Zoletil 100

Adult male mice (10–15-week-old) were anesthetized with Zoletil 100 (tiletamine HCl 50 mg/ml + zolazepam HCl 50 mg/ml; from Virbac, Italy) and Rompun 20 (xylazine 20 mg/ml; from Bayer S. p. A, Italy) dissolved in saline solution (4.1 mg/ml and 1.6 mg/ml, respectively) prior to injection (7.3 ml/kg). Mice were then positioned in a stereotaxic frame (David Kopf Instruments, CA, USA) equipped with a mouse adapter. For behavioral experiments, mice were chronically implanted bilaterally with a 26-gauge guide cannula positioned 1 mm from the dorsolateral striatum, aiming at the same striatal area investigated in vitro, using the following coordinates (from brain surface): AP = +1 mm, ML = ±1.5 mm, DV: −3.25 mm (Robins et al., 2018 (link)). After 5–7 days of post-operative recovery, DHPG (50 μM in NaCl 0.9%) or vehicle (Veh, NaCl 0.9%) treated animals were bilaterally injected through an injector cannula in a total volume 0.5 μl/side at a continuous rate of 0.15 μl/min under the control of a micro-infusion pump. The injector cannula was removed 3 min after the end of infusion to prevent backflow. Mice were returned to their home cage and tested after 10 min.

Magnetic resonance imaging (MRI) was performed using a high-field magnetic resonance tomograph (Agilent Technologies DD2–400 9.4 T 400 MHz; Cheadle, UK) with a volume coil M2M (H1). The mice were anesthetized intraperitoneally with 70 mg/kg Zoletil 100 (Virbac Sante Animale, Carros, France) and intramuscularly with 0.02 mg/kg Xylanite (NITA-Pharm, Saratov, Russia) and then fixed in an upright position inside the magnet tunnel set at 27 °C.
The VnmrJ software was used for brain scanning and data processing. T1 tomograms of the layered frontal brain sections weighted by proton density were obtained using the multi-gradient echo multi slice (MGEMS) pulse sequence with the following parameters: TR = 1000 ms; TE = 1.49 ms; 6 echoes; FOV—20 × 20 mm; matrix—256 × 256; 15 slices; 1 mm slice thickness; 17 min; and 4 s scanning time [21 (link)].

While still in the pen, animals were sedated with an intramuscular injection of 4 mg/kg of Tiletamine+Zolazepam (Zoletil 100^®, Virbac SA, Esplugues del Llobregat, Barcelona, Spain) and 2 mg/kg of Xylacine (Xilagesic 20%^®, Laboratorios Calier SA, Les Franqueses del Vallès, Barcelona, Spain). After loss of reflexes, the pigs were transported on a trolley. A venous catheter was placed in the auricular vein and an arterial catheter was placed in the auricular artery. Pre-oxygenation with 100% oxygen was applied using a facemask while the animals were aseptically washed. In the operating room, intravenous (IV) anesthesia was induced with Propofol (Propofol^®, B Braun Medical SA, Melsungen AG, Germany) at 4 mg/kg. Endotracheal intubation was immediately performed and anesthesia maintained with 60% oxygen and 2% isoflurane (Isoflo, Abbott laboratories Ltd, Saint-Laurent, Québec, Canada) for the duration of the experiment. Ringer lactate (Ringer lactate, B Braun Medical SA) was continuously infused at 10 ml/kg/h and an IV constant-rate infusion of 6 μg/kg/h of Fentanyl (Fentanest^®, Kern Pharma SL, Terrasa, Barcelona, Spain) was also administered for analgesia. In order to prevent an increase in intracranial pressure (ICP), a single bolus of 10% mannitol (Fresenius Kabi España SA, Barcelona, Spain) was infused at 0.5 g/kg immediately before craniotomy.

Zoletil 100 Virbac, Milano, Italy (Tiletamine HCl 50 mg/ml + Zolazepam HCl 50 mg/ml) and Rompun 20 Bayer S.p.A Milano, Italy (Xilazine 20 mg/ml), purchased commercially, are used as anaesthetics, and injected i.p. in a volume of 0.5 ml/kg of each drug.

The following substances and reagents were used in the experiments without additional purification: approved for biomedical use Mowiol^® 28-99 poly(vinyl alcohol) (PVA) (molecular weight of ca. 145 kDa, the deacetylation degree of 99%) was from Merck KGaA (Darmstadt, Germany), the antibiotic Ceftriaxone (CFT) and the injection quality water were from Rafarma Lld. (Lipetsk region, Russian Federation), the antimycotic Fluconazole (FNZ) was from Biocom (Stavropol, Russian Federation). GMF-agar microbiological medium was purchased from NITsF Ltd. (Saint-Petersburg, Russian Federation). The anesthetics for surgery manipulations were Zoletil 100 (Virbac S.A., Carros, France) and diethyl ether (Lenreaktiv, Saint-Petersburg, Russian Federation). Substances and consumables for histology were as follows: formalin, paraffin, hematoxyline, eosine (all BioVitrum, Saint-Petersburg, Russian Federation), ethanol (Ferrain, Moscow, Russian Federation), acetone and xylene (Panreac, Barcelona, Spain), microscope slides (Epredia, Braunschweig, Germany), cover glasses (Assistant, Sondheim, Germany).