Dopaminergic signals in the Nucleus Accumbens, VTA and vmPFC underpin extinction learning from omitted threats

Learning to be safe is central to adjust behaviour when threats are no longer present. Detecting the absence of an expected threat is key for threat extinction learning and behavioural treatment of anxiety related disorders. One possible mechanism underlying extinction learning is a dopaminergic mismatch signal that encodes absent but expected threats. We show that a dopamine-related pathway underlies extinction learning in humans. Dopaminergic enhancement (L-DOPA/Placebo) reduced retention of psychophysiological threat responses, which was mediated by activity in the ventromedial prefrontalcortex during extinction learning. L-DOPA administration enhanced signals at the timepoint of the omitted, but expected threat within the nucleus accumbens, which were functionally coupled with the ventral tegmental area and amygdala. Computational modelling of threat expectancies further revealed prediction-error encoding in nucleus accumbens that was modulated by dopaminergic enhancement. Our results provide a mechanism to augment extinction learning by enhancement of dopaminergic neurotransmission that underlies encoding of absent threats.


Introduction
In order to thrive in dangerous environments, it is important to know when threats are disappearing and situations become safe. As such, safety learning is central for adaptive behaviour and deficits characterize symptoms in a wide range of anxiety related disorders [1][2][3][4]. Yet, the pharmacological mechanism to augment safety learning by encoding the absence of potential threats or aversive outcomes in humans are not completely understood.
Safety learning is often investigated by laboratory protocols of extinction training. Here, a learned predictor (conditioned stimulus, CS) for an aversive outcome (unconditioned stimulus, US) is turning into a safety signal when the expected aversive outcome is omitted. This omission of the expected US after CS presentation is thought to drive extinction or safety learning. However, it is only incompletely understood in humans which neural system detects the omission of the expected aversive outcome and, hence, initiates a shift from threat to safety. Studies in drosophila [5] and rodents [6][7][8][9][10][11][12]12] revealed that the omission of an expected aversive outcomes depends on signals in the dopaminergic system. In rodents, this involved dopaminergic neurons in the ventral tegmental area (VTA), the nucleus accumbens and the medial prefrontal cortex, as well as projections between the VTA and nucleus accumbens [6,9,13,14]. Importantly, these neural regions were also found to underpin the processing of rewarding outcomes. When signalling rewards, this system does not simply detect a rewarding outcome, but codes a difference between the expected reward and the actual outcome in form of an expectancy violation or prediction error [15]. In other words, reward-related response in the VTA, nucleus accumbens and vmPFC reflect outcomes that are better than expected.
Similarly, the omission of an expected aversive US, which could be framed as "better than expected" and it might well be that this dopaminergic signal at the time-point of US omission encodes an expectancy violation that signals the difference between the expected aversive US and the omitted aversive outcome (for review see [16][17][18]).
Even though this idea is not formally tested, it is supported by a functional neuroimaging study in humans. This study provided initial tentative evidence that computational modelling of an prediction error for the omitted aversive outcome during extinction training involves activity in the nucleus accumbens and that this activity was modulated by a genetic variance of the dopamine transporter gene [19]. Additionally, there is cross-species evidence for enhanced extinction memory consolidation by augmented dopaminergic transmission after extinction training (by administration of L-DOPA [20][21][22]). These latter studies suggest that dopaminergic enhancement of extinction memory retrieval is mediated by augmenting activity in the ventral part of the medial prefrontal cortex (vmPFC), a structure that is central for extinction learning and memory retrieval [23][24][25][26]. It is, however, not clear if enhancing of dopaminergic neurotransmission would strengthen extinction learning by modulating vmPFC activity.
In this study, we tested if extinction learning is associated with activity changes in the vmPFC and if such activity is modulated by administration of the dopaminergic precursor L-DOPA. We further tested if the unexpected omission of the US during extinction learning is coded in midbrain pathways that connect the VTA and the nucleus accumbens and if activity within this pathway is modulated by L-DOPA. Based on previous studies [20,22] we hypothesized that L-DOPA treatment would decrease threat responses at retention tests.

