Animals
Wild-type Swiss-Webster (SW) mice (male and female, 12-15 weeks; Charles River) and Esr1-Cre mice (017911, B6N.129S6(Cg)-Esr1tm1.1(cre)And/J; Jackson Laboratory) were crossed with SW females to obtain F1 mice used as experimental mice. Intruders for the resident-intruder (RI) test were 8-12-week-old male or female C57/BL6J mice (Jackson Laboratory). All delivered mice were allowed 1 week of acclimation to the housing facility prior to any experimental protocol. At 8 weeks, mice were separated from their littermates and paired with a member of the opposite sex for sexual experience. Females were paired with castrated SW males (8-12 weeks; Charles River) to prevent pregnancy. 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.
RI Test
Mice were screened using protocols adapted from previous studies3,44. Briefly, cage tops were removed and replaced with Plexiglas covers to monitor trials. A novel C57BL/6J mouse matching the sex of the resident was introduced into each cage and mice were allowed to freely interact for 5 min. After 5 min elapsed, intruder mice were returned to their home cages and, in the case of female resident-intruder trials, cohabiting male mice were returned to their home cages. All videos were recorded for later analysis. Resident behaviors were manually annotated using Observer XT 11.5 (Noldus Interactive Technologies).
Aggression Self-Administration (SA) Test
Aggression SA testing was conducted as previously described45. Briefly, SW resident mice were placed in standard Med Associates operant chambers and underwent 8 trials per day for 16 days in which they could press a lever (FR1) in order to receive access to a subordinate same-sex mouse through a guillotine door into their operant chamber. A house light illuminated the chambers during trials, and an inactive lever was extended at all times. At the beginning of a trial, the house light turned on, and a is lever extended 10 s later. If the resident pressed for a reward, a 2 s tone played and a guillotine door next to the active lever opened for 12 s. A same-sex C57BL/6J intruder manually pushed into the chamber, and the intruder was removed at the end of the trial. If residents did not press, the active lever retracted after 60 s. All chambers also had recessed food pellet receptacles with beam-break registered entries ports. We conducted all Progressive Ratio (PR) tests using the same parameters we used for self-administration training, except for the trial design. Following reward obtention (number of required active lever presses for a given trial), the automatic guillotine door was opened for 10 s and the same-sex intruder (C57BL/6J) was manually pushed inside the main operant chamber. Immediately after the automatic door closed, the active/inactive levers were re-extended. The intruder mouse was removed from the chamber following an attack or after 30 s had elapsed. The PR session was terminated if no reinforced responses occurred after a total duration of 20 min had elapsed. Throughout the sessions, for each trial, the number of responses required to activate the guillotine door and gain access to the intruder was incremented following an exponential progression (R = 5 x e0.12*P - 5, where P is the previous ratio). The breakpoint value corresponds to the total number of rewards (number access to the intruder during the whole session). Esr1-cre mice transfected in the COApl using AAV9-DIO-hM4Di-mCherry or AAV9-DIO-mCherry (see titer, surgeries information and coordinates below) were administered either saline (SAL) or Clozapine-N-Oxide (CNO), counterbalanced over the two days of the PR.
Hidden Food Test
Mice were habituated to and allowed to consume a palatable food (i.e., Reese’s mini peanut butter cup) two days prior to being tested. Mice were then food-restricted the night before the test. Mice were tested under red light and placed in a clean mouse cage. The food was placed 8 cm deep in the bedding of the cage and the time to find the food was recorded with a stop-watch.
Sex Discrimination Test
Male and female C57/BL6J mice were used as target mice and placed on opposite sides of an open field enclosure in mesh cups. Test mice were placed in the open field and allowed to explore the arena for five minutes under red light. Mice were recorded with an overhead Ethovision camera and time spent interacting with each mouse was scored offline manually. To calculate the discrimination ratio, the total time spent interacting with both mice was divided by the difference between time spent with the female and male. A positive discrimination ratio indicates more time spent with the female, a negative ratio indicates more time spent with the male.
Perfusion and brain tissue processing
For iDISCO+, mice were injected with 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 post-fixed for 12 h in the same fixative at 4 °C. For FISH, brains were rapidly removed and flash-frozen in −30 °C isopentane for 5–10 s then kept at −80 °C until sectioning. Slices were sectioned at 15 μm using a cryostat (Leica). Animals injected with H129ΔTK-TT were perfused 48 h after injection.
In Situ Hybridization and Imaging
For FISH, RNAscope Multiplex Fluorescent Kits (Advanced Cell Diagnostics) were used according to the manufacturer’s instructions. Fresh-frozen brains were mounted on slides 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), Vglut1 (SLC17A7-C3, accession no. NM_182993.2) and Esr1 (478201-C2, accession no. NM_007956.5 ) 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 30 s with DAPI and immediately coverslipped with Eco-Mount. All slices were imaged using a Zeiss LSM 780 confocal microscope. Cells from all ISH images were counted blindly across groups. Cells with at least 5 puncta for each probe were considered to be positive for the probe of interest.
