Supplementary Materials and Methods
Cannulation
For intracerebral drug infusion, guide cannulas were stereotaxically implanted 2 mm above the lateral ventricle or bilaterally 2 mmabove the central, medial, or basolateral amygdala (see Table S1 for stereotaxic coordinates). Rats and mice were anesthetized (rats: isoflurane, Forene®, Abbott GmbH & Co. KG, Wiesbaden, Germany; mice: pentobarbital, 0.08 mg/g body weight, Narcoren®, Merial GmbH, Hallbermoos, Germany), injected with an antibiotic (Baytril®, Bayer Vital GmbH, Leverkusen, Germany), and mounted on a stereotaxic frame. The guide cannula (for icv: 21 G,rats: 12 mm; mice: 8 mm length; for local infusions in rats: 23 G, 12 mm; Injecta GmbH, Germany) was fixed to the skull with two jeweller’s screws and dental cement (Kallocryl, Speiko-Dr. Speier GmbH, Muenster, Germany) and closed by a stainless steel stylet (25 G and 27 G, respectively). After surgery, rats and mice were handled daily (stroking, holding, cleaning of stylets) for five days to minimize non-specific stress responses during the experiment.
Intracerebral infusions
Infusions were performed according to the established protocols in our laboratory concerning doses and time-points, which have been proven to have behavioral effects(Bosch and Neumann, 2008; Waldherr and Neumann, 2007). Briefly, rats received either icv or local infusions of a selective OT receptor antagonist(OTR-A; desGly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT;icv: 0.75µg/5µl; local: 0.1 µg/1µlor 1.0 µg/1µl), that is 18-fold more selective for OT receptors over V1a receptors(Manning et al., 2008), synthetic OT (icv: 0.1 µg/5 µl; local: 0.01 µg/1 µl), a selective vasopressin 1a antagonist (V1aR-A; d(CH2)5[Tyr(Me)2]AVP; 0.75µg/5µl or vehicle (sterile Ringer solution) via an infusion cannula (extended 2 mm beyond the guide cannula) connected via polyethylene tubing to a Hamilton syringe. After slow infusion the infusion system was left in place for 30 s. Infusions took placeeither 20 min (icv) or 10 min (local) prior to behavioral testing. Mice underwent the same procedure except that they were given icv infusions with either OTR-A (20µg/2µl) or vehicle.
Administration of pentylenetetrazol in rats
The anxiogenic pentylenetetrazol (PTZ; 15mg/kg, i.p., De Souza et al. (1998)) was injected 15 min (Jones et al., 2002) prior to behavioral testing on the elevated plus-maze or in the social preference test in the neutral arena.
Acute social defeat-induced social avoidance
To induce social avoidance, single housed rats were exposed to an aggressive rat for 30 min 2 h prior to the social preference test in the neutral arena. The experimental rat was introduced into the home cage of an unfamiliar trained aggressive rat. This rat usually attacks an intruder within a minute followed by submissive postures of the intruder rat. Physical interactions were terminated by the experimenter as soon as the intruder showed signs of submissive behavior (to prevent physical damage) or after a period of 10 min to the experimental rat by separating the rats by a wire-mesh screen. After a total time of 30 min, the experimental rat was placed back in its home cage.
Histology
To verify the infusion site, rats and mice were killed using CO2 and ink was infused icv or locally (5µl and 2ul, for rats and mice respectively) before removal of the brain. Icv brains were instantly cut coronally and checked for staining of the ventricle, whereas locally infused brains were frozen in pre-chilled n-methylbutane on dry ice and cut in 40 µm coronal cryostat sections and stained with cresyl violet. Only those animals with correct infusion site were included in the statistical analysis.Accordingly, a total of sixrats had to be excluded from statistical analysis due to misplacement of the cannula(s) (local OT receptor antagonist: meA (2); localOT: ceA (2), meA (2)).
References
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De Souza MM, Schenberg LC, Carobrez AdP, (1998). NMDA-coupled periaqueductal gray glycine receptors modulate anxioselective drug effects on plus-maze performance. Behavioural Brain Research. 90: 157-165.
Jones N, Duxon MS, King SM, (2002). Ethopharmacological analysis of the unstable elevated exposed plus maze, a novel model of extreme anxiety: predictive validity and sensitivity to anxiogenic agents. Psychopharmacology (Berl). 161: 314-323.
Manning M, Stoev S, Chini B, Durroux T, Mouillac B, Guillon G, et al., (2008). Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents. (eds.). Progress in Brain Research. Elsevier. pp. 473-512.
Waldherr M, Neumann ID, (2007). Centrally released oxytocin mediates mating-induced anxiolysis in male rats. Proc Natl Acad Sci U S A. 104: 16681-16684.