Ketamine

HPLC grade methanol, acetonitrile and reagent grade formic acid, hydrochloric acid, potassium dihydrogen phosphate, orthophosphoric acid and sodium chloride were purchased from Merck (Darmstadt, Germany)), xylazine were from Sigma-Aldrich (St. Louis, USA), ketamine was supplied by Vetoquinol (Gorzow Wielkopolski, Poland).

At the end of the experiment, animals were anesthetized with medotomidine (0.05 mg kg^-1, Orion) and ketamine (2.5 mg kg^-1, Vetoquinol, UK) followed by overdose with intraperitoneal administration of pentobarbitone (300 mg kg^-1_, Pentoject, Animal Care). Animals were then transcardially perfused with 0.9% Saline, followed by 4% paraformaldehyde in phosphate buffered solution. The brain was then removed and stored in paraformaldehyde for ≥ 1 week before cryoprotection in sucrose (30% in dH₂O), freezing in dry ice and histological sectioning (cryostat, section thickness: 50μm). Prior to sectioning, the brain was photographed to record the position of electrode penetrations on the cortical surface. Sections were then mounted in 3% gelatin on microscope slides and Nissl stained to visualize the tissue. Electrode tracks were visible as local disruption of tissue, and the pattern of tracks through the tissue was aligned with that observed across the cortical surface of the intact brain. Electrodes that did not enter the Ectosylvian Gyrus were removed from the dataset. We also discarded data from electrodes recorded below the cortical laminae.

The animal experiments were approved by the Ministère de l’Education nationale, de l’Enseignement supérieur et de la Recherche, France (APAFiS no. 4986). The surgical procedure was performed in accordance with the guidelines in the laboratory of the Association for Research in Vision and Ophthalmology within the accredited A67-482-34 and B67-482-34 animal facilities. Nine pigmented rabbits (Bleu de Champagne) aged one year and weighing 3.5–4 kg were anesthetized by a lumbar intramuscular injection of ketamine, 20 mg/kg (Virbac, Carros, France) and xylazine, 3 mg/kg (Bayer Healthcare, Puteaux, France). After local anesthesia of the eyeball with 2–3 drops of oxybuprocaine chlorhydrate (Théa, Clermont-Ferrand, France), PVL in phosphate-buffered saline (PBS) (3 µg/50 µL), purified from ATCC49775 S. aureus^{43 (link)}, was intravitreally injected with a 30-Gauge needle inserted 4 mm behind the corneal limbus. For controls, three eyes were injected with 50-µL PBS using the same technique. After PVL injection, animals were sacrificed at 30 min, 1, 2 and 4 h (two animals: three eyes with PVL and one control eye in each group) first using anesthesia with ketamine-xylazine (as mentioned above), followed by a lethal intravenous injection of 2-mL Pentobarbital Dolethal^® (Vetoquinol, Lure, France) through a 22-Gauge catheter inserted in the marginal auricular vein.

fluoxetine hydrochloride (Tocris Bioscience, Oakville ON, Canada), escitalopram oxalate (Tocris Biosceicne) and ketamine (Narketan 10, Vetoquinol, Lavaltrie, QC, Canada) were diluted in 0.9% NaCl solution and administered IP as follows: acute fluoxetine, 1 dose of 3 mg kg^‒1 (behavioral testing/tissue collection occurred 0.5 h after injection); chronic fluoxetine, 14 doses (daily) of 10 mg kg^‒1 (tissue collection occurred 0.5 h after last injection); repeated citalopram 3 doses of 10 mg kg^‒1 (24, 5 and 0.5 h before tissue collection); ketamine (10 mg kg^‒1) 1 or 24 h before behavioral testing/tissue collection or mPFC whole-cell recordings.

The following drugs were used: 5-chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole hydrochloride (EMD386088) and s-citalopram were synthesized by Adamed (Pieńków, Poland), imipramine (Sigma Aldrich, Germany), moclobemide (Sigma Aldrich, Germany), reboxetine (Ascent, UK), bupropion (Sigma Aldrich, Germany), xylazine (Sigma-Aldrich, USA), ketamine (Vetoquinol, Poland). All drugs were dissolved in distilled water immediately before administration in a volume of 2 mL/kg. All the compounds were administered intraperitoneally (i.p.). EMD386088, imipramine, and s-citalopram were given 30 min before the test, while the remaining compounds were injected 60 min before. Control animals received vehicle according to the same schedule.

Trapped macaques were sedated with 0.3–0.5 mL ketamine (Vetoquinol, UK) via intramuscular injection before blood collection (1–5 mL). The blood samples were transferred into ethylenediaminetetraacetic acid (EDTA) tubes (Becton-Dickinson, Franklin Lakes, NJ, United States). Blood spots on filter paper (Whatman^® No. 1) were also collected. Thick and thin blood films were also prepared for malaria parasite examination by microscopy (BFMP). Each macaque was marked and then handed over to the Wildlife Department for release into the deep forest or areas of less conflict with humans. The blood samples in EDTA tubes were kept at 4 °C during transportation to the Parasitology Laboratory, IMR. Thin blood smears were fixed with methanol. Both thick and thin blood smears were then stained with 3–10% KaryoMAX^® Giemsa Stain (Gibco^®, Life Technologies Carlsbad, CA, United States) for 30–60 min, examined and results were recorded. Blood samples in EDTA tubes were subjected to DNA extraction.

The EMKA invasive measurement of dynamic resistance (EMKA Technologies, Paris, France) in response to increasing doses of methacholine (acetyl-β-methyl-choline chloride, Sigma-Aldrich) (0–25 mg/mL, 10% puff for 10 s) was used to determine lung function of dams on day 21 of lactation (day 42 of the experiment) as performed previously (Verheijden et al., 2014 (link)). Mice were anesthetized by intraperitoneal injection of ketamine (Vetoquinol S.A., France; 125 mg/kg) and medetomidine (Pfizer, Netherlands; 0.4 mg/kg) mixture. Data are expressed as average lung resistance (RL) in cm H2O/mL × sec^–1.