Skin conductance responses (SCRs) for each trial, with CS+ (shown in orange) and CS- (shown in blue) responses paired in blocks. Reinforcement of the CS+ by a US (CS+/US) is indicated by filled squares. The CS- is never reinforced. Bar plots on the top right show early and late mean responses for both CS+ and CS-. On day 1, there was no differentiation between CS+ and CS- in the habituation phase, with significant differentiation emerging during acquisition training. On day 2, there was a loss of differentiation during extinction training. On day 3, during the initial recall test, participants exhibited spontaneous recovery, i.e., a return of differential responses after extinction training. During initial reacquisition, there were again differential responses to the CS+ and CS-, which decreased in reextinction and the unexpected US phase. Mean values are shown with error bars representing the standard error of the mean. CS: Conditioned stimulus; US: Unconditioned stimulus; SCR: Skin conductance response; μS: microSiemens.

Cerebellar cortex, DCN and VTA fMRI activations related to the prediction and presentation of the US. Cerebellar cortex activations are shown on cerebellar flatmaps (SUIT; Diedrichsen & Zotow, 2015), while VTA and DCN activations are displayed on coronal slices progressing from posterior to anterior with MNI y-coordinates indicated on the left-bottom for each slice. A 3D rendering in the bottom right shows the slice locations within the probabilistic DCN and VTA atlases (see methods for atlas generation). A: The event-based CS+ > CS- prediction contrast showed trend-level activations in posterolateral lobules and the VTA; no DCN activations were found. B: Inverse activations were found in the parametric modulation analysis of CS+ x prediction showing activations mainly in the anterior cerebellum, the paravermis, lobule VI and Crus I, and VTA, with trend-level activations in the DCN. C: The presentation of the US elicited widespread cerebellar, DCN and VTA activation. D: The event-based CS+ > CS- contrast in extinction training showed trend-level activations in lobule VIIb, VIIIa and VIIIb and the central VTA, and no activations in the DCN. E: The parametric modulation contrast CS+ x prediction showed activations in lobule VI and Crus I, vermis, and VTA, as well as trend-level activations in the DCN. F: Trend-level activations to the omission of the US in extinction training were shown in the cerebellum, DCN and VTA. VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed nucleus; FN: Fastigial nucleus; CS: Conditioned stimulus; noUSpCS: Omission of unconditioned stimulus post CS; L: left; R: right; MNI: Montreal Neurological Institute standard brain; SUIT: Spatially unbiased atlas template of the cerebellum; TFCE t: threshold-free cluster-enhanced test-statistic.

Cerebellar cortex, DCN and VTA fMRI activations to the unexpected omission of the US. Cerebellar cortex activations are shown on cerebellar flatmaps (SUIT20), while VTA and DCN activations are displayed in coronal slices progressing from posterior to anterior with MNI y-coordinates indicated on the left-bottom for each slice. A 3D rendering in the bottom right shows the slice locations within the probabilistic DCN and VTA atlas (see methods for atlas generation). A-D: Event-based contrasts of the first 3 unexpected omissions showed activations in the cerebellar cortex, DCN and VTA for all four phases. Activations in the DCN during reextinction were trend-level. Activations were most consistent in left lobule VI and Crus I, with DCN activation mainly in the left dentate, while VTA activations were bilateral. E-H: Parametric modulation contrasts related to prediction errors from unexpected US omissions showed activations in all four phases in the cerebellum, DCN and VTA. Activations in reacquisition and reextinction were trend-level. Activations during the recall test in the DCN and VTA were trend- level. Activations were most consistently found in lobule VI and Crus I, in addition activations were found in the vermis, Crus II and lobule VIIb. VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed nucleus; FN: Fastigial nucleus; CS: Conditioned stimulus; noUSpCS: Omission of unconditioned stimulus post CS; L: left; R: right; MNI: Montreal Neurological Institute standard brain; SUIT: spatially unbiased atlas template of the cerebellum; TFCE t: threshold-free cluster-enhanced test-statistic.

