Physiological and Behavioral Measures of Threat Learning
Participants explored a virtual environment (Fig. 1A and B; see Methods for details) that consisted of a mountain landscape and defined two half-zones recognizable by the unique shape of the mountains in the horizon. For each trial, participants freely explored the arena and were instructed to pick up flowers that appeared one at a time in random locations across the environment (approach period). When a participant picked a flower, their position was held stationary for a variable duration (2-8 seconds; stationary period), during which the participant rated the expectancy of receiving a shock (rating of 0-9). Flowers located in one-half of the environment were paired with a shock delivered at the end of the stationary period on 50% of the trials within the danger zone. Flowers in the other half of the environment were never paired with a shock (safe zone). Since all flowers were identical, predictive value (danger or safety) could not be attributed to their physical characteristics.
For analysis, the data were divided by zones (safe, danger) and segregated into 4 learning blocks (10 trials in each). All these physiological and behavioral analyses can be found in the supplementary material.
Skin conductance. We measured participants’ skin conductance level (SCL), as they navigated towards the flower (approach period), and skin conductance responses (SCR), during the stationary period. A 2x4x2 ANOVA (zone x block x group) of both SCL and SCR revealed a significant main effect of zone, due to higher responses in the danger zone as compared to the safety zone (SCL, F(1,49)=16.24, p<0.01; SCR, F(1,49)=4.13, p<0.05; For a full description of skin conductance results, see supplementary material and Figure S1).
Shock expectancy. A 2x4x2 ANOVA (zone x block x group) assessing shock expectancy ratings after picking the flower revealed a significant zone x block interaction (F(3,147)=31.07, p<0.001). This interaction stemmed from consistently (across the 4 blocks) high expectancy ratings of the shock in the danger zone (danger vs safe: t(50)=0.06, p>0.05), while ratings significantly decreased between block 1 and 4 in the safe zone suggesting that there was discrimination learning over blocks for all subjects (block 1 vs 4: t(50)=8.83, p<0.001; For a full description of the results see supplementary material and Figure S1).
fMRI Measures of Threat Learning
Threat appraisal (Anxiety-state): Approaching flowers (approach period) in the danger vs. the safety zone
Each approach period (approaching a flower) began at trial onset, when the flower appeared in the environment and ended when the flower was “collected.” For analyses we excluded the initial orienting period (looking for the flower) of the approach period, only including the last 75% of the approach (active navigation towards the flower).
We compared brain activation (i.e., presumed metabolic activity) between diagnostic groups (ANX vs. HC) as individuals approached flowers located in the danger/safe zones of the environment (see Table S1 for full results from this analysis). To assess discrimination learning, and increase signal-to-noise ratio, trials were divided into two blocks comprising the first- (early) and second-half (late) of the experiment. To directly examine group differences, the first-level analysis contrasted factors of zone (safe vs. danger) and block (early vs. late), whereas the second-level analysis directly compared groups (group: ANX vs HC). Significant peak activation was extracted and analyzed to disentangle the directionality of the results.
An overall group contrast between ANX and HC of approach periods (assessing changes across both zone and block) identified two opposing patterns of activity changes in a range of areas comprising posterior cingulate cortex (PCC; p<0.05 FWE), vmPFC, orbitofrontal cortex (OFC), and bilateral anterior hippocampus (p<0.05 FWE small volume corrected; SVC, Fig. 2A). Compared to the HC group, the ANX group demonstrated a greater increase in activity in these areas from early to late blocks (late > early) of the safe zone, and a decrease in activity from early to late blacks of the danger zone. To further understand these distinct patterns of activity, we next performed direct group comparisons on separate components of the task.
When approaching flowers in the danger zone (danger > safe), the ANX group (ANX > HC) showed greater activation of bilateral insula (p<0.05 FWE) and dmPFC (p<0.05 FWE SVC; Fig. 2B) compared to the HC group. That is, these areas were more responsive in the ANX group when approaching flowers in the danger compared to safe zone. No group differences were observed when looking for areas that showed greater activity when approaching the safe compared to danger zone.
For the HC group, compared to ANX (HC > ANX), approaching flowers in the second half of the experiment, compared with the first half (late > early) showed that, regardless of zone, there was increased activation from early to late blocks in PCC, vmPFC, OFC, and anterior hippocampus (p<0.05 FWE SVC). No other significant results were found (p>0.001).
