DOI: 10.5061/dryad.2v6wwpzz9
title: Anatomical, behavioral, and imaging data for a study on neural circuits for stress modulation of pain

description: Stress is a potent modulator of pain. Specifically, acute stress due to
 physical restraint induces stress-induced analgesia (SIA). However, where
 and how acute stress and pain pathways interface in the brain are poorly
 understood. Here, we describe how the dorsal lateral septum (dLS), a
 forebrain limbic nucleus, facilitates SIA through its downstream targets
 in the lateral hypothalamic area (LHA) of mice. Taking advantage of
 transsynaptic viral-genetic, optogenetic, and chemogenetic techniques, we
 show that the dLS→LHA circuitry is sufficient to drive analgesia and is
 required for SIA. Further, our results reveal that the dLS→LHA pathway is
 opioid-dependent and modulates pain through the pro-nociceptive neurons in
 the rostral ventromedial medulla (RVM). Remarkably, we found that the
 inhibitory dLS neurons are recruited specifically when the mice struggle
 to escape under restraint and, in turn, inhibit excitatory LHA neurons. As
 a result, the RVM neurons downstream of LHA are disengaged, thus
 suppressing nociception. Together, we delineate a poly-synaptic pathway
 that can transform escape behavior in mice under restraint to acute stress
 into analgesia.
description: # Anatomical, behavioral, and imaging data for a study on neural circuits
 for stress modulation of pain
 [https://doi.org/10.5061/dryad.2v6wwpzz9](https://doi.org/10.5061/dryad.2v6wwpzz9) ## Description of the data and file structure All the images are included in the zip file. The image file nos. corresponds to the Fig no. of the images in the paper. ### All the behavior data figure wise is included in the raw PRISM file `SeptumBehaviorData.pzfx` 1\. Number of licks for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dorsal Lateral Septum (dLS) with intraperitoneal (i.p.) Saline or Deschloroclozapine (DCZ) injection (n=7) 2\. Latency of licks for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dLS with i.p. Saline or DCZ injection (n=7) 3\. Number of shakes for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dLS with i.p. Saline or DCZ injection (n=7) 4\. Latency of shakes for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dLS with i.p. Saline or DCZ injection (n=7) 5\. Number of jumps for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dLS with i.p. Saline or DCZ injection (n=7) 6\. Latency of jumps for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dLS with i.p. Saline or DCZ injection (n=7) 7\. Tailflick latency for mice with Gad67-Cre + DIO-hM3Dq-mCherry/DIO-tdTomato viral injection in the dLS with i.p. Saline or DCZ injection (n=7) 8\. Number of licks for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 9\. Latency of licks for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 10\. Number of shakes for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 11\. Latency of shakes for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 12\. Number of jumps for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 13\. Latency of jumps for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 14\. Tailflick latency for mice with Gad67-Cre + DIO-Tettox/DIO-GFP viral injection in the dLS with and without restraint stress (n=7) 15\. Number of licks for mice with Gad67-Cre + DIO-ChR2/DIO-GFP viral injection in the dLS with and without blue light illumination (n=7) 16\. Latency of licks for mice with Gad67-Cre + DIO-ChR2/DIO-GFP viral injection in the dLS with and without blue light illumination (n=7) 17\. Number of shakes for mice with Gad67-Cre + DIO-ChR2/DIO-GFP viral injection in the dLS with and without blue light illumination (n=7) 18\. Latency of shakes for mice with Gad67-Cre + DIO-ChR2/DIO-GFP viral injection in the dLS with and without blue light illumination (n=7) 19\. Number of licks for mice with Gad67-Cre + DIO-ChR2 viral injection in the dLS and optic fiber over the Lateral hypothalamic area (LHA) with and without blue light illumination (n=7) 20\. Latency of licks for mice with Gad67-Cre + DIO-ChR2 viral injection in the dLS and optic fiber over the LHA with and without blue light illumination (n=7) 21\. Number of shakes for mice with Gad67-Cre + DIO-ChR2 viral injection in the dLS and optic fiber over the LHA with and without blue light illumination (n=7) 22\. Latency of shakes for mice with Gad67-Cre + DIO-ChR2 viral injection in the dLS and optic fiber over the LHA with and without blue light illumination (n=7) 23\. Tailflick latency for mice with Gad67-Cre + DIO-ChR2 viral injection in the dLS and optic fiber over the LHA with and without blue light illumination (n=7) 24\. Number of licks for mice with retro-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the dLS with i.p. Saline or DCZ injection (n=7) 25\. Latency of licks for mice with retro-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the dLS with i.p. Saline or DCZ injection (n=7) 26\. Number of shakes for mice with retro-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the dLS with i.p. Saline or DCZ injection (n=7) 27\. Latency of shakes for mice with retro-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the dLS with i.p. Saline or DCZ injection (n=7) 28\. Tailflick latency for mice with retro-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the