dLS neurons are both sufficient and necessary for causing acute restraint-induced analgesia.

(A) Schematic representing the Restraint Stress (RS) assay used to induce stress in mice. (B) cFos expression in the dLS of restrained mice compared to unrestrained control mice (Green-cFos, Red-neurotrace (nuclear dye specific to neuronal nuclei)). (C) Total number of cFos+ cells in the dLS (area marked by dotted lines) in restrained vs unrestrained mice (6.40±1.21 compared to 56.40±3.09, respectively; t-test, ***P=0.0001, n=5). (D) Multiplex In Situ hybridization with VGlut2 and VGat (red-VGat, green-VGlut2), highlighted by arrowheads (left), with zoom-in view of a rare VGlut2+ (green) cell (right). (E) Gad67-Cre and DIO-hM3Dq-mCherry/DIO-tdTomato were injected in the dLS of wild-type mice. (F) i.p. DCZ and not Saline evoked cFos expression (green) in the mCherry-positive cells (red) in the dLSGad67-hM3Dq neurons. (G) Tail-flick latency (seconds) (2.28±0.16 compared to 2.8±0.16, respectively; t-test, **P=0.0056, n=7) post-saline or DCZ administration in dLSGad67-hM3Dq mice, with no significant difference in dLSGad67-tdTomato mice. (H) Licks (8.71±1.76 compared to 4.14±0.74, respectively; t-test, *P=0.0335, n=7), and latency to lick (seconds) (15.57±2.03 compared to 22.86±2.26, respectively; t-test, *P=0.0338, n=7) on the hot-plate test of dLSGad67-tdTomato mice, administered with either i.p. Saline or DCZ, with no significant difference in dLSGad67-tdTomato mice. (I) Gad67-Cre and DIO-Tettox-GFP/DIO-GFP were co-injected in the dLS of wild-type mice. (J) GFP-positive neurons (green) seen in the dLS. (K) Tail-flick latency (seconds) (2.08±0.14 compared to 3.9±0.15, respectively; t-test, ***P=0.0001, n=8) with and without restraint for 1 hour using the RS assay in dLSGad67-GFPmice, with no significant difference in dLSGad67-Tettox-GFP mice. (L) Number of licks (8.57±1.34 compared to 4.57±0.69, respectively; t-test, **P=0.0089, n=7), and latency to lick (seconds) (14.29±1.54 compared to 24.14±1.40, respectively; t-test, ***P=0.0001, n=7) with and without restraint in dLSGad67-GFPmice, with no significant difference in dLSGad67-Tettox-GFP mice.

dLS neurons are engaged by acute stress due to physical restraint.

(A) AAVs encoding Gad67-Cre and DIO-GCaMP6s were co-injected in the dLS, and fiber optic cannulaes were implanted above for recording neural activity. (B) Confirmation of the expression of GCaMP6s (Green) in the dLS neurons. (C) Schematic representation of the fiber photometry system. (D) Representative trace or neural activity from GCaMP6s (green) vs control GFP (blue) mice in the homecage. (E) Neural activity from the dLS while mice were under restraint. (F) Traces of neural activity when mice were allowed to move freely in the homecage (blue), and when they were under restraint (red). Peaks corresponding to neural activity (blue dashes) were seen when mice struggled in the tube. (G) Heatmap depicting neural activity during individual instances of struggles (initiation of struggle indicated by white dotted line). (H and I) Average plots and heatmaps for dLS responses on the hot plate at 32 deg (5 mice, 20 trials; dotted line indicating time point when mice were placed on the hotplate). (J and K) Average plots and heatmaps for dLS engagement on the hot plate at a noxious temperature of 52 deg (5 mice, 20 trials; dotted line indicating time point when mice were placed on the hotplate).

dLS neurons synapse onto a small neuronal population of LHA neurons.

