Methysergide

5-HT_2A binding was analyzed in membrane preparations as previously described (Canal et al., 2010 (link)). Three or 10 days following mTBI or sham treatments, subjects were sacrificed via rapid decapitation and bilateral frontal cortex samples were dissected on ice. Samples were homogenized in 3 ml ice-cold Tris binding assay buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, pH = 7.4), and subsequently centrifuged at 20,000 x g for 20 min at 4°C. The supernatant was decanted, and the pellet was resuspended in a 1.5-ml binding assay buffer. Protein concentrations were measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Membrane preparations (200 µg protein) were incubated with the 5-HT_2A antagonist [³H]ketanserin (PerkinElmer, Waltham, MA) (1 or 10 nM) at 37°C for 60 min. Samples were run in duplicate and nonspecific binding was determined in the presence of 100 µM methysergide (Sigma Aldrich, St Louis, MO). Samples were collected using a Brandel cell harvester and washed with ice-cold phosphate-buffered saline (PBS, pH = 7.4). Samples were incubated in 7 ml scintillation fluid overnight (National Diagnostics) and radioactive counts were measured via scintillation spectrometry (Beckman Coulter).

Diclofenac sodium was obtained from Novartis, Switzerland, whereas methysergide was obtained from Sigma-Aldrich, USA. Yohimbine was purchased from Tocris Bioscience (UK). All the drugs were freshly prepared in a sterile normal saline solution and administered intraperitoneally (i.p).

Citalopram hydrobromide (Tocris), reboxetine mesylate (Tocris), paroxetine hydrochloride hemihydrate (Sigma), imipramine hydrochloride (Sigma), and methysergide (Sigma) were freshly dissolved into saline (NaCl 0.9%) before use. Compounds were dissolved in a final volume of 10mL/kg. α-FMHis (synthesized at Abbott Laboratories, Chicago, IL) was injected i.c.v. at the dose of 5 µg dissolved in 5 µL of saline. All doses were calculated as mg/kg of the free base. Control animals received saline. In reverse dialysis experiments, drugs were diluted in the perfusing Ringer’s solution. All other reagents and solvents were of high performance liquid chromatography (HPLC) grade or the highest grade available (Sigma).

The drugs used were the following: Klamin^® extract (kindly supplied by Nutrigea Research s.r.l., Borgo Maggiore, Republic of San Marino), TTX (Alomone Labs, Jerusalem, Israel), EPPTB (N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide) (Hoffmann-La Roche, Monza, IT, Italy), β-PEA (β-phenylethylamine), L-NAME, 5-HT (Serotonin), methysergide (Sigma-Aldrich, Milano, IT, Italy). All the drugs were dissolved in distilled water. Klamin^® extract was dissolved in 0.5 mL water, and then sonicated (twice for 60 s) immediately prior to add to the bath. Chemicals were prepared as stock solution, which were diluted with Krebs solution on the experiment day.

Serotonin 5-HT_2A receptor competition binding experiments were carried out in membranes from CHO-FA4-5-HT_2A cells prepared in our group. On the day of the assay, membranes were defrosted and resuspended in binding buffer (50 mM Tris-HCl, pH 7.5). Each reaction well of a 96-well plate contained 80 μg of protein, 1 nM [³H]Ketanserin (50.3 Ci/mmol, Perkin-Elmer, Waltham, MA, USA), and different concentrations of the compounds in the range from 0.01 nM to 10 µM. Non-specific binding was determined in the presence of 1 μM methysergide (Sigma Aldrich, Spain). The reaction mixture was incubated at 37 °C for 30 min, after which samples were transferred to multiscreen GF/B 96-well plates (Millipore, Spain) pretreated with 0.5% polyethylenimine (PEI, Sigma Aldrich, Spain), filtered, and washed six times with 250 μL wash buffer (50 mM Tris-HCl, pH 6.6). The filters were dried, 35 μL Universol (MP Biomedicals, Spain) per well were added and radioactivity was detected in a microplate beta scintillation counter (Microbeta Trilux, Perkin-Elmer, Waltham, MA, USA).