Ringer s solution

In vitro investigations were performed with VRC and NO drug substances purchased from Toronto Research Chemicals (Toronto, Canada). For all parts of the study, CMA 60 microdialysis catheters (molar mass cut-off 20 kDa, membrane length 30 mm, M Dialysis AB, Sweden) were used and perfused with Ringer’s solution (B. Braun, Melsungen, Germany). CMA 102 in vitro pumps (M Dialysis AB, Sweden) ensured a constant flow of perfusate.

Rats were anesthetized in an induction chamber with 4–5% oxygenated isoflurane for 3–4 min. They were then moved to a preparation area where the tail vein was catheterized with a winged 27 G catheter (Terumo), and the head was shaved. In all, 2 mg kg⁻¹ Meloxicam (Metacam) and 7 mL kg⁻¹ warmed Lactated Ringers solution (Fresenius Kabi, AG) were subcutaneously injected every 3–4 h. The rat was moved to a stereotaxic frame (David Kopf), and an incision was performed on scalp to expose skull surface. A layer of eye cream was put on the eyes. A craniotomy was performed with a micro-drill above vM1 (Coordinates AP: 0–2.5 mm and ML: 0–2 mm, with respect to bregma), and dura was carefully opened with a 30G needle. During the craniotomy, the skull was frequently flushed with Ringers solution (B. Braun) to prevent heating. After dura removal, a piece of gel foam (Pfizer) was put on brain and Ringers solution was regularly applied to keep the brain moisturized until electrode insertion.

MIC_Cop (4–5 × 10⁷) from naïve SJL/J mice were generated as described and labeled with 20 MBq ¹¹¹Indium (In)-oxine (Mallinckrodt Pharmaceuticals, Dublin, Ireland) in PBS for 15 min at room temperature. After removal of free ¹¹¹In-oxine by washing with 50 mL PBS, the cells were resuspended in 130 µL PBS and 50 µL of cell suspension was injected into the tail vein of recipient mice. Twenty-four hours later, mice were anaesthetized with 1 % (v/v) sevoflurane (Baxter) and, after blood samples had been taken from the vena cava, perfusion with Ringer’s solution (B.Braun, Melsungen, Germany) was conducted until blood was completely washed out from the organ system. Tissue samples of organs were harvested and weighed. Radioactivity of the specimen was measured along with 10 µL-aliquots (n = 3) of the injected suspension in a γ-counter (LB951G, Berthold Technologies, Bad Wildbad, Germany). For each sample, the activity of the ¹¹¹In-tracer in 1 g of tissue was calculated in relation to the originally injected total dose (% ID/g).

Osmotic minipumps (Alzet, model 1002, flow rate 0.25 µl/h, 14 days) were filled with either vehicle (VEH; Ringer’s solution, B. Braun Melsungen AG, Germany), 4 µM or 40 µM OXT (Bachem, Bubendorf, Switzerland) to allow the infusion of 1 ng/h or 10 ng/h of OXT, and were implanted subcutaneously [38 (link)]. On day 13, rats were mildly stressed by 5-min exposure on the elevated platform [4 (link), 53 (link)], and tested in the light-dark box (LDB) [15 (link), 38 (link)] on day 14. Another rat cohort was tested in the LDB on day 14 without prior platform exposure.

Bacteria solutions were obtained from a L. pneumophila Sg 1 Subtype Bellingham cryo standard (produced from strain DMSZ 25214, see Supplementary Information) with a TLC of 4.82 × 10⁷ cells mL⁻¹ and an ILC of 4.77 × 10⁷ cells mL⁻¹. Cryo stocks are a 1:1 mixture of bacteria suspended in Evian water (purchased from local store) and cryo buffer (122 g L⁻¹ K₂HPO₄, 14 g L⁻¹ KH₂PO₄, 85 g L⁻¹ NaCl, 20 g L⁻¹ BSA, and 120 g L⁻¹ Dextran 40 in deionized water, all chemicals from Sigma-Aldrich, St. Louis, USA). The produced cryo stocks were stored at  − 80 °C until further use. Through dilution in Ringer’s solution (B.Braun, Melsungen, Germany), the intended concentrations of bacteria solutions were achieved.

Cell death and forms were determined by AnnexinA5 (Anx5)-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. For analysis of cell death, 1 × 10⁵ cells were transferred in 400 μl of Ringer's solution (B. Braun, Melsungen, Germany) containing 0.2 μg AnxA5-FITC and 0.4 μg PI. After 30 min of incubation at 4 °C in the dark, the samples were analyzed by flow cytometry. AnxA5 protein was expressed and produced in 293 human embryonic kidney cells (FreeStyle 293 Expression System, Life Technologies, Regensburg, Germany) and purified (Life Technologies, GENEART). Labelling with FITC was performed with the FluoroTag FITC Conjugation Kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Double-negative cells were defined as viable, AnxA5⁺/PI⁻ as apoptotic, and AnxA5⁺/PI⁺ as necrotic ones.

Synthetic OXT (Sigma Aldrich Biochemicals, Steinheim, Germany) was infused at a dose of 0.1 µg/5 µl. This dose was carefully chosen to be low enough to avoid stereotyped barrel rotations (as often seen in female Wistar rats at 1.0 µg/5 µl, personal communication, M. Lukas), but high enough to reliably affect social behaviors in male rats [50] (link)). For ICV infusion of either synthetic OXT or VEH (5 µl Ringer’s solution, pH = 7.4, B. Braun AG, Melsungen, Germany), a 25 G infusion cannula extending 2 mm beyond the guide cannula and connected via polyethylene tubing to a Hamilton syringe was inserted into the guide cannula. After slow manual infusion the system was left in place for 10 s. Infusions took place 20 min prior to the FIT. After completion of the experiment, cannula placement was verified post mortem by ICV infusion of 2–3 µl black ink (Pelikan 4001, Hannover, Germany).