Figure 1 | Behavioural and psychophysiological outcome measures a) US expectancy, b) SCR and c) Fear ratings reflect successful acquisition of CS-US
contingencies during acquisition and decreasing responses during extinction training. Retention of CS-US memory was evident during retention test on day 3, as well as initial enhancement of responses after reinstatement within three trials after presentation of the reinstatement USs. We found decreased differential (CS+ -CS-) fear ratings in the retentiontest during day 2 in the L-DOPA group, when compared to placebo controls. Hence, the L-DOPA group rated lower fear when the CSs were presented within a new context. Additionally, differential SCRs (CS+ -CS-) in 3 trials after reinstatement were lower in the L-DOPA, when compared to the Placebo group (see figure 2).

Acquisition of CS-US contingencies on day 1
Participants in both groups learned CS-US contingencies during acquisition training, which was indicated by a CS-type main effect that consisted of enhanced responses to the CS+ as compared to the CS-in all dependent measurements, namely binary (yes/no), trial-wise US  Figure 1 c), see Table S2 for full statistics, means and CI. Unexpectedly, we found an interaction effect in US expectancy between CStype, trial and group-status (i.e., subjects that were allocated to receive Placebo or L-DOPA on the next day: CS-type*trial*group F(2,88)=3.3, p=0.044, hp 2 =0.07). However, follow-up group comparisons of block-wise US expectancy did not support any differences in CS+ or CSresponses (two-tailed independent post-hoc t-tests: ps>0.1, see Table. S3) or CS+/CSdiscrimination between groups (p>0.05, CS discrimination was descriptively lower in the prospective Placebo vs. L-DOPA group, see Table. S3). There was no support for differences between groups in fear ratings or SCRs (group main effect or interaction ps>0.1, see Table   S2).

Extinction learning on day 2
On day 2, participants discriminated between CS+ and CS-, as indicated by a main effect of see Table S4 for full statistics). Responses in all measures decreased over the time course of extinction training (CS-type by block interaction, all ps<0.05, see Table S4, see Figure 1). In particular, trial-wise US expectancy indicated successful extinction learning of the CS-US association, by evidence for differential CS responses in the first two of three blocks (CS+ > CS-, Block 1: p<.001, Block 2 p=.048), but not the last (Block 3: p=0.57, see Table S4). We found no statistical evidence that would support a difference between groups in US expectancy ratings or SCR (see Table S4).
Next, we examined how decreasing US expectancy, which indicates extinction learning is driven by expectancy violation from the omission of the US, by fitting US expectancy ratings with a Rescorla-Wager-Pearce-Hall-Hybrid model [27,28]. The fitted prediction error (as a measure of expectancy violation), associability (as a measure of prediction error-guided surprise) and learning rate did not differ between groups (two-sided independent sample t-test for mean prediction error: t(40)=0.097, p=0.923, mean associability: t(40)=0.015, p=0.988, and mean learning rate: t(40)=0.179, p=0.859, see Table S6). Differential SCRs (CS+ > CS-) were decreased when compared to the Placebo group within 3 trials after the reinstatement procedure. See figure S1 for CS-specific responses. Asterisk indicate a p-value<0.05 for the CS-type by group interaction and a one-sided t-test.

Memory retrieval on day 3
At the retrieval test on day 3, participants discriminated between CSs in all outcome measures Importantly, the SCR analyses of the three trials before and after reinstatement revealed a difference between groups in differential CS responses (CS-type by group interaction F(1,40)=5.443, p=0.025, hp 2 = 0.120, see Table S7 and S8), indicating lower CS discrimination in the L-DOPA group when compared to the Placebo controls after the reinstatement procedure  Figure   2, S1 and Table S8). While our a priory hypothesis was an effect of L-DOPA on the psychophysiological measurements at retrieval-test on day 3, our analyses suggest that L-DOPA administration during extinction training reduced threat responses after reinstatement.

