Social trauma engages lateral septum circuitry to occlude social reward

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Animals

Wild-type C57BL/6J mice, 7–8 weeks old (males, 22–26 g; females, 18–22 g; Jackson Laboratory) were used as experimental mice in CSDS studies; 4–6-week-old C57BL/6J mice (Jackson Laboratory) were used as new intruders in both the RI test and sCPP test; 16–24-month-old male CD-1 (ICR) mice (sexually experienced retired breeders; Charles River Laboratories) were used as aggressors for male CSDS. ERα-Cre mice (017911, B6N.129S6(Cg)-Esr1tm1.1(cre)And/J; Jackson laboratory) were crossed with CD-1 females to obtain F1 males, which were used as aggressors for female CSDS. Nt-Cre (01752, B6;129-Ntstm1(cre) Mgmj/J; Jackson Laboratory) homozygous mice were crossed with WT C57BL/6 J mice, and the F1 generation was used as experimental mice in the CSDS studies. Littermates were randomly assigned to experimental groups. All mice were allowed 1 week of acclimation to the housing facilities before the start of experiments. WT CD-1 and F1 ERα-Cre mice were single housed, Nt-Cre and WT C57BL/6J mice were housed in groups of between three and five. All mice were maintained on a 12/12-h light/dark cycle (07:00–19:00) with ad libitum access to food and water. Housing and experimental rooms were maintained at 20–22 °C and 40–60% humidity. Experiments were conducted during the light phase. Procedures were performed in accordance with the National Institutes of Health Guide for Care and approved by the Use of Laboratory Animals and the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee. Additional information about mice used in this study can be found in the Life Sciences Reporting Summary.

Aggressor screening, CSDS and stCSDS

Female11 and male10 aggressor screening for CSDS and SI tests was performed as previously described. Experimental males were single housed after CSDS, and females were group housed during CSDS but single housed after defeat. Defeat was halted if an intruder showed any signs of injury. An all-male CSDS lasted 10 min per day for 10 days an all-female CSDS lasted 5 min per day for 10 days; stCSDS lasted for 5 and 2 min per day for males and females, respectively, for 10 days.

Chronic variable stress

CVS was modified from our previous work51. Male and female mice were randomly assigned to CTRL and CVS groups. CVS groups underwent 28 days of stress with one stressor per day, the stressors consisting of 1 h foot shock (random shock 60 times in 1 h), 1 h tail suspension and 1 h restraint.

Chronic restraint stress

Male mice were randomly assigned to CTRL and CRS groups. The CRS group underwent 28 days of 1 h restraint stress each day. For the juvenile odour-paired CRS, mice were restrained in a 50 mL restrainer and put in a new cage with bedding from a same-sex C57BL/6J juvenile mouse.

Social interaction test

SI tests were performed 24 h after the last defeat, as described previously10. Mice were habituated in the testing rooms for 1 h before testing and all testing was performed under red-light conditions. SI tests were performed with mice freely exploring in a target-free arena (44 cm (w) × 44 cm (d) × 38 cm (h)) for 2.5 min, followed by another 2.5 min target-present (CD-1 or ERα-Cre mice) session during which target mice were confined in a wire-mesh enclosure (10 cm (w) × 6.5 cm (d) × 38 cm (h)). The ‘interaction zone’ of the test arena encompassed a 14 cm × 24 cm rectangular area projecting 8 cm around the wire-mesh enclosure. The ‘corner zones’ encompassed a 9 cm × 9 cm area projecting from both corner joints opposing the wire-mesh enclosure. We calculated SI ratio as the ratio of time spent in the interaction zone with a CD-1 or F1 ERα-Cre mouse present over time spent with the target absent. All mice with a SI ratio over 1 were classified as RES mice and all with a ratio below 1 as SUS mice. Corner ratio was calculated as the ratio of time spent in the corner zone with an adult CD-1 or F1 ERα-Cre target mouse present over time spent when the target mouse was absent.