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 μm. Cleared brains were processed as previously described using ClearMap13. 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. To compare cell counts between AGG and NON animals in both sexes and hM4Di vs. mCherry, 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 AGG group and 1 for the NON 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. Z-scores 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
Esr1-Cre mice (8-10 weeks old) were anaesthetized with an intraperitoneal injection of ketamine HCl (100 mg kg–1) and xylazine (10 mg kg–1) and positioned on a stereotaxic instrument (David Kopf Instruments). In the lateral portion of the COAp (COApl) (bregma: AP -1.7 mm; ML ±2.8 mm; DV −5.9 mm), 0.3 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 virus delivery, 0.3 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 COApl. We also injected 0.3 μL AAV9-hSyn-DIO-hM4D-mCherry (2.0 × 1012 vg mL–1, no. 44362-AAV9, Addgene) into the COApm (bregma: AP -2.4 mm; ML ±2.0 mm; DV −5.6 mm) For anterograde tracing, 0.3 μL of AAV9-hSyn-DIO-EYFP (2.0 × 1012 vg mL–1, no. 50457-AAV9, Addgene) or 0.2 μl of H129ΔTK-TT (4.0 × 109 vg mL–1, Center for Neuroanatomy with Neurotropic Viruses) was injected unilaterally into the COApl. For optogenetic manipulations, AAV9-Ef1a-DIO eNpHR3.0-EYFP (3.0 × 1012 vg mL–1, no. 26966-AAV9, Addgene) or AAV9-EF1a-DIO-EYFP (3.0 × 1012 vg mL–1, no. 20298-AAV9, Addgene) was injected into the COApl. All AAV injections were performed 3-4 weeks before behavioural experiments. 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 COApl local, fibres were implanted 0.2 mm above the injection site). For optogenetic (eNpHR3.0) experiments of COApl terminal stimulation, cannulae (MFC_200/240-0.22_MF1.25_FLT, 6 mm for VMHvl, 5 mm for CEA) were bilaterally implanted into the VMHvl (from bregma: AP -1.7 mm; ML ±2.5 mm; DV -5.5 mm, 20° angle), or CEA (from bregma: AP -1.5 mm; ML ±2.5 mm; DV -4 mm, 0° angle). For secure fixture of the optic fibre, dental cement (Grip cement; Dentsply) was added to the skull and around the fibres.
DREADD manipulation
CNO (1 mg kg–1, Tocris) was given intraperitoneally 30 min before the RI test, open field, aggression self-administration, hidden food and sex discrimination tests.
Optogenetics manipulation
For yellow (eNpHR3.0) light stimulation, optical fibres (BFP(2)_200/220/900-0.22_4m_FCM-2xMF1.25, Doric Lenses) were connected to a 589 nm yellow 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 constant orange light was used for eNpHR3.0 experiments during the 5 min RI test. 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.
Fibre photometry
Fibre photometry was performed according to the Neurophotometrics manual and published protocols5,44. A fibre-optic patch cord was attached to the implanted cannula with cubic zirconia sleeves covered with black tubing. The opposite end of the 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, mice were tested in the RI test and were presented different odours in a counterbalanced manner. Once connected to the apparatus, mice were allowed to rest and habituate for 3–5 min before starting. For the RI test and odour presentation, we recorded Ca2+ fluorescence during 2 min of baseline activity without an intruder, followed by 5 min of intruder or odor exposure. 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 computed by subtracting the average value during the final minute of baseline recording and then dividing the resulting value by the averaged value during the final minute of baseline. This value was then z-scored by subtracting the average ΔF/F from each value and dividing by the standard deviation. Behavioural data were manually annotated in Ethovision and time stamps were aligned with fluorescence recording.
Weighted correlation network analysis
For network construction, we followed previously published guidelines14. Briefly, a n x n adjacency matrix was created which encodes the connection strengths between pairs of nodes (brain regions), using the power adjacency function: Aij = |sij|β. Aij refers the adjacency matrix and sij refers to the correlation between regions i and j raised to the power of β. The correlation is raised to the power of β in order to reduce the influence of potentially specious correlations. To measure the similarity between the nodes for clustering the topological overlap measure (TOM) was used:
refers to the TOM matrix,
In words, this is the sum of the product of the shared connections between regions i and j plus the direct connection between regions i and j. This value is divided by the denominator to achieve a value between 0 and 1. In order to compare the expression of these modules between sexes within a network, a singular value decomposition (SVD) was computed on each module. The SVD is defined as:
Xn is the TOM matrix for the nth module in the network, U and VT are matrices of orthonormal vectors, and S is a diagonal matrix of eigenvalues that denote how much variation is accounted for by each column of U and V. To determine which modules differed between AGG and NON networks, or whether the mCherry and hM4Di networks conserved module connectivity and density, we used the module preservation function. For comparisons between networks we focused on the following measures for connectivity: Z.cor KIM, Z.cor.KME, Z.cor.KMEall, Z.cor.cor, and Z.cor.MAR. For density measures, we focused on Z.propVarExplained, Z.meanSignAwareCorDat, Z.meanAdj and Zmean.MAR. All Z values were derived by randomly permuting the module labels in the test network and calculating the corresponding preservation metric. The average value of each statistic from the permutations is subtracted from the observed statistic and divided by the standard deviation of the statistic from the permutations. For a detailed explanation of the preservation measures, see table 2. All of the above steps were performed using the WGCNA package in R.
Statistical analysis
All statistical tests and associated information are reported in the Figure legends. All t-tests and two-way ANOVAs were performed using GraphPad Prism software (GraphPad Software Inc.). Two-way repeated-measures ANOVA analysis was followed by Šídák’s multiple-comparisons test for post-hoc analysis. For comparisons between groups for region by region iDISCO+ analysis, 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.