A septo-hypothalamic-medullary circuit directs stress-induced analgesia
- Centre for Neuroscience, Indian Institute of Science, India
- Department of Bioengineering, Indian Institute of Science, India
Figures
Figure 1 with 2 supplements
Dorsal lateral septum (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 a zoom-in view of a rare vglut2+ (green) cell (right). (E) Gad1-Cre and DIO-hM3Dq-mCherry/DIO-tdTomato were injected in the dLS of wild-type mice. (F) i.p. Deschloroclozapine (DCZ) and not Saline evoked cFos expression (green) in the mCherry-positive cells (red) in the dLSGad1-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 dLSGad1-hM3Dq mice, with no significant difference in dLSGad1-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 dLSGad1-tdTomato mice, administered with either i.p. Saline or DCZ, with no significant difference in dLSGad67-tdTomato mice. (I) Gad1-Cre and DIO-Tettox-GFP or DIO-GFP were co-injected in the dLS of wild-type mice. (J) GFP-positive neurons (green) are 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 hr using the RS assay in dLSGad1-GFP mice, with no significant difference in dLSGad1-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 dLSGad1-GFP mice, with no significant difference in dLSGad1-Tettox-GFP mice.
Figure 1—figure supplement 1
The LS is engaged by acute RS and is involved in SIA.
(A) Acute restraint caused elevated cFos expression in the dorsal lateral septum (dLS) and vLS of mice. (B) Blood corticosterone levels in mice administered with saline, Deschloroclozapine (DCZ), or subjected to restraint stress (RS). (C) Sample traces demonstrating time spent by dLSGad1-hM3Dq mice in the lit chamber of the classic light-dark box assay to test stress-induced anxiety. (D) 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). (E) Sample traces demonstrating activity of dLSGad1-hM3Dq mice in an open field test. (F) Percentage of time spent by dLSGad1-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). (G) 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). (H and I) Number and latency (seconds) of jumps post saline or DCZ administration on the hot plate in dLSGad67-tdTomato and dLSGad67-hM3Dq mice. (J) Gad1-Cre and DIO-ChR2-YFP/DIO-GFP were injected in the dLS of wild-type mice to label the dLS neurons with the opsin. Optic fiber cannulae were implanted over the dLS at the same coordinates to deliver blue light. (K) YFP-positive cells (green) were observed in the dLS inhibitory neurons (zoom-in shown in the box). (L) 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 dLSGad1-ChR2 neurons, with no significant difference in dLSGad1-GFP mice. (M) 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 dLSGad1-ChR2 neurons, with no significant difference in dLSGad1-GFP mice.
Figure 1—figure supplement 2
dLS mediated SIA is short lasting as in RS.
(A) Tail-flick latencies (seconds) over an hour of dLSGad1-ChR2 mice post blue light illumination for 30 min (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 min t-test, **p=0.00877, n=4) (2.37 ± 0.06 compared to 2.98 ± 0.06, respectively; Post 30 min t-test, **p=0.00832, n=4). (B) Tail-flick latencies (seconds) of mice with and without restraint at different time points post the restraint stress (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 min t-test, **p=0.0057, n=4) (2.29 ± 0.05 compared to 2.97 ± 0.03, respectively; Post 30 min t-test, **p=0.0061, n=4). (C) AAVs encoding Gad1-Cre, DIO-hM3Dq-mCherry, and DIO-TetTox-GFP or DIO-GFP were injected in the dorsal lateral septum (dLS) of wild-type mice to simultaneously label the dLSGad1 neurons with the excitatory Designer Receptors Exclusively Activated by Designer Drugs (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 dLSGad1-TetToxGFP-hM3Dq mice (2.45 ± 0.07 compared to 2.99 ± 0.05, respectively; t-test, **p=0.00943, n=4), and in dLSGad1-TetTox-hM3Dq mice post saline or Deschloroclozapine (DCZ) administration. (E) Gad1-Cre and DIO-Tettox-GFP/DIO-GFP were injected in the dLS of wild-type mice. (F and G) Number and latency (in seconds) of jumps with and without restraint.
