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+ were 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 of context acclimatization, the 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+ were 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, NC response latency between the CSs differed in 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. Days 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 to photoactivate the SST+ vlPAG cells. The optogenetic manipulation was performed 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 labeling 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 an 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 fiber 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-fiber 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 in 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 inhibition of analgesia induced during defensive states.

Cued fear-conditioned analgesia behavior.

a. During habituation, the freezing levels for the context (BL) and the two tones were low but significantly different (****, P = 0.0003, Friedman test, n = 12 mice). b. Mean temperature at which the NC response was observed in the HP test. The temperature of NC response was higher during CS+ trials when compared to the CS- trials (***, P = 0.0005, Wilcoxon matched-pairs signed rank test, n = 12 mice). c. Schematic representation of how the back and tail temperature of the mice was measured. Every 30-sec three-point temperature was taken for each of the two body parts. The temperature of the mice back and tail were measured by the infrared digital thermographic camera and analyzed offline (see Methods). The average temperature of the mice back (d) and tail (e) while the CS+ or the CS- were presented. There were no differences in body temperature for the different CSs trials (ns, P > 0.05, mixed-effects model, n = 13 mice). Vertical dashed lines correspond to the average time of NC response for the CS+ (red) and CS- (blue) during the standard CFCA protocol.

CFCA depends on associative processes.

a. Top, protocol for the No Tone: naïve mice were submitted to two HP trials without conditioning nor tone presentation. Bottom, protocol for the Tone: naïve mice were submitted to two HP trials paired with an unconditioned tone presentation. b, c. Mean latency and temperature of NC response for the two tests mentioned above and the CFCA. The transient cued-fear induced analgesia (CS+) compared to the basal nociception, the tone, and the CS- trials (***, P = 0.0001, Krustal-Wallis test). Trials between the same type of test (No tone or Tone) were not significantly different (ns, P > 0.05, Bonferroni post-hoc test, see statistical table for all comparisons) d. After fear extinction, there was no difference in the mean temperature response between the two CSs (ns, P = 0.3276 paired t-test, n = 10 mice). e. Protocol for stability training (see methods). Mice were submitted to two rounds of the CFCA paradigm. During retrieval (f, g), average freezing values during CS+ was higher than CS- or baseline (BL) periods (****, P < 0.0001, one-way RM ANOVA, n = 10 mice). h, i. Mean latency and temperature of NC response during CS- and CS+ trials for FC HP test 1 (h) and test 2 (i). CS+ presentation increased the latency and temperature of NC response in FC HP test 1 and test 2 (*, **, and ***, P < 0.05, paired t-test; for details, see Supplementary Table 1).

Two distinct neuronal populations in SST-Cre mice within the PAG.

a. Representative picture of single-molecular fluorescent in situ hybridization for Sst mRNAs in the PAG. Scale bar, 400 µm. b, c. Single-molecular fluorescent in situ hybridization for Sst (green) and Cre (red) mRNAs in the vlPAG (left). Histograms showing the co-expression of Sst/Cre as percentage of Sst-expressing cells (green) and as percentage of Cre-expressing cells (red) in the dlPAG and vlPAG (right). Scale bar, 20 µm. In the dlPAG, 94% of RNA SST-expressing-cells (101 out of 107 cells) expressed Cre RNA, and 84 % of RNA Cre-expressing cells expressed somatostatin RNA (101 out of 20 cells). In the vlPAG, the values are 88% (74 out of 84 cells) and 99% (74 out of 75 cells) for Cre and SST, respectively. d. Single-molecular fluorescent in situ hybridization for Sst (red) and Slc32a1 (green) within the dlPAG (upper panel) and vlPAG (bottom panel). Scale bar, 20 µm. e. Quantification of colocalization within the dlPAG (upper panel) and vlPAG (bottom panel) of Sst+ and Slc32a1+. In the dlPAG, approximately 19% of Slc32a1+ cells (17 out of 80 cells) are Sst+, and 17% (17 out of 88 cells) of Sst+ cells are Slc32a1+. On the contrary, in the vlPAG, approximately 95% of Slc32a1+ cells (183 out of 186 cells) are Sst+, and 61% of Sst+ cells (183 out of 254 cells) are Slc32a1+. f. Single-molecular fluorescent in situ hybridization for Sst (red) and Slc17a6 (green) within the dlPAG (upper panel) and vlPAG (bottom panel). Scale bar, 20 µm. g. Quantification of colocalization within the dlPAG (upper panel) and vlPAG (bottom panel) of Sst+ and Slc17a6+. In the dlPAG, approximately 91% of Slc17a6+ cells (54 out of 61 cells) are Sst+, and 38% of Sst+ cells (54 out of 172 cells) are Slc17a6+. On the contrary, in the vlPAG, approximately 34% of Slc17a6+ cells (43 out of 176 cells) are Sst+ and 33% of Sst+ cells (43 out of 115 cells) are Slc17a6+. White arrows indicate colocalization.

