimulus -specific activation in the 1082 \nbilateral fusiform gyri, superior parietal lobes and inferior frontal gyri, and in the right thalamus, 1083 \ncaudate, middle frontal gyrus, and angular gyrus  (see Supplementary T able 3). A priori defined 1084 \nregions of interest (ROIs) during the spontaneous recovery task were submitted to a Group (CC, Ext) x 1085 \nCS-type (CS+, CS-) x Phase (early, late) rmANOVA but did not reveal any effects. 1086 \n 1087 \nSupplementary Figure 4. During the spontaneous recovery test, stimulus type-specific activation of the inferior temporal 1088 \nand frontal gyri differed between groups. The inferior temporal Gyrus (A) and Inferior frontal gyrus (B)  show increased 1089 \nCS+-specific activation in the CC gro up as compared to the Ext group.  Group F -images thresholded at FWE -corrected 1090 \np<0.05, cluster -forming threshold p=0.001, displayed on the single-subject high-resolution T1 volume provided by the 1091 \nMontreal Neurological Institute (MNI) and parameters estimates from peak voxels. 1092 \nSupplementary Table 3. Peak voxel coordinates and statistics of activations during the spontaneous recovery phase in 1093 \nthe CC group . Clusters were labelled using the AAL atlas. For each cluster, the peak voxel coordinates and regions are 1094 \nreported, and additional regions contained within the cluster are added in italics. Clusters are whole-brain FWE-corrected at 1095 \np<0.05. 1096 \nPeak MNI coordinate \nRegion Cluster x y z Size \n(mm3) \npFWE \n(cluster) \nPeak T-\nvalue \nDirection \nCS-type         \nThalamus R \nParahippocampal Gyrus R \n1 10 -22 -4 2160 <0.001 6.70 \nCS+>CS- \nInferior temporal Gyrus R \nFusiform gyrus R \n2 46 -52 -2 4856 <0.001 6.56 \nInferior frontal gyrus, triangular R 3 58 26 26 4992 <0.001 5.93 \nSuperior parietal lobe R \nAngular Gyrus R \n4 26 -60 50 2048 <0.001 5.36 \nInferior frontal gyrus, orbital part L 5 50 26 -6 1120 0.019 5.31 \nFusiform gyrus L \nLingual gyrus \n6 -38 -80 -18 1176 0.015 5.18 \nCaudate R 7 6 0 10 1952 <0.001 4.87 \nMiddle Frontal gyrus R 8 37 0 44 992 0.03 4.86 \nSuperior parietal lobule L \nAngular gyrus L \n9 -32 -58 58 1480 0.004 4.53 \n 1097 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint \n\n34 \n \nReinstatement test 1098 \nSCRs showed a generalized increase from the spontaneous recovery phase to the reinstatement test 1099 \n(CS-type (CS+, CS -) x Group (CC, Ext)  x Phase (spontaneous recovery test, reinstatement test) 1100 \nrmANOVA, main effect Phase: F( 1,39)=25.758, p<0.001, η²=0.398, last two trials of spontaneous 1101 \nrecovery: 0.22 ±0.04, first two trials of reinstatement: 0.38±0.03 ). Across the last two trials of the 1102 \nspontaneous recovery test and the first two trials of the reinstatement test, differential SCRs differ 1103 \nbetween the counterconditioning and extinction group (interaction effect of stimulus type and 1104 \ngroup: F( 1,39)=4.967, p=0.032, η²=0.113). Yet, there is no evidence for differential reinstatement 1105 \nbetween groups (no CS-type x Phase x Group interaction, p=0.218). Moreover, mean SCRs to CS+ and 1106 \nCS- stimuli do not differ within either group (all p’s>0.12). 1107 \nPDRs showed a generalized decrease from the spontaneous recovery phase to the reinstatement test 1108 \n(CS-type (CS+, CS -) x Group (CC, Ext)  x Phase (spontaneous recovery test, reinstatement test) 1109 \nrmANOVA, main effect of Phase (F( 1,29)=9.104, p=0.005, η²=239)). Mean PDRs decreased from 1110 \nspontaneous recovery to reinstatement (t(30)=3.063, p=0.005, last two tr ials of spontaneous 1111 \nrecovery: 1.04±0.01, first two trials of reinstatement: 1.01±0.01). Given that we did not observe 1112 \nsuccessful reinstatement in either group, our reinstatement test does not inform us about whether 1113 \nCC can lead to a more persistent attenuation of fear as compared to classic extinction. 1114 \nCS+-specific enhancement of recognition memory depends on CS+ category 1115 \nCorrected recognition scores (pHits – pFA) were subjected to a task (acquisition, CC/extinction task) x 1116 \nCS-type (CS+, CS -) x Group (CC, E xt) rmANOVA, including CS+-category (animals, tools) as covariate.  1117 \nAlthough the effect of CS -type differed depending on the category used as CS+ (CS -type x CS+ -1118 \ncategory interaction: F( 1,42)=19.400, p<0.001, η²=0.316) and task (CS-type x CS+ -category x task 1119 \ninteraction: F(1,43)=5.375, p=0.025, η²=0.113), showing a stronger effect for tools as CS+,  this did not 1120 \ndiffer between our experimental groups (CS-type x CS+ -category x Group interaction: F(1,41)=0.050, 1121 \np=0.824; CS-type x task x CS+-category x Group interaction: F(1,41)=0.005, p=0.946). 1122 \nTo further investigate to what extent CC retroactively affected memory for items presented during 1123 \nthe acquisition task, we examined recognition of items presented during acquisition and the 1124 \nCC/extinction tasks separately. Retrospective memory enhancement for the CS+ items compared to 1125 \nCS- items differed depending on the CS+ category during both the acquisition task (CS-type x CS+ 1126 \ncategory interaction: F( 1,42)=29.730, p<0.001, η²=0.414, CS+ category main effect: F( 1,42)=5.346, 1127 \np=0.026, η²=0.113) and the CC/extinction task (CS-type x CS+ category interaction: F( 1,42)=5.121, 1128 \np=0.029, η²=0.1 09), showing a stronger effect for tools as CS+. Importantly,  this effect was 1129 \ncomparable between groups  (CS-type x CS+ category  x Group  interaction: acquisition task 1130 \nF(1,41)=0.016, p=0.806; CC/extinction task F(1,41)=0.019, p=0.890). 1131 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted July 30, 2024. ; https://doi.org/10.1101/2024.07.29.605706doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}