Administration of L-DOPA enhances vmPFC responses reflecting decreasing US expectancy during extinction learning
First, our analyses of neural responses focused on the effect of L-DOPA on extinction learning, were we expected an involvement of the vmPFC that is modulated by L-DOPA. To this end we examined brain regions that increased their activity to a decline of US expectation. In order to examine extinction learning by decreasing US expectancy, we contrasted responses during extinction training to CS+ trials when participants expected no US against CS+ trials in which participants expected an US (i.e., expectation of no US > expectation of a US). We found that decreasing US expectancy was accompanied by more pronounced signalling in the right vmPFC in the L-DOPA group as compared to the placebo group (see Figure 3 a). Thus, administration of L-DOPA augmented vmPFC activity during extinction learning, i.e., when participants decreased their US-expectancy.
Next, we tested if this difference in the right vmPFC activity is related to individual differences in extinction memory retrieval. A previous study indicated that vmPFC activation during extinction learning was associated with retention of extinction memory (measured as differential SCR) 24 hours later [20]. Indeed, we found that higher vmPFC activation is associated with reduced differential SCR, i.e., better individual extinction memory retention, 24 hours later (Pearson correlation: t(40) = -2.18, p-value = 0.035, r=-0.326, see Figure 3 b).
Hence, vmPFC responses during extinction learning were elevated after L-DOPA administration and such enhanced vmPFC activity is associated with stronger extinction memory retrieval 24 hours later. Importantly, there was no difference between groups detectable in SCRs during retrieval test, which might have biased this correlation [29].
However, it might be possible that L-DOPA treatment has an indirect effect on SCR during retrieval test, which is mediated by vmPFC activity during extinction learning. Indeed, we found support for a treatment effect of L-DOPA on SCRs during retrieval test, which was indirectly mediated by vmPFC activity during extinction learning (average causal mediation effect: b=0.0563, 95% confidence intervals = 0.007-0.12, p=0.038, quasi-Bayesian estimation of confidence intervals with 1000 iterations, N=40, see figure 3 for detailed statistics).
As such, L-DOPA strengthens vmPFC activation that accompanies decreasing expectation of the US (i.e., extinction learning), which results in better extinction memory retrieval. Our results thereby provide a direct link between L-DOPA augmented vmPFC signalling and the individual time-course of decreasing expectancy of the aversive outcome, which drives extinction learning and memory retrieval.

Omission of an expected aversive outcome is coded in the nucleus accumbens and modulated by L-DOPA
In the next step, we examined if decreasing US expectancy during extinction learning is driven by the omission of the US in form of an expectancy violation (i.e., prediction error) and if this process is modulated by dopamine. To this end, we used the modelled US expectancy ratings from the Rescorla-Wagner-Pearce-Hall hybrid model that has previously been used to describe computational processes in associative threat learning [27,28,30]. In order to test for signals that reflect expectancy violation, we examined the prediction error term as parametric In addition to neural signaling that aligned with the prediction error term, we further investigated potential differences between groups in neural signals that follow the associability term, which provides a measure of prediction error-guided attention shift. We found that administration of L-DOPA enhanced associability related neural signals in the amygdala at the time-point of US omission, when compared to Placebo (see Figure S2). Our results suggest that dopaminergic enhancement might enhance shifting of attention or surprise that is initiated by unexpected omission of the US during extinction training.

L-DOPA modulates functional connectivity between responses in the nucleus accumbens and the VTA when the US is omitted
Results in animals suggested that processes at the time-point of US omission involve not only the nucleus accumbens, but dopaminergic neurons in the VTA [11] and projections from the VTA to the nucleus accumbens [9], as well as projections from the basolateral complex of the amygdala to the nucleus accumbens [7].
To test if the reported results in the nucleus accumbens at the time-point of the omitted US are functionally connected with other regions in the brain, we employed a condition-specific connectivity analysis with an anatomical nucleus accumbens (bilateral) mask as a seed region.
In line with animal data, we found stronger connectivity between the nucleus accumbens and

