Tiagabine

Indomethacin (Sigma-Aldrich, St Louis, MO, USA) was dissolved in phosphate buffered saline (PBS); tiagabine, bicuculline and CGP 35348 hydrate (Sigma-Aldrich, St Louis, MO, USA) in normal saline (NaCl 0.9%) and enaminones (resynthesized in-house6 (link)9 (link)) in peanut oil. The drugs were freshly prepared before administration and administered intraperitoneally (i.p.) to mice at a volume of 10 ml/kg body mass. Indomethacin and tiagabine were administered to mice 1 hour before subcutaneous (s.c.) administration of formalin (5%; 20 μl). Enaminones were administered 1.5 hours before administration of formalin, taking into consideration the time the enaminone E139 produced significant antinociceptive effect in the hot plate test15 . bicuculline, a GABAA receptor antagonist, and CGP 35348 hydrate, a GABA_B receptor antagonist, were administered 15 minutes before the administration of E139 for the hot plate test.

[³H]GABA (specific activity: 25–40 Ci/mmol) was obtained from PerkinElmer Life Sciences (Llantrisant, United Kingdom). Liothyronine was purchased from Tocris Bioscience (Bristol, United Kingdom). 4-Phenylbutyrate (4-PBA), liothyronine and tiagabine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture media, supplements, and antibiotics were obtained from Invitrogen. SDS was from BioMol GmbH (Hamburg, Germany). Bovine serum albumin (BSA) and Complete TM protease inhibitor mixture were purchased from Roche Applied Science (Mannheim, Germany). Tris and scintillation mixture (Rotiszint^® eco plus) were ordered from Carl Roth GmbH (Karlsruhe, Germany). Anti-green fluorescent protein (GFP) antibodies (ab290 and A-11122) were purchased from Abcam Plc (Cambridge, UK) and Invitrogen (Life Technologies, Carlsbad, CA, United States), respectively. All other chemicals were of analytical grade. The MNCD2: hybridoma, monoclonal antibody (developed by M. Takeichi and H. Matsunami) was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology (Iowa City, IA 52242, USA).

Stock solutions of drugs were freshly prepared and filtered using 0.22 μM solvent resistant filters (Millipore) as follows: topiramate, picrotoxin and tiagabine (Sigma-Aldrich), retigabine, XE991 (both from Alamone) and kynurenic acid (Tocris) were dissolved in DMSO to obtain stock solutions of 10 mM. Bicuculline, d-2-Amino-5-phosphonovaleric acid (D-AP5), 4-Aminopyridine (4-AP), phenobarbital, valproic acid (all from Sigma-Aldrich), 6,7-dinitroquinoxaline-2,3-dione (DNQX), tetrodotoxin (TTX) (both from Tocris) were dissolved in water to obtain stock solutions of 10 mM. Carbamazepine, ethosuximide and diazepam (all from Sigma-Aldrich) were dissolved in ethanol to obtain stock solutions of 10 mM. All drugs were then serially diluted to their final concentrations in cell culture/recording media.