Resident intruder test

The RI test was modified from a previously described protocol52. After defeat and SI, mice were habituated in the testing rooms for 1 h before testing, and all testing was performed under dim light conditions. Experimental mice were kept in their home cage, placed under an Ethovision camera, habituated for 2–3 min with their wired cage top removed and then intruders (mice or objects) were introduced into their home cage and allowed to interact freely for 5 min (RI test for iDISCO+ cohort lasted for 10 min, to maximumly stimulate Fos expression). Social investigation included the amount of active interaction including approach, close following and sniffing. Social avoidance was defined as the escaping from a juvenile mouse of the experimental mouse when approached and investigated by the former. A speed of more than 20cm s–1 was considered an escape. SUS mice typically escaped at speeds of 20–65 cm s–1 immediately to avoid social encounters following juveniles’ approach/investigation. All experimental mice showing aggressive behaviours towards juveniles (around 1%) were excluded from analyses. All RI behaviours were scored blindly by experimenters.

Social conditioning place preference

The sCPP protocol, as published previously53, consisted of three phases: pretest, social conditioning and posttest. Mice were habituated in the testing rooms for 1 h before conditioning or testing. All phases were conducted under red-light and sound-attenuated conditions. The CPP apparatus (Med Associates) has a neutral middle zone that allows for unbiased entry and two conditioning chambers with different walls and floors. On the pretest day, mice were introduced into the middle chamber and allowed to explore freely in all three chambers of the CPP box for 20 min. No group differences in bias for either chamber were found, and conditioning groups were balanced in an unbiased fashion to account for start side preference, as described previously54. The conditioning phase consisted of four consecutive days with two conditioning sessions each day: during the morning paired sessions (08:00–12:00), experimental mice were confined to the assigned chamber for 15 min with a new same-sex juvenile C57BL/6J intruder; during the afternoon unpaired session (13:00–17:00) mice were put into the opposite chamber without a social target for 15 min. For female sCPP, during conditioning the juvenile mice were confined in a wire-mesh cup, which we found was necessary for females to form CPP, whereas males formed a preference only when they were able to freely interact with the juvenile outside the cup. All groups were counterbalanced for conditioning chamber. On posttest day, experimental mice were placed in the middle chamber of the CPP apparatus and allowed to freely explore all chambers for 20 min. Duration spent within either context was used to measure CPP. For chemogenetics experiments, CNO was administered during the full conditioning sessions. Behavioural analysis of sCPP data was performed by assessing (1) subtracted CPP (posttest phase duration spent in the intruder-paired chamber minus test phase duration spent in the intruder-unpaired chamber, accounting for test session behaviour only); and (2) group and individual durations in both pretest and posttest sessions.

Novel-object recognition and object location test

Novel-object recognition (NOR) and object location tests were performed as previously described55. Male mice were habituated in the testing room for 1 h before testing and then placed in the middle of an empty plexiglass open field (45 cm (w) × 45 cm (d) × 38 cm (h)) under dim light for 10 min (habituation phase). Twenty minutes after the habituation phase, mice were placed in the same open field with two objects (A and B) and allowed to explore for 10 min. Mice were then placed back in their home cage for 20 min before being put back into the open field with object B replaced by a new object, C. Mice were allowed to explore for 10 min. Following the NOR test, mice were transferred back to their home cage for 20 min before being returned to the open field, in which the location of object A was changed and the time spent interacting was recorded. Time spent with the new versus familiar object or location was recorded and scored by Ethovision software.

Elevated plus maze

The EPM was performed as previously described11. Male mice were habituated in the testing room for 1 h before testing and then placed in the middle of the plexiglass EPM under red light for 5 min. Each arm of the maze measured 12 × 50 cm2. Behaviour was tracked using Noldus Ethovision (Noldus Interactive technologies). Total time spent in the open and closed arms was measured.

Open field test and locomotor measures

Open field test was performed as previously described11. Male mice were habituated in the testing room for 1 h before testing and then placed in the middle of the plexiglass arenas (44 × 44 × 35 cm3) under red light for 10 min. Behaviour was tracked using Noldus Ethovision (Noldus Interactive technologies) to record the total distance moved, as well as the time spent in the centre (22 × 22 cm2) versus outer zones.

Marble burying test

The marble-burying test was performed as previously described56. Male mice were habituated in the testing room for 1 h before testing. Fresh, unscented mouse corncob bedding (depth 5 cm) was put in standard rat cages (26 cm (w) × 48 cm (d) × 20 cm (h)) with filter-top covers, and another cage was inserted onto the surface of the bedding to create parallel lines on the bedding surface that could be used for marble placement. Standard glass toy marbles (1.6 cm diameter) were placed gently on the surface of the bedding in five rows of four. Marbles were washed in 70% ethanol, rinsed in distilled water and dried before each use. Mice were introduced into the corner of the cage to explore for 30 min with the filter-top covered on the cage. A marble was scoredas buried if two-thirds of its surface area was covered by bedding. A 2-day, DREADD-manipulated marble-burying test was performed using a within-subjects design; mice were given either CNO or vehicle in a counterbalanced way, and thus they received CNO or vehicle on the first day and the alternative on the second day.

Real-time place preference

The RTPP experiments was performed as previously described54: mice were placed in the centre of an arena (44 cm (w) × 44 cm (d) × 35 cm (h)) with a central divider and allowed to explore freely for 20 min. The time spent on each side was recorded using Noldus Ethovision (Noldus Interactive technologies). For the first 10 min of the test, one side of the open field was paired with 20 ms pulses of 15 Hz blue-light stimulation (473 nm, 7–10 mW, 1 s on, 1 s off). For the second 10 min of the test, laser stimulation was paired with the opposite side of the arena; this was done to minimize inherent bias toward one side of the arena. There was a 1-min interval between the two phases. Total time spent in the unstimulated and stimulated sides was calculated and analysed.

Perfusion and brain tissue processing

For immunohistochemistry and iDISCO+, mice were euthanized by injection of 10% chloral hydrate and perfused transcardially with ice-cold 1× PBS (pH 7.4), followed by fixation with cold 4% paraformaldehyde in 1× PBS. Brains were postfixed for 12 h in the same fixative at 4 °C. For immunohistochemistry, coronal sections were prepared on a vibratome (Leica) at 50 μm to assess viral placement and for immunohistochemistry. For ISH, mouse brains were rapidly removed and flash-frozen in −30 °C isopentane for 5–10 s then kept at −80 °C until sectioning at 15 μm using a cryostat (Leica). Animals injected with AAV viruses were perfused at least 4 weeks after injection; animals injected with H129ΔTK-TT were perfused 48 and 70 h after injection.

IHC, ISH and confocal microscopy

For Fos IHC, slices were incubated for 2 h in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in PBS) then incubated overnight in primary antibody (mouse anti-Fos, 1:1,000 (Santa Cruz Biotechnology, C-10)) at 4 °C. Slices were then washed in PBS for 3 × 20 min and incubated in secondary antibody (Cy2 (no. 711-225-152), Cy3 (no. 711-165-152), Cy5 (no. 711-175-152), AffiniPure Donkey Anti-Rabbit IgG (H+L), 1:1,000 (Jackson ImmunoResearch)) for 2 h at room temperature, then washed in PBS for 3 × 20 min before staining with DAPI (1 μg mL–1, Sigma) for 20 min. Sections were then mounted with Eco-Mount (Life sciences) and coverslipped (Fisher). For Fos analysis, magnification of ×20 and tile-scan function were used to acquire the entire region of interest. Analysis of Fos-positive cells was performed using Fiji (NIH)57. For representative images of viral infection, images were acquired at ×10 magnification using the tile-scan function. For ISH, RNAscope Multiplex Fluorescent Kits (Advanced Cell Diagnostics) were used according to the manufacturer’s instructions. Briefly, fresh-frozen brains were slide mounted at 15 μm thickness, fixed for 15 min in cold 4% PFA and dehydrated serially with 50, 70 and 100% EtOH/H2O for 2 min each, followed by 20 min Protease IV (RNAscope) treatment. Proprietary probes (Advanced Cell Diagnostics) for Fos (316921, accession no. NM_010234.2); Sst (404631-C2, accession no. NM_009215.1), Gad67 (400951-C2, accession no. NM_008077.4), Oxtr (412171-C2, accession no. NM_001081147.1), Drd3 (447721-C, accession no. NM_007877.1) or Crhr2 (413201-C2, accession no. NM_009953.3); Nt (420441-C3, accession no. NM_024435.2) were hybridized at 40 °C for 2 h then subjected to a series of amplification steps at 40 °C (1-FL, 30 min; 2-FL, 15 min; 3-FL, 30 min; 4-FL, 15 min). Reagent Alt-A was used for the fourth amplification step, with Channel 1 at 488 nm, Channel 2 at 550 nm and Channel 3 at 647 nm. Finally, slides were treated for 1 min with DAPI and immediately coverslipped with Eco-Mount. All slices were imaged using a Zeiss LSM 780 confocal microscope. Cells and Fos from all ISH and IHC images were counted blindly across groups.