Figure 2 with 1 supplement
Dorsal lateral septum (dLS) neurons are engaged by acute restraint.
(A) AAVs encoding Gad1-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 degrees (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 degrees (5 mice, 20 trials; dotted line indicating time point when mice were placed on the hotplate).
Figure 2—figure supplement 1
Mild stressors and not noxious thermal stimuli engages dLS neurons.
(A and B) Average plot and heat map of dorsal lateral septum (dLS)GCaMP6s transients for home cage activity. (C and D) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 16 trials) during immobilization. (E and F) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 18 trials) during tail suspension.
Figure 3 with 2 supplements
Dorsal lateral septum (dLS) neurons synapse onto excitatory lateral Hypothalamus (LHA) neurons.
(A) Gad1-Cre and DIO-GFP were 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) Gad1-Cre and DIO-ChR2-YFP were injected in the dLS of wild-type mice to express the excitatory opsin ChR2 in the dLSGad1 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 dLSGad1-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 dLSGad1-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-localized 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 deschloroclozapine (DCZ) administration in dLSpre-LHA-Gad1-hM3Dq mice, with no significant difference seen in dLSpre-LHA-Gad1-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-LHA-Gad1-hM3Dq mice. (J) AAVTransyn-Cre was injected in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato in the LHA of wild-type mice. Presence of mCherry-positive cells (red) co-localized 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-dLS-hM3Dq mice, with no significant difference seen in dLSpost-dLS-Gad1-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-dLS-tdTomato mice, with no significant difference seen in dLSpost-dLS-Gad1-tdTomato mice. (M) AAVTransyn-Cre injected were in the dLS and DIO-hM3Dq-mCherry in the Habenula (Hb) of wild-type mice to express the excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Presence of mCherry-positive cells (red) in the Hb. (N) Tail-flick latency post saline or DCZ administration in Hbpost-dLS-hM3Dq mice. (O) Number and latency of licks (in seconds) post saline or DCZ administration in Hbpost-dLS-hM3Dq mice. (P) AAVTransyn-Cre were 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-dLS-GFP mice, with no significant difference seen in LHApost-dLS-TetTox 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-dLS-GFP mice, with no significant difference seen in LHApost-dLS-TetTox mice.
Figure 3—figure supplement 1
Axonal arborizations of the dLS neurons are found across LHA.
(A) AAVs encoding Gad1-Cre and DIO-SynRuby injected in the dorsal lateral septum (dLS) of wild-type mice. (B) Ruby expression was seen at the injection site and post-synaptic region (LHA), marked region on lateral Hypothalamus (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 4 X and 10 X magnifications showing projections from the dLS, post Gad1-Cre, and DIO-GFP injections in the dLS of wild-type mice.
Figure 3—figure supplement 2
CNO delivery via microparticle enables long-term activation of dLS neurons.
