Xylazine

Rats were anesthetized (ketamine hydrochloride, 75 mg/kg, i.m. and xylazine, 5 mg/kg, i.m.; Biowet, Poland) and implanted with a silastic catheter in the external right jugular vein, as described previously (Bystrowska et al. 2014 (link)). Meloxicam (Metacam, Boehringer IIngelheim; 5 mg/kg, s.c.) was used to reduce post-operative pain during 3 days after surgery. Catheters were flushed daily with 0.2 ml of saline solution containing cefazolin (100 mg/ml; Biochemie GmbH, Austria) and heparin (100 U/ml; Biochemie GmbH, Austria) to prevent catheter non-patency as a result of blood clotting during the recovery period (7 days).

Animals were anesthetized with ketamine HCl (75 mg/kg, i.m.; Biowet, Poland) and xylazine (5 mg/kg, i.m.; Biowet, Poland) cocktail and chronically implanted with a Silastic catheter in the external jugular vein, as described previously (Filip et al. 2006 (link)). During 3 days after surgery, meloxicam (Metacam, Boehringer Ingelheim; 5 mg/kg, s.c.) was used to reduce post-operative pain.
Rats were allowed 7–9 days to recover from surgery before the start of the experiments. Catheters were flushed daily with 0.2 ml of saline solution containing heparin (100 U/ml, Biochemie, Austria) and 0.1 ml of a cephazolin solution (100 mg/ml Biochemie GmbH, Austria) to prevent catheter non-patency. Catheter patency was tested periodically with the short-acting barbiturate anesthetic methohexital (10 mg/kg, i.v.), which induced the loss of consciousness within 5 s. No problems with catheter patency were reported in the tested rats.

For intracerebroventricular (i.c.v.) injection of compounds, mice were anesthetized with ketamine (100 mg/kg i.p.) and xylazine (10 mg/kg i.p.; Biowet, Poland) and stereotaxically, bilaterally implanted with the guide cannulae (8 mm); (coordinates relative to bregma: 1 mm lateral, 0.2 mm posterior and 3,7 mm ventral). After 14 days of recovery, mice were subjected to injection and behavioral studies. Compounds were applied to each ventricle for 1 min, followed by a 1 min diffusion time. Mice received one injection each through both ventricles (1 μL per ventricle) using an infusion cannula (9 mm) 15 min before i.p. hyperforin (2.5 mg/kg) administration. 1 h after hyperforin treatment TST was conducted.

Methcathinone [MC, 2-(methylamino)-1-phenyl-1-propanone] and 3-fluoromethcathinone [3-FMC, 2-(methylamino)-1-(3-fluorophenyl)-1-propanone] were purchased in the form of hydrochloride salts from Cayman Chemical (Ann Arbor, MI, USA). Isotonic solution of saline (0.9% NaCl) for injections was purchased from Polska Grupa Farmaceutyczna (Łódź, Poland). Selective D₁-dopamine receptor antagonist, SCH 23390 (SCH, 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine), in the form of hydrochloride salt, was purchased from Sigma Aldrich (Poznań, Poland). All chemicals used for high-performance liquid chromatography (HPLC) were purchased from Merck (Warsaw, Poland). Ketamine hydrochloride and xylazine used for anesthesia were purchased from Biowet (Puławy, Poland).

The animal epilepsy model was induced by stereotactic, intracerebral injection of KA (KA; Sigma-Aldrich, Poznan, Poland). KA is a substance with a selective neurotoxic activity that is specific to hippocampal neurons. KA was injected once in the amount of 0.5 μg in 1 μL of PBS per mouse. Prior to injection, each mouse was anaesthetized with mixture of ketamine (1.15 mg; Biowet, Pulawy, Poland) and xylazine (0.1 mg; Biowet, Pulawy, Poland). After complete anaesthetization, mice were placed in a stereotactic frame (David Kopf Instruments, Los Angeles, CA, USA), the skin on the head was cut and a small hole in the skull was drilled. KA was administered with a Hamilton syringe (32G needle) (Hamilton Company, Bonaduz, GR, Switzerland). The site of injection (A—2 mm; L—1.2 mm; D—2.5 mm) was selected using the stereotactic atlas “The Mouse Brain in Stereotaxic Coordinates”, Second Edition, by George Paxinos and Keith B.J. Franklin. Clinical signs of epilepsy were observed for a few hours after the surgical procedure. Tissue samples were collected 24 and 72 h after model induction. Brains from uninjected mice and from mice injected in the same way but with PBS only were used as controls. The brain hemisphere with the site of injection is denoted as ipsilateral hemisphere (ipsi), whereas the opposite one is the contralateral hemisphere (contra).

Neurotensin (NT) and neurotensin antagonist SR 142948 were purchased from Tocris Bioscience (Bristol, UK). 1-fluoro-2,4-dinitrobenzene (DNFB), dinitrobenzene sulfphonic acid (DNS), TX-100 and protease inhibitor cocktail were obtained from Sigma-Aldrich (Poznań, Poland). Pentobarbital sodium, ketamine and xylazine were acquired from Biowet (Puławy, Poland).