Comparable fear levels prior to the FC HP test.

After retrieval, fear conditioning levels between the opsins and their respective GFP groups were tested to ensure equivalent fear levels. The discrimination index (see methods) between the GFP and ChR2 (a, ns, P = 0.6052 unpaired t-test, n = 16 mice) or ArchT (b, ns, P = 0.4575, unpaired t-test, n = 23 mice) were not significantly different. All the mice discriminated equally between CSs. The conditioning index (see methods) between the GFP and ChR2 (c, ns, P = 0.2333, unpaired t-test n = 16 mice) or ArchT (d, ns, P = 0.1320, unpaired t-test, n = 23 mice) were also not significantly different. All mice displayed a similar high freezing level to the CS+.

Optogenetic manipulation of SST+ vlPAG cells does not affect locomotion or produce aversion.

a. Photoactivation of SST+ vlPAG cells abolished the analgesic effect induced during defensive states (**, P = 0.0035, opsin x CS - two-way RM ANOVA, n = 7 GFP, n = 9 ChR2). The mean temperature of NC response for the CS+ was significantly different between the ChR2 and GFP groups (*, P = 0.0322, Bonferroni post-hoc test). For the ChR2 group, the mean temperature of NC response during CS+ was equivalent to the CS- (ns, P = 0.1783, Bonferroni post-hoc test). On the contrary, the mean temperature of NC response between the CSs differed for the GFP group (****, P < 0.0001, Bonferroni post-hoc test). b. Photoinhibition of SST+ vlPAG cells increased the analgesic effect for the ArchT group when compared to the GFP (**, P = 0.0035, opsin x CS - two-way RM ANOVA, n = 12 GFP, n = 11 ArchT). The mean temperature of NC response for the CS- was significantly different between the ArchT and GFP groups (****, P < 0.0001, Bonferroni post-hoc test). For the GFP group, the mean temperature 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). c. The photoactivation of SST+ vlPAG cells was performed during the ON epoch (blue shaded area), and the average distance traveled during the ON epoch was not different from the OFF epochs when comparing the two opsins (ns, P = 0.5121, opsin x light - two-way RM ANOVA, n = 7 GFP, n = 9 ChR2). d. The photoinhibition of SST+ vlPAG cells was performed during the ON epoch (yellow shaded area), and the average distance traveled during the ON epoch was not different from the OFF epochs when comparing the two opsins (ns, P =0.4047, opsin x light - two-way RM ANOVA, n = 12 GFP, n = 11 ArchT). e. Real-time place-preference location plot from a representative animal while submitted to optogenetic activation of SST+ vlPAG cells in the left compartment throughout the 15-min session. f. There was no difference between ChR2-expressing mice and the control group in the time spent in the stimulated and non-stimulated compartments (ns, P = 0.1812, Mann-Whitney test).