dLS with i.p. Saline or DCZ injection (n=7) 29\. Number of licks for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the LHA with i.p. Saline or DCZ injection (n=7) 30\. Latency of licks for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the LHA with i.p. Saline or DCZ injection (n=7) 31\. Number of shakes for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the LHA with i.p. Saline or DCZ injection (n=7) 32\. Latency of shakes for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the LHA with i.p. Saline or DCZ injection (n=7) 33\. Tailflick latency for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the LHA with i.p. Saline or DCZ injection (n=7) 34\. Number of licks for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry injection in the Habenula with i.p. Saline or DCZ injection (n=7) 35\. Latency of licks for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry injection in the Habenula with i.p. Saline or DCZ injection (n=7) 36\. Number of shakes for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry injection in the Habenula with i.p. Saline or DCZ injection (n=7) 37\. Latency of shakes for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry injection in the Habenula with i.p. Saline or DCZ injection (n=7) 38\. Tailflick latency for mice with AAV1-transyn-Cre injection in the dLS and DIO-hM3Dq-mCherry injection in the Habenula with i.p. Saline or DCZ injection (n=7) 39\. Number of licks for mice with AAV1-transyn-Cre injection in the dLS and DIO-Tettox-GFP/DIO-GFP injection in the LHA with and without restraint stress (n=7) 40\. Latency of licks for mice with AAV1-transyn-Cre injection in the dLS and DIO-Tettox-GFP/DIO-GFP injection in the LHA with and without restraint stress (n=7) 41\. Number of shakes for mice with AAV1-transyn-Cre injection in the dLS and DIO-Tettox-GFP/DIO-GFP injection in the LHA with and without restraint stress (n=7) 42\. Latency of shakes for mice with AAV1-transyn-Cre injection in the dLS and DIO-Tettox-GFP/DIO-GFP injection in the LHA with and without restraint stress (n=7) 43\. Tailflick latency for mice with AAV1-transyn-Cre injection in the dLS and DIO-Tettox-GFP/DIO-GFP injection in the LHA with and without restraint stress (n=7) 44\. Number of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the Rostral ventromedial Medulla (RVM) with i.p. Saline or DCZ injection (n=7) 45\. Latency of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the RVM with i.p. Saline or DCZ injection (n=7) 46\. Number of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the RVM with i.p. Saline or DCZ injection (n=7) 47\. Latency of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the RVM with i.p. Saline or DCZ injection (n=7) 48\. Tailflick latency for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry/DIO-tdTomato injection in the RVM with i.p. Saline or DCZ injection (n=7) 49\. Number of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM4Di-mCherry injection in the RVM with i.p. Saline or DCZ injection (n=7) 50\. Number of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM4Di-mCherry injection in the RVM with i.p. Saline or DCZ injection (n=7) 51\. Latency of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM4Di-mCherry injection in the RVM with i.p. Saline or DCZ injection (n=7) 52\. Latency of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM4Di-mCherry injection in the RVM with i.p. Saline or DCZ injection (n=7) 53\. Tailflick latency for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM4Di-mCherry injection in the RVM with i.p. Saline or DCZ injection (n=7) 54\. Number of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry bilaterally injected in the parabrachial nucleus (PBN) with i.p. Saline or DCZ injection (n=7) 55\. Latency of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry bilaterally injected in the PBN with i.p. Saline or DCZ injection (n=7) 56\. Number of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry bilaterally injected in the PBN with i.p. Saline or DCZ injection (n=7) 57\. Latency of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry bilaterally injected in the PBN with i.p. Saline or DCZ injection (n=7) 58\. Tailflick latency for mice with AAV1-transyn-Cre injection in the LHA and DIO-hM3Dq-mCherry bilaterally injected in the PBN with i.p. Saline or DCZ injection (n=7) 59\. Number of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-Halorhodopsin-YFP/DIO-GFP injection in the RVM with and without yellow light illumination (n=7) 60\. Latency of licks for mice with AAV1-transyn-Cre injection in the LHA and DIO-Halorhodopsin-YFP/DIO-GFP injection in the RVM with and without yellow light illumination (n=7) 61\. Number of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-Halorhodopsin-YFP/DIO-GFP injection in the RVM with and without yellow light illumination (n=7) 62\. Latency of shakes for mice with AAV1-transyn-Cre injection in the LHA and DIO-Halorhodopsin-YFP/DIO-GFP injection in the RVM with and without yellow light illumination (n=7) 63\. Tailflick latency for mice with AAV1-transyn-Cre injection in the LHA and DIO-Halorhodopsin-YFP/DIO-GFP injection in the RVM with and without yellow light illumination (n=7) 64\. Bar graphs indicating percentage time spent in the light side of the light-dark box with i.p. Saline or DCZ injection (n=6) 65\. Bar graphs indicating percentage time spent in the centre of the open-field box with i.p. Saline or DCZ injection (n=6) 66\. Bar graphs indicating the fold change in the blood corticosterone levels in mice with i.p. Saline or DCZ injection and post-restraint stress 67\. Number of licks for mice with Gad67-Cre + DIO-hM3Dq viral injection and CNO Beads injection in the dLS with over a period of 10 days post Beads injection. Saline, i.p. CNO and Washout (Saline2) measurements done in the same mice before beads injection provided for comparison (n=7) 68\. Latency of licks for mice with Gad67-Cre + DIO-hM3Dq viral injection and CNO Beads injection in the dLS with over a period of 10 days post Beads injection. Saline, i.p. CNO and Washout (Saline2) measurements done in the same mice before beads injection provided for comparison (n=7) 69\. Tailflick latency for mice with Gad67-Cre + DIO-hM3Dq viral injection and CNO Beads injection in the dLS with over a period of 10 days post Beads injection. Saline, i.p. CNO and Washout (Saline2) measurements done in the same mice before beads injection provided for comparison (n=7) 70\. Number of licks for mice with Gad67-Cre + DIO-hM3Dq viral injection in the dLS and CNO Beads injection in the LHA with over a period of 10 days post Beads injection. Saline, i.p. CNO and Washout (Saline2) measurements done in the same mice before beads injection provided for comparison (n=7) 71\. Latency of licks for mice with Gad67-Cre + DIO-hM3Dq viral injection in the dLS and CNO Beads injection in the LHA with over a period of 10 days post Beads injection. Saline, i.p. CNO and Washout (Saline2) measurements done in the same mice before beads injection provided for comparison (n=7) 72\. Tailflick latency for mice with Gad67-Cre + DIO-hM3Dq viral injection in the dLS and CNO Beads injection in the LHA with over a period of 10 days post Beads injection. Saline, i.p. CNO and Washout (Saline2) measurements done in the same mice before beads injection provided for comparison (n=7) ### The photometry data is included Figure wise in the raw PRISM file `SeptumPhotometryData.pzfx` 1\. Average trace comprising of 20 trials across 5 mice with Gad67-Cre + DIO-GCaMP6s-GFP viral injections in the dorsal Lateral Septum (dLS) recorded using fiber photometry during the Immobilisation experiments. 2\. Average trace comprising of 20 trials across 5 mice with Gad67-Cre + DIO-GCaMP6s-GFP viral injections in the dLS recorded using fiber photometry during the Tail suspension experiments. 3\. Average trace comprising of 20 trials across 5 mice with Gad67-Cre + DIO-GCaMP6s-GFP viral injections in the dLS recorded using fiber photometry during the Hotplate experiments at 32 degrees. 4\. Average trace comprising of 20 trials across 5 mice with Gad67-Cre + DIO-GCaMP6s-GFP viral injections in the dLS recorded using fiber photometry during the Hotplate experiments at 52 degrees. 5\. Average trace comprising of 20 trials across 5 mice with Gad67-Cre + DIO-GCaMP6s-GFP viral injections in the dLS recorded using fiber photometry during the struggle bouts in the Restraint Stress assay. 6\. Representative trace comparing of a single trial from a mouse with Gad67-Cre + DIO-GCaMP6s-GFP in the dLS with a mouse with Gad67-Cre + DIO-GFP in the dLS recorded using fiber photometry in the homecage. 7\. Plot comparing the area under the curve of neural activity from the dLS, LHA and RVM. 8\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the dLS and DIO-GCaMP6s-GFP in the Lateral Hypothalamic area (LHA) recorded using fiber photometry during the Immobilisation experiments. 9\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the dLS and DIO-GCaMP6s-GFP in the LHA recorded using fiber photometry during the Tail suspension experiments. 10\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the dLS and DIO-GCaMP6s-GFP in the LHA recorded using fiber photometry during the Hotplate experiments at 32 degrees. 11\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the dLS and DIO-GCaMP6s-GFP in the LHA recorded using fiber photometry during the Hotplate experiments at 52 degrees. 12\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the dLS and DIO-GCaMP6s-GFP in the LHA recorded using fiber photometry during the struggle bouts in the restraint stress assay. 13\. Representative trace from one mouse with AAV1-transsyn-Cre in the dLS and DIO-GCaMP6s-GFP in the LHA recorded using fiber photometry for comparison of neural activity in the restrain stress assay and during free movement in the homecage. 14\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the LHA and DIO-GCaMP6s-GFP in the Rostral ventromedial medulla (RVM) recorded using fiber photometry during the struggle bouts in the restraint stress assay. 15\. Representative trace from one mouse with AAV1-transsyn-Cre in the LHA and DIO-GCaMP6s-GFP in the RVM recorded using fiber photometry during the restraint stress assay. 16\. Representative trace from one mouse with AAV1-transsyn-Cre in the LHA and DIO-GCaMP6s-GFP in the RVM recorded using fiber photometry during the Hotplate experiment at a gradient temperature setting of 32-52 degrees. 17\. Average trace comprising of 20 trials across 5 mice with AAV1-transsyn-Cre in the LHA and DIO-GCaMP6s-GFP in the RVM recorded using fiber photometry during the Tailflick experiments.