iDISCO+ staining, imaging and ClearMap analysis

The iDISCO+ staining protocol was modified from http://www.idisco.info. Fixed whole brains were incubated with the primary Fos antibody (no. 226003, 1:1,000, Synaptic Systems) and secondary donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (no. A-31573, 1:1,000, Thermo Fisher Scientific) for 7 days each. A LaVision lightsheet microscope with zoom body was used for half-brain sagittal scanning, with dynamic focus and a step size of 4 um. Cleared brains were processed as previously described using ClearMap12. Fos+ cells were quantified using the cell detection module, with cell detection parameters optimized and validated based on the intensity and shape parameters of the signal. The autofluorescence channel was aligned to the Allen Institute’s Common Coordinate Framework using the Elastix toolbox. Brain areas were collapsed into their parent region (for example, the rostroventral, caudodorsal and ventral lateral septum were combined into the ‘lateral septal nucleus’). These decisions were made before analysis. To compare cell counts in RES and SUS animals, a negative binomial regression was applied using the glm.nb function from the MASS package in R. Group classifications were dummy coded (0 for the SUS group and 1 for the RES group). The maximum-likelihood coefficients α and β were determined through iterative reweighted least squares. A significant β means that group status is related to cell count number at the specified region of interest. The z-values in Extended Data Fig. 2i correspond to this β coefficient, normalized by its sample standard deviation. P values were corrected for multiple comparisons using the Benjamini–Hochberg procedure to decrease false discovery rate. Q-values below 0.05 were considered significant.

