Overview of the in vivo experiments

Female Wistar rats were split into three different housing conditions: GH females were kept in groups of three to five animals per cage, IS females were singly housed for 8 days, and IST females were also kept singly for 8 days, but from day 5 on, they underwent three consecutive FITs for aggression-training. On day 9, GH females were singly housed 4 h prior to the behavioral experiments. One hour after lights went off, GH, IS, and IST rats were either exposed to the FIT, whereas control rats were left undisturbed in their home cage (behavioral data displayed in Fig. 1). Immediately after the FIT, rats were deeply anesthetized using intraperitoneal (i.p.) urethane (25%, 1.2 ml/kg) to allow CSF collection via puncture of the Cisterna cerebromedullaris. After decapitation, brain and trunk blood were collected for receptor binding and hormonal measurements, respectively (data displayed in Fig. 2). For radioimmunoassays, animals were again split into two groups according to the peptide measured, i.e., half of the subjects were used for OXT radioimmunoassay (RIA), whereas the other half was used for AVP RIA. Here we again counterbalanced levels of aggressin to avoid uneven groups. In addition, samples coming from rats that either we could not extract or had shown blood in their CSF were not included in the assays.

For pharmacological experiments, female rats were split into GH and IST conditions. All females underwent surgery for i.c.v. or local cannula implantation. IST females were left undisturbed for recovery for 3 (i.c.v.) or 5 (local) days. The aggression training was performed as described above, except for the fact that residents received a sham-infusion before the FIT to get used either to the i.c.v. or local infusion procedure. GH animals were kept single-housed overnight for recovery and brought back to their original groups the next day until the start of the experiments when they were transferred into an observation cage and single-housed for 4 h before the FIT. All animals were handled daily to get used to the infusion procedure. Typically, a cross-over, within-subjects design was used for all i.c.v. agonist experiments, whereas a between-subjects design was used for the local infusions and i.c.v. antagonists experiments. Additionally, to guarantee that all groups had similar average levels of aggression before pharmacological manipulations, we allocated IST rats into the treatment groups according to their average levels of aggression expressed in the third session of training.

OXT and AVP release within the LS was monitored in GH and IST females before and during FIT exposure. After 4 days of training, IST rats had their microdialysis probes implanted into the LS. After 1 day of recovery, IST subjects received the 5th training FIT to confirm their previous levels of aggression. On the following day, both GH and IST animals underwent the microdialysis procedure. In brief, rats were connected to a syringe mounted onto a microinfusion pump via polyethylene tubing and were perfused with sterile Ringer’s solution (3.3 μl/min, pH 7.4) for 2 h before sampling of microdialysates to establish an equilibrium between inside and outside of the microdialysis membrane. One hour after lights went off, three consecutive 30-min dialysates were collected (baseline samples 1 and 2 represented in Figs. 4 and ​and55 as an average of both time-points) and during an ongoing FIT (sample 3). Dialysates were collected into Eppendorf tubes containing 10 μl of 0.1 M HCl, were immediately frozen on dry ice, and stored at −20 °C until subsequent quantification of AVP and OXT by radioimmunoassay (for behavioral analyses see Supplementary Data ²–³). Microdialysis data are displayed here as a percentage of release from baseline (Peptide content of sample 3/average Peptide content in sample1 + sample 2 × 100).

Chemo- and optogenetic experiments targeting the OXT system were only performed in GH rats. After stereotaxic virus infusion into the PVN and SON, animals were kept single-housed for 1 week to recover. Thereafter, they were again group-housed for 2 weeks until either the experiment took place (chemogenetic) or they had their optical fiber implanted (optogenetic). Similarly to the pharmacologal experiments, chemogenetic rats were kept in groups and isolated shortly before the dark phase (see above). Subjects received an i.p. infusion of clozapine-N-oxide dihydrochloride (CNO, 2 mg/kg) 45 min before the FIT. They were brought back to their original groups directly after the FIT. Importantly, for those experiments, we had three control groups: (i) subjects infected with a control rAAV1/2 OXTpr-mCherry into the PVN and SON, which received CNO (virus control), non-infected rats who received either (ii) CNO or (iii) saline infusions (drug control). Since there was no difference among the levels of aggression displayed by those three groups (not shown) they were pooled together in a single control group depicted in gray in Fig. 3g. In the optogenetic experiments, after optical fiber implantation rats were single-housed for three days for recovery and to avoid damaging the fiber. Similarly to the microdialysis experiments, both controls and ChR2 animals were connected to the optogenetic cables two hours before the experiment to get used to the cables. In this experiment, the FIT lasted 12 min. Blue-light stimulation (30 ms pulses of 30 Hz delivered for 2 min; in analogy to Knobloch et al., 2012) was delivered at the 2nd and again 8th minute after the beginning of the FIT. Here, controls consisted of animals infected with a control rAAV1/2 OXTpr-mCherry into the PVN and SON. For both, chemo- and optogenetic experiments, animals were tested twice at different phases of their estrous cycle: once in proestrus/estrus, and once in metestrus/diestrus. All animals were transcardially perfused with paraformaldehyde (PFA 4%) for histological verification of the specificity of viral infections after the last test.

Although the i.c.v. and local pharmacology experiments showed robust serenic effects of AVP (Figs. 3 and ​and5),5), we decided to not genetically manipulate the AVP system centrally or locally due to the following reasons: (i) AVP is one of the main players in brain physiology and stimulation of several cell bodies in different brain regions especially in the hypothalamus could disturb homeostasis. In fact, high doses of i.c.v. AVP are known to elicit barrel rotations in rats⁶⁶. (ii) AVP neurons are widespread in several nuclei in the rat brain such as the medial amygdala, the BNST, PVN, and SON⁶⁷, which makes the infusion of all targeted neurons challenging in terms of animal welfare. (iii) We (data not shown) and others⁶⁸ have found low specificity of the AVP directed virus to infect intra- and extrahypothalamic parvocellular AVP neurons, which are known to project to the LS⁶⁷.

To compare neuronal activity patterns in the dLS and vLS of IST and GH rats after exposure to the FIT, we used female Venus-VGAT rats (10–14 weeks old). Immediately after FIT exposure rats were deeply anesthetized with isoflurane (ForeneH, Abbott GmbH & Co. KG, Wiesbaden, Germany), followed by CO_2, transcardially perfused, and brains were harvested for subsequent immunohistochemistry stainings (behavioral data in Supplementary Data ²–³).