(A) Gad67-Cre and DIO-GFP injected in the dLS of wild-type mice. (B) GFP-positive cells seen in the dLS. (C) GFP-positive terminals from the dLS were seen in the Hippocampus (Hipp), Habenula (Hb) and Lateral Hypothalamus (LHA), marked region on LHA zoomed-in on the bottom. (D) Gad67-Cre and DIO-ChR2-YFP injected in the dLS of wild-type mice to express the excitatory opsin ChR2 in the dLSGad67 neurons. The fiber was implanted in the LHA to facilitate terminal activation. ChR2-YFP-positive terminals were observed in the LHA. (E) Tail-flick latency (seconds) (2.49±0.04 compared to 2.30±0.03, respectively; t-test, ***P=0.0001, n=7) with (ON) and without (OFF) blue light illumination in dLSGad67-ChR2 mice (F) Licks (12.86±1.50 compared to 5.57±0.75, respectively; t-test, ***P=0.001, n=7), and latency to lick (seconds) (12.43±0.72 compared to 22.29±1.34, respectively; t-test, ***P=0.0002, n=7) on the hot plate with (ON) and without (OFF) blue light illumination in dLSGad67-ChR2 mice. (G) AAVRetro-Cre was injected in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato in the dLS of wild-type mice. Presence of mCherry-positive cells (red) co-localised with cFos-positive cells (green) in the dLS (overlap between red and green cells shown in zoom-in box). (H) Tail-flick latency (seconds) (2.58±0.12 compared to 3.32±0.10, respectively; t-test, ***P=0.0005, n=7) post saline or DCZ administration in dLSpre-LHAGad67-hM3Dqmice, with no significant difference seen in dLSpre-LHAGad67-tdTomato mice. (I) Number of licks (11.14±1.45 compared to 6.29±0.89, respectively; t-test, *P=0.0147, n=7), and latency to lick (seconds) (11.29±1.51 compared to 16.43±1.39, respectively; t-test, *P=0.0277, n=7) post saline or DCZ administration in dLSpre-LHAGad67-hM3Dq mice.. (J) AAVTransyn-Cre injected in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato in the LHA of wild-type mice. Presence of mCherry-positive cells (red) co-localised with cFos-positive cells (green) in the LHA. (K) Tail-flick latency (seconds) (2.97±0.07 compared to 4.12±0.22, respectively; t-test, ***P=0.0003, n=7) post saline or DCZ administration in LHApost-dLShM3Dq mice, with no significant difference seen in dLSpost-dLSGad67-tdTomato mice. (L) Number of licks (4.71±0.94 compared to 10.00±1.60, respectively; t-test, *P=0.0149, n=7), and latency to lick (seconds) (23.00±3.11 compared to 9.43±1.93, respectively; t-test, **P=0.003, n=7) post saline or DCZ administration LHApost-dLStdTomato mice, with no significant difference seen in dLSpost-dLSGad67-tdTomato mice. (M) AAVTransyn-Cre injected in the dLS and DIO-hM3Dq-mCherry in the Habenula (Hb) of wild-type mice to express the excitatory DREADD. Presence of mCherry-positive cells (red) in the Hb. (N) Tail-flick latency post saline or DCZ administration in Hbpost-dLShM3Dq mice. (O) Number and latency of licks (in seconds) post saline or DCZ administration in Hbpost-dLShM3Dq mice. (P) AAVTransyn-Cre injected in the dLS and DIO-Tettox-GFP/DIO-GFP bilaterally in the LH of wild-type mice. Presence of GFP-positive cells (green) in the LHA. (Q) Tail-flick latency (seconds) (2.38±0.04 compared to 3.00±0.04, respectively; t-test, ***P=0.0001, n=7) with and without restraint in LHApost-dLSGFP mice, with no significant difference seen in LHApost-dLSTetTox mice. (R) Number of licks (9.43±0.92 compared to 4.00±0.53, respectively; t-test, ***P=0.0003, n=7), and latency to lick (seconds) (13.29±0.89 compared to 24.86±3.47, respectively; t-test, **P=0.0072, n=7) with and without restraint in LHApost-dLSGFP mice, with no significant difference seen in LHApost-dLSTetTox mice.

LHApost-dLS neurons are glutamatergic in nature.

(A) AAV encoding Transsyn-FlpO injected in the dLS; fDIO-tdTomato, and DIO-GFP injected in the LHA of VGlut2-Cre transgenic mice. (B) Overlapping red and green cells (yellow, seen in the zoom-in box marked by arrowheads) were seen in the LHA. (C) AAVTransyn-Cre injected in the dLS of Ai14 transgenic mice. (D) The LHA was labeled with probes against VGlut2 using in situ hybridization. Co-localisation of tdTomato-positive (red) and GFP-positive (green) cells in the LHA (overlapping areas marked by arrowheads), zoom-in of overlap on right. (E) AAVs encoding Gad67-Cre and DIO-Synaptophysin-GFP injected in the dLS and DIO Gephyrin-tagRFP in the LHA of VGlut2-Cre transgenic mice. (F) Closely apposed green synaptophysin and red gephyrin puncta seen in the LHA (zoom-in of overlap on right).