Discussion
Our results provide evidence that dopaminergic processes are involved in threat extinction learning. Dopaminergic enhancement during extinction learning augmented extinction memory at a later test, which was mediated by extinction learning specific vmPFC responses (i.e., reflecting decreasing US expectancy). Decreased US expectancy in extinction learning was further driven by dopaminergic activity within the nucleus accumbens that signalled the omission of expected aversive outcomes. This activity in the nucleus accumbens, when the US was omitted, was functionally coupled with the midbrain SN/VTA complex, as well as the amygdala. Additionally, we found weak statistical support for decreased fear ratings in the retrieval test before extinction training (the CS+ was presented within a new context) in the L-DOPA group when compared to Placebo, as well as reduced SCRs after the reinstatement one day after extinction learning.
The main finding is that L-DOPA reduced retrieval of conditioned threat responses (SCR) by a mediation through enhanced vmPFC activity. In detail, we found that enhancement of dopaminergic transmission by the administration of L-DOPA (as compared to Placebo) during extinction learning enhanced individual neural signalling in the vmPFC that reflects the reduction of US expectation (i.e., extinction learning).
These enhanced vmPFC responses were found to mediate the effect of L-DOPA on extinction memory retention, measured as reduced differential SCRs 24 hours later.
Besides the implication of the vmPFC in safety signal processing [31] and threat extinction in humans [23,24,26,32,33], our results align specifically with a previous finding of vmPFC activity pattern during extinction memory consolidation that mediates the effect of L-DOPA on extinction memory retention [20]. Our results extend this finding on memory consolidation by providing a link between dopaminergic effects on vmPFC activity that is specific for individual extinction learning (i.e., decreasing US expectancy) and augmentation of extinction memory. L-DOPA might have the potential to improve (otherwise low, see parameter estimates in the Placebo group, figure 3 B) vmPFC activity during extinction learning.. Hence, rather than enhancing extinction learning per se, L-DOPA administration seems to augment vmPFC responses that accompany decreased US expectancy. This suggests a benefit of L-DOPA for extinction learning processes. Our findings would fit to previous results that link the benefit of neuropharmacological intervention in extinction learning to decreasing threat expectancies [34][35][36]36]. Thereby, L-DOPA might have the potential for a psychopharmacological treatment that augments threat extinction learning instead of dampening overall threat responses, like classic anxiolytics. Additionally, we found that L-DOPA administration during extinction training reduced differential SCRs after reinstatement, which aligns with a finding of decreased SCRs after reinstatement by L-DOPA administration that followed extinction training in women diagnosed with posttraumatic stress disorder [37].
A second set of our results implicate that decrement of US expectancy in extinction training involves a dopaminergic coding of expectancy violation in form of a prediction eror at the time-point of US omission. Our study was intentionally not designed to disentangle details of expectancy violation coding of omitted USs, but rather to provide a scenario of safety learning. Nevertheless, we provide evidence for a role of the nucleus accumbens in processing of US omissions, which is in line with a function of the nucleus accumbens in rodents [6,13,38] in particular during extinction learning [7,8,14,39]. Our results furthermore align with a previous neuroimaging study reporting an association between prediction error signals in the nucleus accumbens during extinction training and a genetic variant of the dopamine transporter [19]. Our results point moreover to a dopaminergic modulation of surprise in the amygdala that is evoked by US omission, which fits well to previous reports of associability coding in the amygdala during threat learning [27,30].
We further show that signals in nucleus accumbens at the time-point of US omission were functionally coupled with activation in the amygdala and the SN/VTA, which were enhanced by administration of the dopaminergic precursor L-DOPA. This finding mirrors findings in animals implying neurons in the VTA, as well as projections from the VTA to the nucleus accumbens, in the encoding of the omission of an expected US [9,11]. Furthermore, our results would align with studies in animals that provided evidence for amygdala to nucleus accumbens projections that underlie extinction of threat responses [7].
We highlight that our fMRI study in human volunteers is only suited to draw inferences on blood-oxygen-level-dependent signals as a function of L-DOPA administration and hence might imply that activity changes are related to dopaminergic neurotransmission, but invasive studies in animals are needed to confirm that these results are related to dopamine release. We further found that L-DOPA administration did not decrease conditioned responding across all outcome measures that reflect different threat processing, such as US expectancy, psychophysiological arousal (SCR) and affective (fear) ratings. In comparison to the effects of L-DOPA on consolidation of extinction memories [20][21][22]34], the reported effect on these outcome measures are rather weak, which might point towards a larger effect of dopaminergic enhancement on consolidation processes. Nevertheless, the effect of L-DOPA to decrease differential SCRs by the mediation of vmPFC activity during extinction learning converges with a previous finding [20] and might suggest that L-DOPA enhances learned decrement of US expectancy (and accompanying vmPFC activity) during extinction training, rather than blunting responses, per se.
In sum, our results thereby provide a neuropharmacological mechanism that augments the neural substrates underling extinction learning in humans [34][35][36]36], which could provide a promising novel strategy to augment behavioural treatments of anxiety related disorders [37]. The study (including sample size approximation) was approved by local ethics committee in Hamburg (Ärztekammer Hamburg). Full participation of this study was remunerated with 120,-EURO. individually adjusted prior to acquisition training (day 1) to a level of maximal tolerable pain (mean 8.1 ± 0.5 mA, range 2.5-21.0 mA) and participants were asked to rate the aversiveness of the US between 0 ("I feel nothing") and 10 ("maximally unpleasant"; rating: mean 7.1 ± 0.1, range 4.0-8.0).