(A) Absorption spectra for the vehicle Dimethysulfoxide (DMSO) (green) and clozapine N-Oxide (CNO) dissolved in DMSO (red). (B) Cy3 fluorescent beads as seen under the microscope at 100 X with oil immersion. (C) Cy3 fluorescent beads were injected in the dorsal lateral septum (dLS) and images under the fluorescence microscope at 4 X and 10 X magnifications (marked region on dLS shown in zoom-in on the bottom). (D) Gad1-Cre and DIO-hM3Dq-mCherry were stereotaxically injected in the dLS of wild-type mice to express the excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADD). 3 wk later, CNO beads were injected stereotaxically in the dLS over the same coordinates. cFos expression was muted before MP-CNO delivery, increased after 4 d, while reduced on the eighth d 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; Day 2 One-way ANOVA test, **p=0.00765, n=7) (2.28±0.16 compared to 3.10 ± 0.22, respectively; Day 4 One-way ANOVA test, ***p=0.0001, n=7) over a course of 8 d post CNO beads injection. (F) Number of licks (10.43 ± 1.09 compared to 4.14 ± 1.14, respectively; Day 2 One-way ANOVA test, **p=0.00821, n=7) (10.43 ± 1.09 compared to 4.57 ± 1.04, respectively; Day 4 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; Day 2 One-way ANOVA test, *p=0.0328, n=7) (15.14 ± 1.77 compared to 22.57 ± 1.76, respectively; Day 4 One-way ANOVA test, *p=0.0412, n=7) over a course of 8 d post CNO beads injection. (G) Gad1-Cre and DIO-tdTomato were injected in the dLS of wild-type mice. 3 wk later, CNO beads were injected stereotaxically in the dLS over the same coordinates. (H) Tail-flick latency (seconds) over the course of 8 d post-CNO beads injection in dLSGad1-hM3Dq mice. (I) Number and latency of licks (seconds) post-CNO beads injection in dLSGad1-hM3Dq mice. (J) Gad1-Cre and DIO-hM3Dq were injected in the dLS of wild-type mice. 3 wk later, CNO beads were injected into the lateral Hypothalamus (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; Day 2 One-way ANOVA test, ***p=0.0001, n=7) (2.49 ± 0.01 compared to 2.97 ± 0.08, respectively; Day 4 One-way ANOVA test, ***p=0.0001, n=7) over a course of 8 d post CNO beads injection. (L) Number of licks (10.83 ± 2.33 compared to 2.17 ± 0.79, respectively; Day 2 One-way ANOVA test, *p=0.0435, n=7) (10.83 ± 2.33 compared to 2.17 ± 1.22, respectively; Day 4 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; Day 4 One-way ANOVA test, **p=0.00456, n=7) over a course of 8 d post CNO beads injection.
Figure 4 with 1 supplement
Lateral Hypothalamus (LHA)post-dLS neurons are glutamatergic.
(A) AAV encoding AAVTranssyn-FlpO injected in the dorsal lateral septum (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 were injected in the dLS of Ai14 transgenic mice. (D) The LHA was labeled with probes against vglut2 using in situ hybridization. Co-localization 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 Gad1-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 were seen in the LHA (zoom-in of overlap on right).
Figure 4—figure supplement 1
LHA neurons post-synaptic to dLS are excitatory and do not colocalize with PV.
(A) Allen Brain Atlas images showing the anatomical location of the lateral Hypothalamus (LHA) next to the para subthalamic nucleus (PSTN) and vglut/vgat expressing cells in the LHA. (B) In situ hybridization using probes against VGlut2 and VGat was 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.
Figure 5 with 1 supplement
Lateral Hypothalamus (LHA)post-dLS neurons are acutely engaged during the initial struggle due to physical restraint.
(A) AAVTransyn-Cre was 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 (5 mice, 12 trials). (E) Heat maps depicting neural activity in LHApost-dLS (left) and dorsal lateral septum (dLS)Gad1 neurons (right) during struggle in the restraint stress (RS) assay (5 mice, 12 trials; dotted lines indicating initiation of struggle in restraint stress RS assay).
Figure 5—figure supplement 1
LHA neurons downstream of dLS are activated by acute stressors.
(A and B) Average plot and heatmap (individual trials) of Z-Score traces (5 mice, 12 trials) during immobilization of Lateral Hypothalamus (LHA)post-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 dorsal lateral septum (dLS) responses on the hot plate at 32 degrees of LHApost-dLS neurons (5 mice, 12 trials; dotted line indicating time point when mice were placed on the hot plate). (G and H) Average plots and heatmaps for dLS responses on the hot plate at 52 degrees of LHApost-dLS neurons (5 mice, 12 trials; dotted line indicating time point when mice were placed on the hot plate).
Figure 6 with 1 supplement
Rostral ventromedial medulla (RVM) ON-cells are downstream of the dorsal lateral septum (dLS)→Lateral Hypothalamus (LHA) pathway.