LHApost-dLS neurons are acutely engaged during the initial struggle due to physical restraint.

(A) AAVTransyn-Cre injected in the dLS and DIO-GCaMP6s in the LHA of wild-type mice to record neural activity from the LHApost-dLS neurons. (B) GCaMP6s-positive cells (green) and tissue injury from the fiber implant seen in the LHA, zoom-in of the marked area on the right. (C) Sample trace of neural activity when mice were allowed to move freely in the homecage (blue), and when they were under restraint (red). Peaks corresponding to neural activity (blue dashes) were seen when mice struggled in the tube. (D) Cumulative plots for calcium transients when mice struggled under restraint (5mice, 12 trials). (E) Heat maps depicting neural activity in LHApost-dLS (left) and dLSGad67 neurons (right) during struggle in the RS assay (5 mice, 12 trials; dotted lines indicating initiation of struggle in RS assay).

RVM ON-cells are downstream of the dLS→LHA circuitry.

(A) AAVTransyn-Cre injected in the dLS and DIO-GFP in the LHA of wild-type mice to label the LHApost-dLS neurons. (B) GFP-positive cell bodies in the LHA (green, left; zoom-in of LHA in the box on bottom). Projections of the LHpost-dLS neurons in the PBN, and RVM. (C) Presence of fluorescence (black) in RVM post tissue clearing of the same brains expressing GFP in the LHApost-dLSneurons. (D) AAVTransyn-Cre injected in the LHA and DIO-GFP in the RVM of wild-type mice to label the RVMpost-LHA neurons with GFP. (E) GFP-positive cell bodies (black, marked by arrowheads) seen in the RVM. (F) Projections from the RVMpost-LHA were observed in the lumbar spinal cord. (G) AAVTransyn-Cre injected in the dLS, and DIO-G and DIO-TVA-GFP in the LHA of wild-type mice. Three weeks later, the delG-Rabies-mCherry virus was injected into the RVM. (H) Starter cells (yellow) observed in the LHA. mCherry-positive cell bodies (red) seen in the dLS (marked region zoomed-in on the right with overlapping cells marked with arrowheads). (I) AAVRetro-FlpO injected in the RVM, fDIO-post-GRASP injected in the LHA, and Pre-GRASP in the dLS of wild-type mice. (J) Axon terminals from the dLS neurons (Green) were observed around the LHApre-RVM (red) cell-bodies (zoom-in of overlaps on the right).

RVMpost-LHA neurons fire during nocifensive behaviors on the hotplate and respond to RS-mediated struggle.

(A) AAVTransyn-Cre was injected in the LHA with DIO-GCaMP6s in the RVM of wild-type mice (top). A fiber was implanted on the RVM over the same coordinates to perform fiber-photometry (bottom). (B) GCaMP6s-positive cells in the RVM (green) to demonstrate successful expression of GCaMP6s in the RVMpost-LHA neurons. (C) Z-Score of calcium dynamics recorded from the RVMpost-LHA neurons while the mice are restrained and struggling (blue dashes) in a falcon tube. (D) Average plot (individual trials) of Z-Score traces (4 mice, 16 trials) during struggle bouts of RVMpost-LHA neurons. (E) (Left to right) Heatmaps depicting neural activity patterns during individual instances of struggle in the falcon tube, licks, and shakes on the hot plate at 52 degrees, respectively (4 mice, 16 trials). (F) Z-Score of calcium dynamics recorded from the RVMpost-LHA neurons while the mice are on the hotplate set at a gradient from 32-52 degrees. (G) Average plot (individual trials) of Z-Score traces (4 mice, 16 trials) during tail flick assay of RVMpost-LHA neurons.

Activation of RVMpost-LHA neurons resulted in thermal hyperalgesia, while inhibition was anti-nociceptive.