Stereotaxic surgery and viral gene transfer

Nt-Cre mice (4–5 weeks old) were anaesthetized by intraperitoneal injection with a mixture of ketamine HCl (100 mg kg–1) and xylazine (10 mg kg–1) and positioned on a stereotaxic instrument (David Kopf Instruments). In the LS (from bregma: AP +0.7 mm; ML ±0.4 mm; DV −3.0 mm), 0.5 μL of virus was bilaterally infused using 33-gauge Hamilton needles over 5 min, with needles left in place for 5 min after injection. For DREADD virus delivery, 0.5 μl of AAV8-hSyn-DIO-hM3D-mCherry (2.0 × 1012 vg mL–1, no. 44361-AAV8, Addgene), AAV9-hSyn-DIO-hM4D-mCherry (2.0 × 1012 vg mL–1, no. 44362-AAV9, Addgene) or AAV9-hSyn-DIO-mCherry (2.0 × 1012 vg mL–1, no. 50459-AAV9, Addgene) was injected into the LS. For anterograde tracing, 0.5 μL of AAV9-hSyn-DIO-EYFP (2.0 × 1012 vg mL–1, no. 50457-AAV9, Addgene) or 0.15 μl of H129ΔTK-TT (4.0 × 109 vg mL–1, Center for Neuroanatomy with Neurotropic Viruses) was injected unilaterally into the LS. For retrograde tracing of LS downstream regions, 0.5 μL of retrograde AAV-DIO-EGFP/tdTomato (2.0 × 1012 vg mL–1, nos. 50457-AAVrg and 28306-AAVrg, Addgene) was injected into the medial part of the NAc (from bregma: AP +1.5 mm; ML ±0.5 mm; DV −4.4 mm), AHN (from bregma: AP −0.7 mm; ML ±0.5 mm; DV −5.0 mm) or PAG (from bregma: AP −4.2 mm; ML ±0.2 mm; DV −2.5 mm). For CTB injection, 0.5 μL of Alexa Fluor 488-conjugated Cholera Toxin Subunit B (1.0 mg mL–1, no. C-34775, Thermo Fisher) was injected into the NAc (from bregma: AP +1.5 mm; ML ±0.5 mm; DV −4.4 mm), 0.5 μL of Alexa Fluor 555-conjugated Cholera Toxin Subunit B (1.0 mg mL–1, no. C-34776, Thermo Fisher) was injected into the AHN (from bregma: AP −0.7 mm; ML ±0.5 mm; DV −5.0 mm) and 0.5 μL of Alexa Fluor 647-conjugated Cholera Toxin Subunit B (1.0 mg mL–1, no. C-34778, Thermo Fisher) was injected into the PAG (from bregma: AP −4.2 mm; ML ±0.2 mm; DV −2.5 mm). For optogenetics, 0.5 μL of AAV9-EF1a-DIO-EYFP (3.0 × 1012 vg mL–1, no. 27056-AAV9, Addgene), AAV9-Ef1a-DIO eNpHR3.0-EYFP (3.0 × 1012 vg mL–1, no. 26966-AAV9, Addgene) or AAV9-EF1a-DIO-ChR2-EYFP (3.0 × 1012 vg mL–1, no. 20298-AAV9, Addgene) was injected into either the LS (cell body stimulation) or downstream regions (terminal stimulation). For CreOff virus injection, AAV-EF1a-Flpo (2.0 × 1012 vg mL–1, no. 55637-AAV1, Addgene) and AAV-nEF-Coff/Fon-ChR2(ET/TC)-EYFP (2.0 × 1012 vg mL–1, no. 137141-AAV8, Addgene) were mixed 1:1 and injected into the LS. All AAV injections were performed 3 weeks before perfusion or behavioural experiments. For aggressors used in female CSDS, we targeted the VMHvl of ERα-Cre F1 mice as described previously11,58. For FP, 0.5 μL of AAV9-CAG-FLEX-G6s/EGFP virus (2.0 × 1012 vg mL–1, no. 100842-AAV9, 51502-AAV9 Addgene) was injected unilaterally into the LS. For optogenetic (ChR2) and FP experiments, cannulae (ChR2: MFC_200/240-0.22_3mm_MF1.25_FLT; FP: MFC_200/250-0.57_3mm_MF1.25_FLT) were implanted at the same time as viral delivery (for LS local, fibres were implanted 0.2 mm above the injection site). For optogenetic (ChR2 and eNpHR3.0) experiments on NTLS terminal stimulation, cannulae (MFC_200/240-0.22_MF1.25_FLT, 5 mm for NAc/AHN, 3 mm for PAG) were implanted into the NAc (from bregma: AP +1.5 mm; ML ±1.5 mm; DV −4.4 mm, 15° angle), the AHN (from bregma: AP −0.7 mm; ML ±1.5 mm; DV −4.8 mm, 10° angle) or PAG (from bregma: AP −4.2 mm; ML ±0.2 mm; DV −2.3 mm). For secure fixture of the optic fibre, dental cement (Grip cement; Dentsply) was added to the skull and around the fibres.

DREADD manipulation

For ERα-Cre mice (used for female CSDS), CNO (1 mg kg–1, Tocris) was given intraperitoneally 30 min before CSDS11. For OFT, EPM and the marble-burying, SI and RI tests, CNO was given 30 min before the test; for sCPP, CNO was given 30 min before each conditioning session.