(A) AAVTransyn-Cre was 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 the 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-dLS neurons. (D) AAVTransyn-Cre was 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) are seen in the RVM. (F) Projections from the RVMpost-LHA were observed in the lumbar spinal cord. (G) AAVTransyn-Cre was injected in the dLS, and DIO-ΔG and DIO-TVA-GFP in the LHA of wild-type mice. Three weeks later, the ΔG-Rabies-mCherry virus was injected into the RVM. (H) Starter cells (yellow) were 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).
Figure 6—figure supplement 1
dLS mediated SIA is opioid dependent.
(A) Number and latency to lick before and after restraint stress (RS) in wild-type mice i.p. injected with the mu-opioid receptor antagonist naltrexone. (B) Gad1-Cre and DIO-hM3Dq were injected in the dorsal lateral septum (dLS) of wild-type mice to express the excitatory DREADD, hM3Dq in the dLSGad1 neurons. (C) Number and latency (in seconds) of licks post-saline or deschloroclozapine (DCZ) administration in dLSGad1-hM3Dq mice post i.p. Naltrexone administration. (D) AAVTransyn-Cre was injected in the lateral hypothalamus (LHA), and DIO-SynRuby was injected in the rostral ventromedial medulla (RVM) of wild-type mice. (E) Red cell bodies expressing SynRuby were observed in the RVM (zoom-in of the marked 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 are seen in the dorsal horn of the lumbar spinal cord (* marking distinct neurons).
Figure 7
Rostral ventromedial medulla (RVM)post-LHA neurons fire during nocifensive behaviors on the hotplate and respond to restraint stress (RS)-mediated struggle.
(A) AAVTransyn-Cre was injected in the lateral hypothalamus (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 to 52 degrees. (G) Average plot (individual trials) of Z-Score traces (4 mice, 16 trials) during tail flick assay of RVMpost-LHA neurons.
Figure 8 with 1 supplement
Activation of rostral ventromedial medulla (RVM)post-LHA neurons resulted in thermal hyperalgesia, while inhibition was anti-nociceptive.
(A) AAVTransyn-Cre injected in the lateral hypothalamus (LHA) and DIO-hM3Dq-mCherry/DIO-tdTomato in the RVM of wild-type mice. (B) Deschloroclozapine (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 was injected in the LHA and DIO-hM4Di in the RVM of wild-type mice to express the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (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 was 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 was injected in the LHA and DIO-eNPHR3.0-YFP/DIO-GFP in the RVM of wild-type mice to label the RVMpost-LHA neurons. Optic fiber cannulae were implanted over the RVM at the 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-LHA-eNPHR3.0-YFP mice, with no significant difference seen in RVMpost-LHA-DIO-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-LHA-eNPHR3.0-YFP mice, with no significant difference seen in RVMpost-LHA-GFP mice.
Figure 8—figure supplement 1
A poly-synaptic circuit mechanism for dLS mediated SIA.
(A) DIO-SynGFP injected in the lateral hypothalamic area (LHA), and DIO-PSD95-tagRFP in the rostral ventromedial medulla (RVM) of VGlut2-Cre transgenic mice. (B) Confocal images of the LHAVGlut2 terminals (green), and RVMVGlut2 dendrites (red) were observed under 10 X magnification. (C) Confocal images under 20 X magnification of the same regions (closely apposed green and red cells marked with arrowheads, zoom-in on the right). (D) 40 X Airyscan super-resolution 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 area under the curve (AUC) of the photometry recordings during the struggle bouts from dLSGad1, LHApost-dLS, and RVMpost-LHA neurons. (F) Diagrammatic representation of the dorsal lateral septum (dLS)-centric restraint stress (RS)-induced stress-induced analgesia (SIA) pathway that originates from dLS and terminates in the spinal cord via LHA and RVM.
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A septo-hypothalamic-medullary circuit directs stress-induced analgesia
eLife 13:RP96724.
https://doi.org/10.7554/eLife.96724.3