(A) AAVTransyn-Cre injected in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato in the RVM of wild-type mice. (B) DCZ induced c-Fos (green) expression in the RVMpost-LHA-hM3Dq (red) neurons (zoom-in shown on the right with overlapping cells marked using arrowheads). (C) Tail-flick latency (seconds) (3.22±0.08 compared to 2.69±0.09, respectively; t-test, ***P=0.0001, n=7) post saline or DCZ administration in the RVMpost-LHA-hM3Dq mice, with no significant difference in RVMpost-LHA-tdTomato mice. (D) Number of licks (4.57±1.27 compared to 8.43±0.87, respectively; t-test, **P=0.0062, n=7), and latency to lick (seconds) (23.57±7.12 compared to 12.29±2.68, respectively; t-test, *P=0.0466, n=7) post saline or DCZ administration in the RVMpost-LHA-hM3Dq mice, with no significant difference in RVMpost-LHA-tdTomato mice. (E) AAVTransyn-Cre injected in the LHA and DIO-hM4Di in the RVM of wild-type mice to express the inhibitory DREADD in the RVMpost-LHA neurons. (F) hM3Dq-mCherry expressing neurons (red) in the RVM. (G) Tail-flick latency (seconds) (2.40±0.11 compared to 3.38±0.23, respectively; t-test, **P=0.0025, n=7) post saline or DCZ administration in RVMpost-LHA-hM4Di mice. (H) Number of licks (12.00±0.62 compared to 6.86±0.67, respectively; t-test, ***P= 0.0001, n=7), and latency to lick (seconds) (12.14±1.42 compared to 18.86±0.86, respectively; t-test, **P=0.0016, n=7) post saline or DCZ administration in RVMpost-LHA-hM4Di mice. (I) AAVTransyn-Cre injected in the LHA and DIO-hM4Di in the PBN (bilaterally) of wild-type mice to express the inhibitory DREADD, hM4Di in the LPBNpost-LHA neurons. (J) hM4Di-mCherry (red) expressing neurons in the PBN. (K) Tail-flick latency (seconds) post-saline or DCZ administration in the PBNpost-LHA-hM4Di mice. (L) Number and latency of licks (seconds) post-saline or DCZ administration in the PBNpost-LHA-hM4Di mice. (M) AAVTransyn-Cre injected in the LHA and DIO-eNPHR3.0-YFP/DIO-GFP in the RVM of wild-type mice to label the RVMpost-LHAneurons. Optic fiber cannulae was implanted over the RVM at same coordinates to deliver yellow light. The summary of the fiber placements are graphically represented (right corner). (N) YFP-positive cells expressing eNpHR3.0 in the RVMpost-LHA (green) neurons in the RVM. (O) Tail-flick latency (seconds) (2.48±0.04 compared to 3.00±0.03, respectively; t-test, ***P=0.0001, n=7) with (ON) and without (OFF) yellow light illumination in RVMpost-LHAeNPHR3.0-YFP mice, with no significant difference seen in RVMpost-LHADIO-GFP mice. (P) Number of licks (11.14±1.01 compared to 5.00±0.49, respectively; t-test, ***P=0.0001, n=7), and latency to lick (seconds) (12.71±0.52 compared to 22.86±1.18, respectively; t-test, ***P=0.0001, n=7) with (ON) and without (OFF) yellow light illumination in RVMpost-LHAeNPHR3.0-YFP mice, with no significant difference seen in RVMpost-LHADIO-GFP mice.

(A) Blood corticosterone levels in mice administered with saline, DCZ, or subjected to RS. (B) Sample traces demonstrating time spent by dLSGad67-hM3Dq mice in the lit chamber of the classic light-dark box assay to test stress-induced anxiety. (C) Time spent in the light box post-saline or DCZ administration (35.83±2.59 compared to 26.17±1.35, respectively; t-test, **P=0.00578, n=6). (D) Sample traces demonstrating activity of dLSGad67-hM3Dq mice in an open field test. (E) Percentage of time spent by dLSGad67-hM3Dq mice in the centre of the open field (55.33±3.21 compared to 23.17±2.36, respectively; t-test, ***P=0.0001, n=6). (F) Percentage response of mice to the von Frey Filament Test (63.89±11.72 compared to 19.44±10.01, respectively; t-test, *P=0.0365, n=5). (G and H) Number and latency (seconds) of jumps post saline or DCZ administration on the hot plate in dLSGad67-tdTomato and dLSGad67-hM3Dq mice. (I) Gad67-Cre and DIO-ChR2-YFP/DIO-GFP injected in the dLS of wild-type mice to label the dLSvgatneurons with the opsin. Optic fiber cannulae was implanted over the dLS at the same coordinates to deliver blue light. (J) YFP-positive cells (green) observed in the dLSvgat neurons (zoom-in shown in the box). (K) Tail-flick latency (seconds) (2.27±0.05 compared to 3.03±0.03, respectively; t-test, ***P=0.0001, n=5) with (ON) and without (OFF) blue light illumination in dLSGad67-ChR2 neurons, with no significant difference in dLSGad67-GFP mice. (L) Number (10.43±1.00 compared to 2.86±0.51, respectively; t-test, ***P=0.0001, n=5) and latency of licks (seconds) (10.57±0.57 compared to 20.14±0.74, respectively; t-test, ***P=0.0001, n=5) with (ON) and without (OFF) yellow light illumination in dLSGad67-ChR2 neurons, with no significant difference in dLSGad67-GFP mice.

