Fear modulation of pain behavior.

a. Schematic of the setup and the CFCA paradigm. The paradigm consists of a discriminative auditory fear conditioning test followed by a pain sensitivity assay (for details, see Methods). b. Average freezing values for CS+ was higher than CS- or baseline (BL) periods during retrieval (****, P < 0.0001, one-way RM ANOVA, n = 12 mice). c. Example of the infrared digital thermographic camera view of a mouse during the FC HP test. After 1 min context acclimatization, plate temperature steadily increases at 6 °C per min (HP start). CSs started 130 s after the HP start and continued until mice displayed an NC response (licking the hindpaw or jumping). d. Example of assessed hindpaw licking NC response. e. Latency of NC response on the FC HP test increased during CS+ compared to CS- (***, P = 0.0005, Wilcoxon matched-pairs signed rank test, n = 12 mice). f. Left: dynamics of freezing probability at CS onset and NC response for both CS- and CS+ (freezing periods for each individual mouse are displayed by rows in grey; error bars display S.E.M; bin size = 0.5 s). Right: average freezing values during 10 s after CS onset and before NC response for CS+ were higher than CS- only at CS onset (***, P onset = 0.0015, paired t-test, n = 12 mice). g. Mean freezing values during fear extinction, 24 CS+ were presented across two separate extinction sessions (joined by dashed lines). Mice acquired the CS+-US association (1st CS+ block vs. BL/CS- ****, P < 0.0001, Friedman test, n = 10 mice), followed by a rapid extinction (5th & 6th block of CS+ vs BL/CS-ns, P > 0.05, one-way RM ANOVA). h. After extinction, there was no difference in the NC response latency for both CSs (ns, P > 0.05, paired t-test n = 10 mice). i. Average NC response latencies for the different HP tests. No tone - mice submitted to the HP test without conditioning nor tone presentation. Tone - mice submitted to the HP test paired with unconditioned tone presentation. CS-/CS+-mice were submitted to the CFCA protocol. (No tone/Tone/CS- vs. CS+ **, P < 0.0001, Krustal-Wallis test). For exact P values and test statistics on this and all subsequent figures, see Supplementary Table 1.

SST+ cells in vlPAG mediate the fear modulation of pain behavior.

a. Sst-IRES-Cre mice were bilaterally injected with viruses that expressed ChR2 (right, upper), ArchT (right, bottom), or GFP (control) in SST+ vlPAG cells. b. Schematics of the stimulation protocol used for photoactivation of the SST+ vlPAG cells: blue light delivered at 2 Hz with 5 ms pulse duration during CSs presentation until NC response. For photoinhibition experiments, green light was delivered continuously. c, e. Average freezing values during retrieval for ChR2-(c) and ArchT-infected mice (e) and their respective GFP controls. The opsin and respective control groups were pooled together because no difference was found in the conditioning level (see Supplementary Figure 4). Average freezing values during CS+ was higher than CS- or baseline (BL) periods (****, P < 0.001, one-way RM ANOVA, (c) n = 16 mice and (e) n = 23 mice). d. Photoactivation of SST+ vlPAG cells abolished the analgesic effect induced by fear (*, P = 0.0112, opsin x CS - two-way RM ANOVA, n = 7 GFP, n = 9 ChR2). The NC response latency for the CS+ was significantly different between the ChR2 and GFP groups (*, P = 0.0344, Bonferroni post-hoc test). For the ChR2 group, the NC response latency during CS+ was equivalent to the CS- (ns, P = 0.3876, Bonferroni post-hoc test). On the contrary, the NC response latency between the CSs differed for the GFP group (***, P = 0.0003, Bonferroni post-hoc test). f. Photoinhibition of SST+ vlPAG cells increased the analgesic effect for the ArchT group when compared to the GFP (*, P = 0.0037, opsin x CS - two-way RM ANOVA, n = 12 GFP, n = 11 ArchT). The NC response latency for the CS- and CS+ was significantly different between the ArchT and GFP group (CS-: ****, P < 0.0001, Bonferroni post-hoc test; CS+: *, P = 0.0265, Bonferroni post-hoc test). For the GFP group, the latency of NC response was higher for the CS+ trials when compared to the CS- trials (***, P = 0.0003, Bonferroni post-hoc test), yet this was not the case for the ArchT group (ns, P > 0.999, Bonferroni post-hoc test).

Activation of SST+ vlPAG cells reduced fear expression

a. Protocol for optogenetic manipulation during fear retrieval. Day 1 and 2 were done as described previously for the CFCA paradigm. During retrieval, there were 12 CS+ presentations divided into three blocks. b. The stimulation protocol used for photoactivation of the SST+ vlPAG cells. The optogenetic manipulation was done during the 2nd block of the CS+ presentation (analogously to CFCA manipulations, Figure 2b). c. Light inhibition of SST+ vlPAG cells did not modulate freezing levels (ArchT ns, P = 0.9396, opsin x CS two-way RM ANOVA, n = 6, GFP, n = 8). d. Photoactivation of the SST+ vlPAG cells had no effect on the GFP group, but it transiently decreased the freezing levels for the ChR2 group (***, P < 0.0001, opsin x CS two-way RM ANOVA, n = 9 GFP, n = 8 ChR2).