Functional connectivity analysis using PPI and DCM. A,B: Trend-level activations in the cerebellum found by PPI analysis using the VTA as a seed region, shown on cerebellar flatmaps 20. To highlight activations, a zoomed cutout of lobule VI and Crus I is also shown. A: Trend-level connectivity with VTA is found in lobule VI and Crus I for extinction training, the recall test, reacquisition and reextinction using PPI of the unexpected US omission contrasts from the parametric modulation analysis with a VTA seed. B: Summary of trend-level PPI results by summing all contrasts in A. C-E: DCM analysis showed significant modulation of cerebellar cortex (CB) to VTA connections with prediction errors during unexpected omission events. C: DCM model is shown, modulations act on both the CB/DCN to VTA as well as the VTA to CB/DCN connections. Events, i.e., US post CS+, no US post CS+, no US post CS-, CS+ and CS- for each phase, are provided as inputs to both nodes. Colors of bars are determined by the posterior probability calculated in the PEB analysis (very strong: P>0.99; strong: 0.95<P<0.99; moderate: 0.75<P<0.95; weak: P<0.75). Very strong and strong results are considered significant, moderate results are considered trend-level. D: Significant modulations during extinction training, the recall test and reacquisition. The strongest result was found during extinction training in the connection from the cerebellum to VTA. E: Trend-level results showed connectivity from the DCN to VTA in extinction, and from the VTA to DCN in reextinction. PPI: Psychophysiological interaction; DCM: Dynamic causal modeling; PEB: Parametric Empirical Bayes; PE: Prediction error; P: Posterior probability; VTA: Ventral tegmental area; CB: Cerebellar region in lobule VI and Crus I consistently active during unexpected US omissions; US: unconditioned stimulus; L: left; R: right; SUIT: spatially unbiased atlas template of the cerebellum; t: test-statistic.

A: Overview of the three-day fear conditioning paradigm. Day 1 consists of habituation and acquisition training, day 2 consists of extinction training, and day 3 consists of the recall test, reacquisition, reextinction and the unexpected US phase. In acquisition training, the CS+ is reinforced 100% of the time, while reacquisition and the unexpected US phases contain 3 unexpected US omissions and 3 unexpected US presentations, respectively, with respective reinforcement rates of 83.3% and 21.4%. Extinction, the recall test, reacquisition and reextinction are marked in purple to indicate that these phases are of interest when studying unexpected US omissions. Extinction, recall test and reextinction show a gradient, as in these phases the US omissions are gradually less unexpected (as opposed to distinct unexpected US omission events during the partially reinforced reacquisition phase). 5B: Schematic of participant lying in the MRI scanner. EDA was measured on the left-hand hypothenar eminence (EDA; blue and purple cables). The US was applied to the first dorsal interosseous muscle area of the right hand; red cable). The button box was held in the right hand. Dielectric bags and cushions were included to reduce susceptibility artifacts and head movement. The participants viewed a screen placed at the end of the scanner bore through mirrors, and the eyes were simultaneously imaged and tracked by an infrared camera. 5C: Mean absolute prediction error values for each trial estimated from SCRs, with CS+ and CS- responses paired in blocks. Reinforcement of the CS+ by a US (CS+/US) is indicated by filled squares. Prediction and prediction error values were estimated over 200 iterations. CS: Conditioned stimulus; US: Unconditioned stimulus; EDA: Electrodermal activity.

Modeling. Parameters the grid search was run for.

Pupil size responses for each trial, with CS+ and CS- responses paired in blocks. Bar plots on the top right show early and late mean responses for both CS+ and CS-. Reinforcement of the CS+ by a US (CS+/US) is indicated by filled squares. On day 1, there was no differentiation between CS+ and CS- in the habituation phase, with significant differentiation emerging during acquisition training. On day 2, there was a loss of differentiation during extinction training. On day 3, during initial recall, participants exhibited spontaneous recovery, i.e., a return of differential responses after extinction training. During initial reacquisition, there were again differential responses to the CS+ and CS-, which decreased in reextinction and the unexpected US phase. Mean values are shown with error bars representing the standard error of the mean. CS: Conditioned stimulus; US: Unconditioned stimulus; PSR: Pupil size response.

