dPAG single unit recordings during risky foraging.

(A) Rats underwent pre-robot, robot, and post-robot sessions, successfully securing pellets in pre- and post-robot trials, and failing during robot trials due to robot interference. (B) Tetrode implantation in dPAG with a photomicrograph of the tip (arrowhead). (C) Outbound foraging time increased significantly in the robot session (Χ2 = 64.00, P < 0.0001, Friedman test; Ps < 0.05 for all comparisons, Dunn’s test). ***P < 0.001 compared to pre-robot and post-robot sessions. #P < 0.05 compared to the pre-robot session. (D) The pellet success rate significantly decreased during robot session (Χ2 = 84.00, P < 0.0001, Friedman test; Ps < 0.0001 for all comparisons, Dunn’s test). ***P < 0.001 compared to pre-robot and post-robot sessions. (E) Cell type proportions revealed that 23.4% cells responded to robot activation (robot cells). (F) Representative dPAG robot cell raster/event histograms aligned with robot activations. (G) Population activity of robot cells around the time of robot activation (t = 0) with 0.1 s and 1 s bins. Firing rates of the robot cells were higher during robot session (0-3 s blocks; Friedman test, all Χ2s > 6.952, all Ps < 0.05; Dunn’s test, all Ps < 0.05). Shaded areas indicate SEM. **P < 0.01 compared to pre-robot session. #, ##, and ### denote P < 0.05, P < 0.01, and P < 0.001 respectively, compared to post-robot session.

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dPAG optical stimulation evokes fear.

(A) Virus injection, optrode implantation in dPAG, and light stimulation during single unit recordings in anesthetized rats. (B) Raster plots and peri-event time histograms for 20-Hz light stimulations (10-ms width, left; 2-s duration, center). 48% of 25 units had increased firing during 2-s light stimulation (right). (C) Virus injection, expression, and optic fiber placement in dPAG. (D) Stimulation testing: baseline trials at 75-cm distance (Long) without light; stimulation trials with 2-s light as the rat approached (∼25 cm) Long pellet; light applied as the rat approached 25-cm (Short) pellet if Long pellet unsuccessful. (E) Representative trajectories for EYFP- and ChR2-expressing rats during stimulation testing. (F) Rat behaviors during light stimulation trials. (G) ChR2 rats showed increased latency to procure pellet upon opto-stimulation (OnL) compared to EYFP rats (OffL, Z = 1.013, P = 0.311; OnL, U = 0.0, P < 0.001; Mann-Whitney U test). (H-J) ChR2 group exhibited increased latency to procure pellet compared to EYFP group based on stimulation intensity (H; Us < for all intensities < 3.5, Ps for all intensities < 0.025; Mann-Whitney U test), frequency (I; Us for 10 Hz and 20 Hz < 2.5, Ps for 10 and 20 Hz < 0.014; Mann-Whitney U test), and duration (J; Us for all durations < 4.5, Ps for all durations < 0.032; Mann-Whitney U test). *, **, and *** denote P < 0.05, P < 0.01, and P < 0.001, respectively.

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dPAG optical stimulation and amygdala recordings.