(A) Tail-flick latencies (seconds) over an hour of dLSGad67-ChR2 mice post blue light illumination for 30 minutes (2.33±0.05 compared to 2.74±0.01, respectively; Immediate t-test, *P=0.0321, n=4) (2.34±0.06 compared to 2.85±0.02, respectively; Post-10 minutes t-test, **P=0.00877, n=4) (2.37±0.06 compared to 2.98±0.06, respectively; Post 30 minutes t-test, **P=0.00832, n=4). (B) Tail-flick latencies (seconds) of mice with and without restraint at different time points post the RS assay (2.27±0.04 compared to 2.73±0.03, respectively; Immediate t-test, *P=0.0437, n=4) (2.30±0.05 compared to 2.87±0.06, respectively; Post-10 minutes t-test, **P=0.0057, n=4) (2.29±0.05 compared to 2.97±0.03, respectively; Post 30 minutes t-test, **P=0.0061, n=4). (C) AAVs encoding Gad67-Cre, DIO-hM3Dq-mCherry, and DIO-TetTox-GFP or DIO-GFP injected in the dLS of wild-type mice to simultaneously label the dLSGad67 neurons with the excitatory DREADD, and TetTox. Overlapping yellow cells representing hM3Dq (red), and TetTox (green) co-expressing neurons in the dLS. (D) Tail-flick latency (in seconds) in dLSGad67-TetToxGFP-hM3Dq mice (2.45±0.07 compared to 2.99±0.05, respectively; t-test, **P=0.00943, n=4), and in dLSGad67-TetTox-hM3Dq mice post saline or DCZ administration. (E) Gad67-cre and DIO-Tettox-GFP/DIO-GFP injected in the dLS of wild-type mice. (F and G) Number and latency (in seconds) of jumps with and without restraint.

(A and B) Average plot and heat map of dLS GCaMP transients for home cage activity. (C and D) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 16 trails) during immobilization. (E and F) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 18 trials) during tail suspension.

(A) AAVs encoding Gad67-Cre and DIO-SynRuby injected in the dLS of wild-type mice. (B) Ruby expression was seen at the injection site and post-synaptic region (LHA), marked region on LHA shown in zoom-in on the right with synapses marked with arrowheads. (C) Rostral to caudal serial images of the LHA taken on a fluorescence microscope at 4X and 10X magnifications showing projections from the dLS, post Gad67-Cre and DIO-GFP injections in the dLS of wild-type mice.

(A) Absorption spectra for the vehicle Dimethysulfoxide (DMSO) (green) and CNO dissolved in DMSO (red). (B) Cy3 fluorescent beads as seen under the microscope at 100X with oil immersion. (C) Cy3 fluorescent beads injected in the dLS and images under the fluorescence microscope at 4X and 10X magnifications (marked region on dLS shown in zoom-in on the bottom). (D) Gad67-Cre and DIO-hM3Dq-mCherry stereotaxically injected in the dLS of wild-type mice to express the excitatory DREADD. 3 weeks later, CNO beads were injected stereotaxically in the dLS over the same coordinates. cFos expression was muted before MP-CNO delivery, increased after 4 days, while reduced on the 8th day of delivery (4.40±1.69 compared to 61.65±3.99, respectively; Control vs Day 4 One-way ANOVA test, ***P=0.0001, n=5), 61.65±3.99 compared to 8.69±5.31, respectively; Day 4 vs Day 8 One-way ANOVA test, **P=0.0048, n=5). (E) Tail-flick latency (seconds) (2.28±0.16 compared to 2.95±0.16, respectively; Day2 One-way ANOVA test, **P= 0.00765, n=7) (2.28±0.16 compared to 3.10±0.22, respectively; Day4 One-way ANOVA test, ***P=0.0001, n=7) over a course of 8 days post CNO beads injection. (F) Number of licks (10.43±1.09 compared to 4.14±1.14, respectively; Day2 One-way ANOVA test, **P=0.00821, n=7) (10.43±1.09 compared to 4.57±1.04, respectively; Day4 One-way ANOVA test, **P=0.0056, n=7), and latency to lick (seconds) (15.14±1.77 compared to 22.57±1.97, respectively; Day2 One-way ANOVA test, *P=0.0328, n=7) (15.14±1.77 compared to 22.57±1.76, respectively; Day4 One-way ANOVA test, *P=0.0412, n=7) over a course of 8 days post CNO beads injection. (G) Gad67-Cre and DIO-tdTomato injected in the dLS of wild-type mice. 3 weeks later, CNO beads were injected stereotaxically in the dLS over the same coordinates. (H) Tail-flick latency (seconds) over the course of 8 days post-CNO beads injection in dLSGad67-hM3Dq mice. (I) Number and latency of licks (seconds) post-CNO beads injection in dLSGad67-hM3Dq mice. (J) Gad67-Cre and DIO-hM3Dq injected in the dLS of wild-type mice. 3 weeks later, CNO beads were injected in the LHA to enable terminal activation of LHApost-dLS neurons. (K) Tail-flick latency (seconds) (2.49±0.01 compared to 3.05±0.02, respectively; Day2 One-way ANOVA test, ***P=0.0001, n=7) (2.49±0.01 compared to 2.97±0.08, respectively; Day4 One-way ANOVA test, ***P=0.0001, n=7) over a course of 8 days post CNO beads injection. (L) Number of licks (10.83±2.33 compared to 2.17±0.79, respectively; Day2 One-way ANOVA test, *P=0.0435, n=7) (10.83±2.33 compared to 2.17±1.22, respectively; Day4 One-way ANOVA test, *P =0.0387, n=7), and latency to lick (seconds) (10.50±1.69 compared to 31.33±4.98, respectively; Day2 One-way ANOVA test, *P=0.0245, n=7) (10.50±1.69 compared to 40.50±7.36, respectively; Day4 One-way ANOVA test, **P=0.00456, n=7) over a course of 8 days post CNO beads injection.