Next, psychophysiological interactions (PPI) analyses were performed for each participant group separately to identify brain regions in which connectivity changed during the danger vs. safe contrast. PPI examined brain connectivity of each significant cluster (i.e., seed ROI) from the approaching flowers period. In the HC group, a positive association between the dmPFC seed (ROI, MNI coordinated: 9, 26, 45) and bilateral insula (p<0.001 Bonferroni corrected) was found in the contrast (dange > safe On the other hand, in ANX, a negative correlation between the dmPFC-OFC and dmPFC-vmPFC (p<0.001 Bonferroni corrected) was found in the same contrast (danger > safe). No other PPI analysis revealed significant results.
Overall, these results suggest that when approaching flowers in the dangerous zone, ANX showed reduced activation in vmPFC, PCC, and anterior hippocampus while showing greater activation in the insula and dmPFC. These findings are further highlighted by negative connectivity between the dmPFC and vmPFC areas. On the other hand, HC displayed greater activation as a function of time in the vmPFC, OFC, PCC, and anterior hippocampus, regardless of the zone suggesting appropriate contextual learning.
Threat anticipation (Fear-state): Held stationary in the danger vs. the safety zone
We next examined changes in brain activation when participants’ positions were held stationary after picking flowers and anticipating a potential shock. We again examined the effects of zone (danger vs. safe zone) and block (early vs. late blocks) in the first level analysis. The second level analysis consisted of the group comparison (ANX vs. HC; see Table S2 for full results). Analyses followed the same model as for the approach period.
An overall group contrast between ANX and HC during stationary periods (assessing changes across both zone and block) identified two opposing patterns of activity changes in a range of areas comprising PCC (p<0.05 FWE), vmPFC, and OFC (p<0.05 FWE SVC). Compared to the HC group, the ANX group demonstrated a greater increase in activity in these areas from early to late blocks (late > early) of the safe zone, and a decrease in activity from early to late blacks of the danger zone. To further understand these distinct patterns of activity, we next performed direct group comparisons on separate components of the task.
When held stationary after picking a flower located in a zone of the environment associated with danger (danger > safe), the ANX group compared to HC (ANX > HC) showed greater activation in dmPFC, dACC, bilateral insula, caudate, thalamus, amygdala, and midbrain areas, including the periaqueductal gray (PAG; p<0.05 FWE SVC; Fig. 3A). A group contrast (ANX > HC) of stationary periods (irrespective of danger or safety) showed increased dmPFC activation from early to late block (late > early) in ANX compared with HC (p<0.05 FWE SVC).
For the HC group, compared to ANX (HC > ANX), flowers located in a zone of the environment associated with safety (safe > danger) generated greater activation in the PCC (p<0.05 FWE), vmPFC, OFC, and anterior hippocampus (p<0.05 FWE SVC; Fig. 3B). For HC, compared to ANX (HC > ANX), we also found increased activation during the last half of learning (late > early) regardless of zone in vmPFC and OFC (p<0.05 FWE SVC). No other significant results were found (p>0.001).
Given the reported group differences during stationary periods, PPI analyses were next performed for each group separately to identify brain regions in which connectivity changed during danger vs. safe. PPI examined brain connectivity of each significant cluster (i.e., seed ROI) from the stationary period. PPI analyses used dmPFC (MNI coordinates: 0, -8, 71) and each amygdala side as seed regions (MNI coordinates: (R) 26, -2, -15, (L) -20, 2, -15). ANX showed increased functional connectivity between the dmPFC-bilateral insula, left amygdala-bilateral insula, and right amygdala-bilateral insula in danger compared to safe zones (p<0.001 Bonferroni corrected). HC showed increased functional connectivity between the dmPFC-OFC, left amygdala-bilateral insula, right amygdala-bilateral insula, left amygdala-vmPFC, right amygdala-vmPFC, left amygdala-OFC, and right amygdala-OFC in danger compared to safe zone during the stationary period (p<0.001 Bonferroni corrected). No other PPI analyses revealed significant results.
In summary, during the stationary period in the danger zone (after collecting a flower) ANX, compared to HC, demonstrated reduced activation in vmPFC and PCC over time, with greater activation in the insula, amygdala, and PAG. This was further highlighted by increased connectivity among the dmPFC, amygdala, and insula while lacking any significant connectivity from vmPFC areas. On the other hand, HC’s recruitment of the vmPFC, OFC, and PCC was seen as a function of time, where those areas were recruited during the stationary periods in both the safe and dangerous zones. Furthermore, HC had significant connectivity of the vmPFC and OFC to areas such as the dmPFC and amygdala.