(A) Virus injection in dPAG and tetrode array implantation targeting BLA. Light stimulation during single-unit recordings in freely-moving rats. (B) Stimulation testing sessions: in pre- and post-stim trials, rats freely procured pellets; in stim trials, optical stimulation prevented procurement of pellets. (C,D) During dPAG stimulation, animals showed increased outbound foraging time (C; Χ2 = 117.8, P < 0.0001, Friedman test; Ps < 0.0001, Dunn’s test) and decreased success rate (D; Χ2 = 154.0, P < 0.0001, Friedman test; Ps < 0.0001, Dunn’s test). ****P < 0.0001 compared to pre-robot and post-robot sessions. ####P < 0.0001 compared pre-robot session. (E) Subset of BLA units (10.0%) responsive to optical stimulation (Stim cells; left), and a representative (center) and all stimulation-responsive (nonStim cells; right) raster plots with PETHs. (F) Subset of animals underwent additional robot trials following the post-stim session. (G) Increased outbound foraging time during robot session compared to post-stimulation session (t(15) = 6.655, P < 0.0001; paired t-test). ****P < 0.0001. (H) Twenty-two BLA units were dPAG stimulation-responsive. (I) Representative raster plots of dPAG stimulation-responsive and -nonresponsive units. (J) Proportions of robot vs. non-robot cells differed between stimulation-responsive and -nonresponsive units (Χ2 = 11.134, P < 0.001; Chi-squared test). ***P < 0.001. (K) PETHs of stim and non-stim cells during stimulation and robot sessions. (L) Relationship between maximal firing rates during first 500 ms subsequent to robot activation and maximal firing rates during first 500 ms after stimulation onset (r(85) = 0.405, P < 0.001; Pearson correlation). (M) Population CCs with significant synchrony during robot session were higher than other sessions. Dotted vertical lines indicate 0-100 ms window for testing significance. Grey, blue, and dark yellow ***P < 0.001 compared to pre-stimulation, stimulation, and post-stimulation sessions, respectively. (N) Among synchronized BLA cell pairs, those including dPAG stimulation-responsive cell(s) (stim pairs; 61.5%) showed increased correlated firings (area under the curve, AUC, during 0-100 ms window) during the robot session compared to other sessions. In contrast, synchronized BLA cell pairs that consisted of stimulation non-responsive cells only showed no AUC differences across sessions. Grey *, blue **, and dark yellow **P < 0.05 compared to pre-stimulation, P < 0.01 compared to stimulation, and P < 0.001 compared to post-stimulation sessions, respectively. (O) Comparing CCs during testing windows (0-50 ms and 50-100 ms) between stim and non-stim pairs, stim pairs exhibited higher correlated firing than non-stim pairs during the 0-50 ms block (t(21.99) = 2.342, P = 0.0286; t-test), while displaying decreased correlated firings in the second block (50-100 ms; U = 42, P = 0.045; Mann-Whitney U test). *P < 0.05 compared to the non-stim pairs.

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PVT’s hypothesized role in dPAG to amygdala signaling and anti-predatory behavior model.

(A) CTB retrograde tracer and AAV-CaMKII-EYFP used to trace dPAG signals to the BLA. (B) Representative images of AAV and CTB expressions in dPAG and BLA, respectively. (C) Robot encounters hindered pellet procurement compared to foraging-only rats (Base, U = 26, P = 0.6926; Test, U = 0, P = 0.0001; Mann-Whitney U test). ***P < 0.001 compared to the foraging-only group. (D) Terminal expressions of AAV injected into the dPAG cell bodies were predominantly observed in the midline nuclei of the thalamus. (E) PVT showed higher c-Fos-positive cells in robot-experienced rats (n = 10) compared to foraging-only control rats (n = 6) (U = 4.0, P = 0.0027; Mann-Whitney U test), while other midline thalamic areas showed no differences (t(14)s < 1.611, Ps > 0.129; t-test). **P < 0.01 compared to the foraging-only group. Values were normalized to the mean of the corresponding control group. (F) Representative photomicrographs of PVT c-Fos staining from foraging-only (upper) and robot-experienced (bottom) rats. (G) Representative microphotographs of AAV, CTB, c- Fos, and triple staining in PVT comparing foraging-only and robot-experienced animals. (H) Robot exposure increased the percentage of CTB labelled PVT neurons expressing c-Fos (t(9) = 3.171, P = 0.0113), while CTB density levels were comparable between the two groups (robot-experienced, n = 5; Foraging-only, n = 6; t(9) = 0.9039, P = 0.3896; t-test). Values were normalized to the mean of the corresponding control group. (I) Proposed model: Predator surge detection via visual pathways, e.g., superior colliculus (Furigo et al. 2010, Rhoades et al. 1989), leads to dPAG activation, signaling through PVT to excite BLA. The BLA then projects to regions controlling escape responses, such as dorsal/posterior striatum (Li et al. 2021, Menegas et al. 2018) and ventromedial hypothalamus (Silva et al. 2013, Kunwar et al. 2015, Wang, Chen, and Lin 2015).