(A) Allen Brain Atlas images showing the anatomical location of the LHA next to the parasubthalamic nucleus (PSTN) and vglut/vgat expressing cells in the LHA. (B) In situ hybridization using probes against VGlut2 and VGat performed on LHA sections of wild-type mice (red: vglut2, green: vgat) (zoom-in of overlapping cells shown on right with overlap marked using arrowheads). (C) Parvalbumin staining in the LHA sections.

(A and B) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 12 trials) during immobilization of LHApost-dLS neurons. (C and D) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 12 trials) during tail suspension of LHApost-dLS neurons. (E and F) Average plots and heatmaps for dLS responses on the hot-plate at 32 deg of LHApost-dLS neurons (5 mice, 12 trials; dotted line indicating time point when mice were placed on the hotplate). (G and H) Average plots and heatmaps for dLS responses on the hot-plate at 52 deg of LHApost-dLS neurons (5 mice, 12 trials; dotted line indicating time point when mice were placed on the hotplate).

(A) Number and latency to lick before and after RS in wild-type mice i.p. injected with the mu-opioid receptor antagonist naltrexone. (B) Gad67-Cre and DIO-hM3Dq injected in the dLS of wild-type mice to express the excitatory DREADD, hM3Dq in the dLSGad67 neurons. (C) Number and latency (in seconds) of licks post-saline or DCZ administration in dLSGad67-hM3Dq mice post i.p. Naltrexone administration. (D) AAVTransyn-Cre injected in the LHA, and DIO-SynRuby injected in the RVM of wild-type mice. (E) Red cell bodies expressing SynRuby were observed in the RVM (zoom-in of maeked region shown on the right). (F) Red puncta of RVMpost-LHA axon terminals observed in the LHA (zoom-in of marked region shown on the right). (G) Axon terminals of the RVMpost-LHA neurons seen in the dorsal horn of the lumbar spinal cord (* marking distinct neurons).

(A) DIO-SynGFP injected in the LHA, and DIO-PSD95-tagRFP in the RVM of VGlut2-Cre transgenic mice. (B) Confocal images of the LHAVGlut2 terminals (green), and RVMVGlut2 dendrites (red) observed under 10X magnification. (C) Confocal images under 20X magnification of the same regions (closely apposed green and red cells marked with arrowheads, zoom-in on the right). (D) 40X Airyscan superresolution image demonstrating close apposition between the red, and green puncta (closely apposed green and red cells marked with arrowheads, zoom-in on the right). (E) Bar graph depicting the AUC of the photometry recordings during the struggle bouts from dLSGad67, LHApost-dLS, and RVMpost-LHA neurons. (F) Diagrammatic representation of the dLS-centric RS-induced SIA pathway that originates from dLS and terminates in the spinal cord via LHA and RVM.