Manipulation of SST+ vlPAG cells alters spinal cord-related pain signals.

a. Schematics of in vivo anesthetized experiments. Extracellular local field potential and single-unit recordings in the spinal cord DH were performed during optogenetic stimulation of SST+ vlPAG cells with concomitant noxious electrical paw stimulation. b. Example of average nociceptive field potentials in the lumbar SC before (OFF), during (ON), and after (OFF) photoactivation of SST+ vlPAG cells (top). Photoactivation induces a significant increase in the nociceptive fields (bottom; **, P = 0.0001, one-way RM ANOVA, n = 8 mice). c. Analogous to panel b, but for photoinhibition of SST+ vlPAG cells. Photoinhibition induces a significant decrease in the nociceptive fields (bottom; *, P = 0.0275, one-way RM ANOVA, n = 5 mice). d. Example of WDR single-unit activity before and during photoactivation of SST+ vlPAG cells (top). Photoactivation of SST+ vlPAG cells induces a significant and global increase in WDR response to both Aδ- and C-mediated nociceptive fibers (bottom; **, PAδ fiber = 0.0022, paired t-test, n = 11 cells; **, PC fiber < 0.0033, paired t-test, n = 11 cells). e. Analogous to panel d, but for photoinhibition of SST+ vlPAG cells. Photoinhibition induces a significant and specific inhibition of WDR response to Aδ- and C-fibers (bottom; *, PAδ fiber = 0.0241, ****, PC fiber < 0.0001, n = 14 cells, Wilcoxon matched-pairs signed rank test). f. Example traces of single-unit recordings of WDR cells with subthreshold electrical stimulation accompanied by photoactivation of SST+ vlPAG cells (top). The photoactivation of SST+ vlPAG cells elicits WDR response to A- and C-mediated peripheral fibers (bottom; *, PAδ fiber = 0.0156, n = 10 cells; **, PC fiber = 0.0020, n = 10 cells, Wilcoxon matched-pairs signed rank test).

SST+ vlPAG-RVM-DH pathway activation removes analgesia.

a. Schematics of possible SST+ vlPAG circuits mediating analgesia by long-range projections directly to the SC or, alternatively, by projecting to the SC via the rostral ventromedial medulla (RVM). b. Sst-IRES-Cre mice were injected with an anterograde AAV Cre-dependent GFP virus in the vlPAG (left). Example of SST fibers labelling at the level of the RVM (middle). Higher magnification (right) reveals putative axonic buttons in the RVM (examples indicated by white arrows). c. Sst-IRES-Cre mice were injected concomitantly with anterograde AAV Cre-dependent GFP virus in the vlPAG and retrograde fluorogold in the lumbar DH of the SC. d. Fluorogold positive cells (red) cross SST+ vlPAG fibers in the RVM (green). Higher magnification (right) shows close contacts between the putative SST+ button and fluorogold+ cells or fibers (white arrows). e. Sst-IRES-Cre mice were injected in the vlPAG with AAV Cre-dependent ChR2 virus and optic fiber placed above the lumbar SC. Single-unit recordings of WDR cells while photoactivation of the SST+ vlPAG SC fibers and electrically stimulating the paw in anesthetized mice (left). Photoactivation of vlPAG projections to SC did not affect nociceptive transmission (right; ns, P = 0.3683, paired t-test, n = 11 cells). f. Same experimental design as in panel e, except that the optic fiber was placed above the RVM (left). Photoactivation of SST+ vlPAG inputs to the RVM induces a significant increase in WDR response to both Aδ- and C-mediated nociceptive fibers stimulation (middle; *, PAδ fiber = 0.0346, Paired t-test, n = 10 cells; **, PC fiber = 0.0020, Wilcoxon matched-pairs signed rank test, n = 10 cells). Removal of analgesia by activation of SST+ vlPAG-RVM induces a significant increase in WDR response to C-fibers responses (right; **, P = 0.0078, Wilcoxon matched-pairs signed rank test, n = 9 cells).

SST+ vlPAG-RVM-SC pathway activation abolishes analgesia induced by CFCA.

a. Sst-IRES-Cre mice were bilaterally injected with an AAV expressing ChR2 or GFP in the vlPAG, and optic fibers were implanted above the RVM (left). Representative example of SST+ vlPAG terminals in the RVM (right). b. Photoactivation of SST+ vlPAG-RVM pathway did not modulate freezing levels (ns, P = 0.6443, opsin x CS two-way RM ANOVA; CS+ 2nd block, GFP vs. ChR2 ns, P > 0.9999, Bonferroni post-hoc test, n = 7 GFP, n = 6 ChR2). c. Average freezing values during retrieval for pooled ChR2 and GFP-infected mice (groups were pooled together since no difference was found in the conditioning level). Average freezing values during CS+ were higher than CS- or baseline (BL) periods (****, P < 0.0001, one-way RM ANOVA, n = 14 mice). d. Photoactivation of SST+ vlPAG-RVM pathway abolished the analgesic effect modulated by fear (**, P = 0.0063, opsin x CS - two-way RM ANOVA, n = 7 GFP, n = 7 ChR2). For the ChR2 group, the NC response latency during CS+ was equivalent to the CS- (ns, P > 0.9999, Bonferroni post-hoc test). On the contrary, the NC response latency between the CSs differed for the GFP group (****, P < 0.0001, Bonferroni post-hoc test). e. Working model schematics for SST+ control analgesia induced during defensive states. Activation of SST+ cells in the vlPAG is associated with the inhibition of fear-induced freezing behavior. Selective activation of RVM projecting SST+ vlPAG cells is associated with an inhibition of analgesia induced during defensive states.