SST+ vlPAG cells photoactivation during conditioning does not impact fear learning.

a. Protocol for optogenetic manipulations during fear conditioning. On Day 2, mice received 5 CS-US associations. The US was either optogenetic stimulation alone or optogenetic stimulation plus footshock. b. Average freezing levels during the CS-US association of optogenetic stimulation alone. There was no difference in the overall freezing levels for the CS-and CS+ between ChR2 and GFP (ns, P = 0.4804, opsin x CS - two-way RM ANOVA, n = 6 GFP, n = 7 ChR2). c. During retrieval, there was no effect on freezing upon activation of SST+ vlPAG cells as an US (ns, P = 0.4804, opsin x CS effect - two-way RM ANOVA, n = 6 GFP, n = 7 ChR2). d. Average freezing levels during the CS-US association of optogenetic stimulation plus footshock. There was no difference in the overall freezing levels for the CS+ and CS- between ChR2 and GFP (ns, P = 0.8663, opsin x CS effect - two-way RM ANOVA, n = 6 GFP, n = 7 ChR2). e. During retrieval, there was no difference in the fear expression by the activation of the SST+ vlPAG cells (ns, P = 0.8967, opsin x CS effect - two-way RM ANOVA, n = 6 GFP, n = 7 ChR2).

Optogenetic effect observed during CFCA is not due to alteration of somatostatin levels nor mediated by VIP+ vlPAG cells.

a. Sst-IRES-Cre heterozygotic mice received a bilateral injection of opsins in the vlPAG, and optic fibers were implanted above the vlPAG. b. A representative example of expression patterns of ChR2 within SST+ vlPAG cells. c. Average freezing values during retrieval. ChR2 and GFP groups were pooled together because no differences were found in the conditioning level (data not shown). The average freezing values during CS+ were higher than CS- or baseline (BL) periods (****, P < 0.0001, Friedman test, n = 13 mice). d. Optogenetic activation of SST+ vlPAG cells abolished the analgesic effect induced by defensive states (**, P = 0.0048, opsin x CS - two-way RM ANOVA, n = 6 GFP, n = 7 ChR2). The mean latency of NC response for the CS+ was significantly different between the ChR2 and GFP groups (*, P = 0.0300, Bonferroni post-hoc test, n = 6 GFP, n = 7 ChR2). For the ChR2 group, the latency of NC response during CS+ was equivalent to the CS- (ns, P = 0.3342, Bonferroni post-hoc test). On the contrary, the latency of NC response between the CSs was different for the GFP group (****, P = 0.0001, Bonferroni post-hoc test). e. VIP-IRES-Cre mice received a bilateral injection of opsins in the vlPAG, and optic fibers were implanted above the vlPAG. f. Representative example of the expression pattern of ArchT within VIP+ vlPAG cells. g. ArchT and GFP groups were pooled together because no differences were found in the conditioning level (data not shown). The average freezing values during CS+ were higher than CS- or BL periods (****, P < 0.0001, one-way RM ANOVA, n = 10 mice). h. Optogenetic inhibition of VIP+ cells did not change the analgesic effect for the ArchT group when compared to the GFP (ns, P = 0.5455, opsin x CS effect - two-way RM ANOVA, n = 5 GFP, n = 5 ArchT). The latency of NC response was higher for the CS+ trials compared to the CS- trials for both GFP and ArchT (***, P < 0.01, Bonferroni post-hoc test, n = 5 GFP, n = 5 ArchT).

Optogenetic activation of SST+ vlPAG cells did not affect tactile sensitivity.

a. Perievent stimulus time histogram indicating the spiking latency of a purely tactile neuron recorded in the spinal cord in response to gentle brushing of the skin. b. Representative spiking activity latency of a purely tactile neuron recorded in the spinal cord in response to gentle brushing of the skin with or without activation of ChR2 expressing SST+ vlPAG cells. c. The activation of ChR2 expressing SST+ vlPAG cells of tactile neurons recorded in the spinal cord in response to gentle brushing of the skin had no effect on the average firing activity (Paired t-test, P = 0.6929, n = 5 cells).

Optogenetic manipulation of GFP expressing vlPAG cells did not affect analgesia.

a. Single-unit recordings of WDR cells in the lumbar spinal cord during optogenetic manipulation of GFP expressing SST+ vlPAG inputs to the RVM. b. Photostimulation of SST+ vlPAG inputs to the RVM has no effect.

Optical fiber placement for VIP-Cre.

Anatomical location of the tip of the optic fiber for VIP-Cre mice (n = 10).

Optical fiber placement for SST-Cre mice.

Anatomical location of the tip of the optic fiber for SST-Cre mice (n = 39).

Summary of statistical results associated with the Figures and Supplementary Figures