Stimulus material
Additional US intensity ratings were acquired after fear acquisition training (between 0 and 100 day 1: mean 68.65 ± 3.0, range 20-100) and at the end of return of fear testing (day 3: mean 49.91 ± 3.9, range 0-100). There were no differences between the Placebo and the L-DOPA group in any of these parameters (all P > .167; see supplements Table. S1).

Experimental Procedure
Using a three-day paradigm, acquisition training (day 1) and extinction training (day 2, approx. 24 h after acquisition) were conducted in the fMRI scanner, while retrieval test (day 3, approx. 24 h after extinction), including reinstatement, were employed within the psychophysiological laboratory.
Acquisition training took place in context A, extinction in context B and retrieval test (including reinstatement procedure) in a 50/50-mixture of context A and B in order to examine contextual generalization [40]. Twenty-four hours after acquisition training participants received doubleblind, randomized and placebo-controlled 150mg L-DOPA (including 37.5 mg Benserazide) before the extinction learning session (the CS+ was no longer followed by an aversive outcome). L-DOPA administration thereby affected extinction training, while acquisition training, as well as retention and reinstatement-test were conducted drug-free.
Acquisition training (day 1) A short habituation phase preceded acquisition training (6 trials: 3 CS+, 3 CS-) without any presentation of the US. Subsequent acquisition training consisted of 24 trials for each CS (in context A). The CS+ was followed by a US in 75% of the trials, whereas the CS-was never followed by a US. Participants were not informed about the conditioning contingencies or the learning element beforehand. were classified as aware and 5 were classified as unaware (no differences between groups, χ 2 -Test, p = .634).

Study medication
Study medication included an oral administration of 150mg L-DOPA (including 37.5 mg Benserazide) in a double-blind and placebo-controlled protocol 60min before extinction learning. Participants were allocate into the placebo or L-DOPA group before day 1 in a restricted randomization procedure that allocated 5 subjects to the L-DOPA and 5 subjects to the placebo group for each group of 10 participants. The dose of 150mg has been found effective in previous studies to enhance the consolidation of extinction memories in humans [20][21][22].

US expectancy
On each CS trial presentation, participants had to rate their US expectancy as a binary choice (key press for yes/no) without any scale presented to avoid any distraction. Participants were excluded from the analyses (day-wise) if less than one third of all data points were missing [excluded participants: N(day 1) = 0, N(day 2) = 3, N(day 3) = 3].

Fear ratings
At the beginning as well as at the end of each experimental day, participants were asked to rate the fear/stress/tension level that was elicited by each CS. On day 1 the first rating was conducted after habituation phase and before acquisition training. Ratings were performed on a computerized Visual Analogue Scale [VAS, 0 (none) -100 (maximal)] using keys with the right hand. Rating values had to be confirmed by a key press (otherwise missing data, N(day 1) = 0, N(day 2) = 3, N(day 3) = 4 ). All rating values were range-corrected (divided by the maximal rating value on that day).

Skin conductance
Skin conductance responses (SCRs) was measured via self-adhesive Ag/AgCl electrodes placed on the palmar side of the left hand on the distal and proximal hypothenar. Data were recorded with a BIOPAC MP-100 amplifier (BIOPAC Systems Inc, Goleta, California, USA) using AcqKnowledge 4 software. For data analysis, SCR signal was down-sampled to 10 Hz and responses were manually scored between 0.9 to 4.0 s after CS onset using a custom-made computer program.
Non-reactions were scored as zero and trials with obvious electrode artefacts were scored as missing data. Afterwards, amplitudes were logarithmized and range-corrected (SCR/SCRmax CS
For acquisition and extinction training, these ANOVAS included CS-type (2) and the effect of time (fear ratings: 2 ratings, SCR and US expectancy: 3 blocks, each average across 8 trials).
Pharmacological group was entered as a between subject factor. For day 3, we analyzed the first block separately as the retrieval test and the reinstatement analyses included two comparisons of trials before and after the reinstatement USs [42]. First, we compared responses averaged across the whole block (8 trails) before and after reinstatement. Since reinstatement effect are transient and only detectable over a few trials, we added a second, more detailed analysis, which compared responses averaged across the 3 trails before and after reinstatement, based on previous findings indicating that transient reinstatement effects can be found up to 3 trials after the US presentation [43]. In all analyses, an α-level of p<0.05 was adopted and sphericity correction (Greenhouse-Geisser) was applied.
Follow-up post-hoc test on measurement on day 2 and 3 were performed as one-sided independent t-test to examine the hypothesis of L-DOPA responses < Placebo responses. During data acquisition, preprocessing and initial analyses, the experimenter were masked to the drug conditions.