Optogenetics manipulation

For blue (ChR2) and orange (eNpHR3.0) light stimulation, optical fibres (BFP(2)_200/220/900-0.22_4m_FCM-2xMF1.25, Doric Lenses) were connected to either a 473 nm blue laser diode (no. BCL-473-050-M, Crystal Laser) or a 589 nm orange laser diode (no. MGL-III-589-50mW, Opto Engine LLC) using a patch cord with a FC/PC adaptor (no. MFP_200/240/900-0.22_4m_FC-MF1.25, Doric Lenses). A function generator (no. 33220A, Agilent Technologies) was used to generate 20 ms blue-light pulses at 15 Hz, 1 s on/1 s off for all ChR2 experiments. Constant orange light was used for eNpHR3.0 experiments during the 5 min resident intruder test. For sCPP studies, orange light was delivered in a 4 min on/1 min off pattern. The intensity of light delivered to the brain was 7–10 mW. These parameters are consistent with previously validated and published protocols24. For all optogenetics tests, experimental mice were habituated to patch cords for 2 days before testing in RI. For RI experiments, mice were tested over 2 days, counterbalanced under laser-on and -off conditions. For social CPP tests, light was provided during the social conditioning session. For the RTPP test, blue-light delivery was controlled by TTL from Noldus Ethovision (Noldus Interactive technologies).

Ex vivo electrophysiology

AAV9-hSyn-DIO-EYFP (0.5 ul, 2.0 × 1012 vg mL–1, Addgene) was injected bilaterally into the LS of 4-week-old male Nt-Cre mice. Two to three weeks after injection, the mice underwent CSDS. Before slice preparation, all mice were exposed to a 4–6-week-old, same-sex juvenile intruder for 5 min. About 20 min after the RI test, mice were anaesthetized using isoflurane. The brain was rapidly extracted and coronal sections (250 µm) sliced using a Compresstome (no. VF-210-0Z, Precisionary Instruments) in cold (0–4 °C) sucrose-based artificial cerebrospinal fluid (SB-aCSF) containing (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 MgCl2, 23 NaHCO3, 75 sucrose and 25 glucose. After 60 min at 32 °C for recovery, slices were maintained in oxygenated (95% CO2/5% O2) aCSF containing (in mM): 130 NaCl, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.2 MgCl2, 23 NaHCO3 and 11 glucose at room temperature for the remainder of the day, and transferred to a recording chamber continuously perfused at 2–3 mL min–1 with oxygenated aCSF. Patch pipettes (4–7 MΩ) were pulled from thin-walled borosilicate glass using a micropipette puller (no. P-97, Sutter Instruments) and filled with a K gluconate (KGlu)-based intrapipette solution containing (in mM): 116 KGlu, 20 HEPES, 0.5 EGTA, 6 KCl, 2 NaCl, 4 ATP and 0.3 GTP and 2 mg mL–1 biocytin (pH adjusted to 7.2). Cells were visualized using an upright microscope with an IR-DIC lens and illuminated with a white light source (Scientifica). A 470 nm LED (no. pE-300ultra, Cooled) illumination through the microscope objective was used for visualization of eYFP+ cells (using a bandpass filter cube, Olympus). Excitability was measured in current-clamp mode by injection of incremental steps of current (0–100 pA, +10 pA at each step). For recording of optically evoked inhibitory postsynaptic currents (oIPSCs), AAV9-EF1a-DIO-ChR2-eYFP (0.5 µL, 3.0 × 1012 vg mL–1, Addgene) was injected bilaterally into the LS of 4-week-old male Nt-Cre mice. At 5–8 weeks post injection, coronal brain slices of NAc/AHN were prepared as described above and NAc/AHN neurons were recorded in voltage-clamp mode using an internal solution containing (in mM): 120 Cs-methanesulfonate, 10 HEPES, 10 Na-phosphocreatine, 8 NaCl, 5 TEA-Cl, 4 Mg-ATP, 1 QX-314, 0.5 EGTA and 0.4 Na-GTP. NTLS terminals were stimulated through the microscope x40 objective (15 Hz, 5 ms per pulse, 470 nm; no. pE-300ultra, CoolLed). oIPSCs were recorded at 0 mV in the presence of tetrodotoxin (TTX, 1 μM, Tocris) to probe monosynaptic effects. oIPSCs were blocked by bath application of gabazine (no. SR-95531, 10 μM, Tocris) confirming the GABAergic nature of the synaptic contact. Whole-cell recordings were performed using a patch-clamp amplifier (Axoclamp 200B, Molecular Devices) connected to a Digidata 1550 LowNoise acquisition system (Molecular Devices). Signals were low-pass filtered (Bessel, 2 kHz) and collected at 10 kHz using the data acquisition software pClamp 11 (Molecular Devices). Electrophysiological recordings were extracted and analysed using Clampfit (Molecular Devices). All groups were counterbalanced by days after defeat. All recordings were performed blind to experimental condition.