Non-parametric ANOVA results for pupil size responses

Non-parametric ANOVA results for skin conductance responses

Self-reports results summary

Median and interquartile range of arousal, fear, US expectancy, and valence ratings post habituation, acquisition training, extinction training, recall test and at the end of day 3.

Non-parametric ANOVA results for self-reports

Results of non-parametric ANOVA-type statistic for arousal, fear, valence, and US expectancy ratings.

FMRI activations related to the prediction and presentation of the US. TFCE and FWE corrected.

FMRI activation clusters (p<0.05, FWE and TFCE corrected) related to the prediction and presentation of the US in the cerebellar cortex, DCN and VTA (Figure 2). Up to three maxima in each cluster are shown separated by at least 8 mm. Coordinates are indicated in MNI space (x,y,z). The cluster size is shown by number of voxels (volume of 1 voxel = 3.375 mm3). US: Unconditioned stimulus; CS: Conditioned stimulus; VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed Nucleus; FN: Fastigial nucleus; MNI: Montreal Neurological Institute standard brain; TFCE t: Threshold-free cluster-enhanced t- statistic; pFWE: family-wise error corrected p-value.

FMRI activations related to the prediction and presentation of the US. Uncorrected.

FMRI activation clusters (p<0.05, uncorrected) related to the prediction and presentation of the US in the cerebellar cortex, DCN and VTA (Figure 2). Up to three maxima in each cluster are shown separated by at least 8 mm. Coordinates are indicated in MNI space (x,y,z). The cluster size is shown by number of voxels (volume of 1 voxel = 3.375 mm3). US: Unconditioned stimulus; CS: Conditioned stimulus; VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed Nucleus; FN: Fastigial nucleus; MNI: Montreal Neurological Institute standard brain; t: t-statistic; punc: uncorrected p-value.

FMRI activations related to the unexpected omission of the US. TFCE and FWE corrected.

FMRI activation clusters (p<0.05, FWE and TFCE corrected) related to the unexpected omission of the US in the cerebellar cortex, DCN and VTA (Figure 3). Up to three maxima in each cluster are shown separated by at least 8 mm. Coordinates are indicated in MNI space (x,y,z). The cluster size is shown by number of voxels (volume of 1 voxel = 3.375 mm3). US: Unconditioned stimulus; CS: Conditioned stimulus; VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed Nucleus; FN: Fastigial nucleus; MNI: Montreal Neurological Institute standard brain; TFCE t: Threshold-free cluster-enhanced t-statistic; pFWE: family-wise error corrected p-value.

FMRI activations related to the unexpected omission of the US. Uncorrected.

FMRI activation clusters (p<0.05, uncorrected) related to the unexpected omission of the US in the cerebellar cortex, DCN and VTA (Figure 3). Up to three maxima in each cluster are shown separated by at least 8 mm. Coordinates are indicated in MNI space (x,y,z). The cluster size is shown by number of voxels (volume of 1 voxel = 3.375 mm3). US: Unconditioned stimulus; CS: Conditioned stimulus; VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed Nucleus; FN: Fastigial nucleus; MNI: Montreal Neurological Institute standard brain; t: t-statistic; punc: uncorrected p-value.

FMRI activations related to PPI connectivity during unexpected US omissions with a VTA seed. Uncorrected.

FMRI PPI positive and negative activation clusters (p<0.05, uncorrected) related to connectivity with the VTA during unexpected omission of the US in the cerebellar cortex and DCN (Figure 4). Up to three maxima in each cluster are shown separated by at least 8 mm. Coordinates are indicated in MNI space (x,y,z). The cluster size is shown by number of voxels (volume of 1 voxel = 3.375 mm3). US: Unconditioned stimulus; CS: Conditioned stimulus; VTA: Ventral tegmental area; DCN: Deep cerebellar nuclei; DN: Dentate nucleus; IN: Interposed Nucleus; FN: Fastigial nucleus; MNI: Montreal Neurological Institute standard brain; t: t- statistic; punc: uncorrected p-value.