Hybrid model
To examine how decreasing US expectancy is driven by expectancy violation from the omission of the US, we fitted trial-wise US expectancy ratings with a Rescorla-Wager-Pearce-Hall-Hybrid model, which is the same model employed in previous neurocomputational studies of aversive learning in humans [27,28]).
In order to examine associative threat learning processes, which can be described by classical formal learning theory such as the Rescorla-Wagner (R-W) (Rescorla and Wagner, 1972a) and Pearce "#$ = " + α * " * PE The predicted "values" (v) on the next trial t+1 are based on the "value" at the current trial t and on prediction errors (PE) scaled by the learning rate α and the current associability ηt.
Prediction errors (PE) are calculated as the difference between the current predicted values (vt) and the received outcomes (RO).

= − "
The current associability ηt is updated according to the absolute prediction error (PE) and the associability of the preceding trial ηt-1 with the free scaling parameter ω.
The model employs a softmax function with a free "inverse temperature" parameter β to generate trial-by-trial probabilities (p) for the binary US expectancy ratings.

= 1 1 + :=/?
The model thus contains three free parameters: (i) the learning rate α, (ii) the scaling parameter ω for the associability η, and (iii) the inverse temperature parameter β. These three free parameters were initialized in the fitting procedure as 0.5, 0.5, and 4, respectively. The starting point for the initial "value" v0 was set to 0.75, i.e., the probability for a US following a CS+ in the acquisition phase. The starting point for the initial associability η0 was set to 1, which assumes that the associability is initially fully dependent on the prediction error (PE).We fitted model parameters using maximum likelihood estimation (MLE). Specifically, we used the non- were defined as separate regressors modeling the predicted time courses of experimentally induced brain activation changes as a stick function. Furthermore, CS+ regressors included a parametric modulation of individual US expectancy ratings in order to examine dopaminedependent differences in neural representation in decreasing US expectancy during extinction learning. Additionally, parametrical modulation of the omitted US was applied to examine neural responses that are related to changes in expectancy-violation over trials. Therefore, the modelled prediction error-term (as a measure of expectancy violation, averaged across the whole sample) and the orthogonalized associability-term (as a measure of prediction error-guided surprise, averaged across the whole sample) was entered trial-wise.
In a next step, subject-and regressor-specific parameter estimate images of interest were normalized to a sample-customized DARTEL template [48] smoothed with an isotropic full-width at half-maximum Gaussian kernel of 4 mm. These estimates were then included into separate random-effects group analysis using SPM's "full factorial" model, which permits correction for available, therefore we defined both ROIs as in a previous study that revealed an effect of L-DOPA treatment in both, the SN/VTA and vmPFC ROI [49]. The SN/VTA complex was defined by [50].
The vmPFC ROI was defined as a box of 20 × 16 × 16 mm at x=0 y=42 z=-12 . Correction for multiple comparisons within these ROIs was performed by using family-wise error correction based on the Gaussian Random Fields as implemented in SPM.

Connectivity analysis
Psycho-physiological interaction (PPI, as implemented in SPM12) was used to examine functional connectivity differences of responses in the nucleus accumbens towards the omitted US between groups. Extracted eigenvariates of nucleus accumbens (bilateral ROI mask) were used as the seed region, deconvolved and multiplied with the condition specific onsets of the omitted US. The product (PPI) was entered as a regressor into an individual GLM for each participant, controlling for the time-course of the nucleus accumbens, the onset regressor and movement as nuisance regressors. Parameter estimates of the omitted US-PPI were then contrasted between groups.

Mediation analysis
To test if the effect of L-DOPA vs Placebo on differential SCRs at retrieval test on day 3 was mediated by the activity in the vmPFC that aligned with decreasing US expectancy, we employed a mediation analysis (R Studio, Version 1.2.1335, package "mediation"). This analysis was based on a prior analysis that revealed that effects of L-DOPA on extinction memory retention (differential SCRs during retention test) were mediated by vmPFC activity during consolidation [20]. Please note that US intensity was not calibrated again on day 2 (extinction) or day 3 (return of fear).        Note.For all tests, the alternative hypothesis specifies that L-DOPA group is less than Placebo