Fibre photometry

Fibre photometry was performed according to the Neurophotometrics manual and published protocols59. A fibre-optic patch cord (no. MFP_200/240/900-0.48_3m_FC-MF1.25, Doric Lenses) was attached to the implanted cannula with cubic zirconia sleeves covered with dark-coloured, shrinkable tubing. The other end of the fibre-optic cable was coupled to a Neurophotometrics LED port. The open-source Bonsai programme was used to control the system; 470 and 415 nm LED lights were used for GCaMP6s signal and autofluorescence measurement. Light at the fibre tip ranged from 40 to 80 μW and was constant across trials over testing days. Simultaneous recording of 40 fps from both 470 and 415 nm channels was achieved phase to phase and visualized via Bonsai. Three weeks after virus injection and ferrule implantation, when mice were around 8 weeks old, they underwent CSDS and SI; they were then habituated to the patch cord for 2 days and Ca2+ fluorescence was recorded during the RI test, social CPP conditioning session, stress and food reward tests. Once connected to the apparatus, mice were allowed to rest and habituate for 3–5 min before starting. For the RI test, we recorded Ca2+ fluorescence during 2 min of baseline activity without an intruder, followed by 5 min of intruder exposure. The food reward was performed in an open field, and peanut butter cups were placed in the arena close to the corners. All food-biting events were scored manually. MATLAB custom-coding was used for analysis of signal. The 415 nm channel served as the control channel and was subtracted from the GCaMP6s channel to eliminate autofluorescence, bleaching and motion effects. Change in fluorescence (ΔF/F) was calculated as the percentage of mean fluorescence signal of GCaMP6s signal. In general, these motion artefacts had very little effect on overall GCaMP6s signal. Behavioural data were aligned with fluorescence recording data by dividing behavioural video frames with GCaMP6s signal frames. For analysis of LS GCaMP6s activity during discrete behaviours in the RI test, average ΔF/F (%) in the 2 s before and after a discrete event (passive social investigation) were compared. A passive social investigation was determined to occur at the moment of the intruder-initiated passive social approach.

Statistical analysis

All statistical details can be found in the figure legends, including type of statistical analysis used, P values, n, what n represents, degrees of freedom and t or F values. All t-tests, one-way ANOVA and repeated two-way ANOVA were performed using GraphPad Prism software (GraphPad Software Inc.). One-way ANOVA analysis was followed by Tukey’s multiple-comparisons test, and two-way repeated-measures ANOVA analysis was followed by Šídák’s multiple-comparisons test. Statistically significant differences are indicated in each figure (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). For detailed P values please see Source data. Analyses of Fos staining, ISH data and behavioural videos during the RI test were performed blinded to experimental conditions. Sample sizes were chosen according to previous experiments. For Extended Data tables and the Supplementary table, P values were corrected for multiple comparisons using the Benjamini–Hochberg procedure to reduce false discovery rate. Q-values below 0.05 were considered significant for all Extended Data tables.

Statistics and reproducibility

Figure 2c and Extended Data Fig. 3i were repeated in three separate cohorts per sex, with all showing similar results. Figure 5a,b (right) was repeated in three seperate male cohorts (n = 6) and in one female cohort (n = 2), with all showing similar results. Extended Data Fig. 3j was repeated twice in both sexes, with both showing similar results. Extended Data Fig. 6a was repeated in four separate cohorts in both sexes, with all showing similar results. Extended Data Figs. 8a,d (right) and 10e were repeated twice in males only, with both cohorts showing similar results. Extended Data Fig. 8b was repeated three times, with all showing similar results.

Image visualization

Brain slice schematics in Figs. 2g,i,j, 3a, 4a and 5a,b and Extended Data Figs. 4d,f,g, 7c, 8a,b,d and 10d were adapted from the Allen Brain Atlas Reference using Adobe Illustrator.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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