Way 100635 maleate

ME mice (pharmacology total n = 15, saline: n = 6; WAY-100635: n = 3; ketanserin: n = 3; ondansetron: n = 3) received daily injections (i.p.), at 2 pm exactly, of a specific 5-HT receptor antagonist at a dose based on prior literature, and specifically during the 3-week period of cross-modal plasticity from week 5 to 7 post-ME (Fig. 1d). Systemic drug delivery was chosen because a long-term treatment with a locally implanted slow release system (e.g. Alzet minipump) would lead to cortical tissue damage and scar formation, potentially influencing the local neuromodulator levels [68 (link)]. Drugs used were the 5-HTR_1A antagonist WAY-100635 maleate (Abcam Ab120550, 1 mg/kg, 0.2 mL i.p.) [37 (link), 36 (link), 69 (link), 70 (link)], the 5-HTR_2A antagonist ketanserin tartrate (R&D Systems, Tocris Bioscience, 5 mg/kg, 0.2 mL i.p.) [71 (link)–74 (link)] and the 5-HTR_3A antagonist ondansetron hydrochloride (R&D Systems, Tocris Bioscience, 5 mg/kg, 0.2 mL i.p.) [75 (link)]. Control animals were housed under the same standard conditions and received saline injections (0.9%, i.p.) following the same injection regimen. All animals were sacrificed by cervical dislocation. Brains were rapidly removed and stored as described above.

Human primary BMVECs were obtained from Cell Systems and cultured in a Complete Serum-Free Medium Kit With RocketFuel™ (SF-4Z0-500, Cell Systems, WA, USA). The HBPCT was established as described previously [27 (link)] and maintained in Dulbecco’s Modified Eagle Medium (DMEM) high glucose with 10% fetal bovine serum at 33 °C These cell lines were passaged every 7 days at 1 to 5 dilution using 0.25% trypsin-EDTA (209-16941, Wako, Tokyo, Japan). For tube formation, 1 × 10⁴ HBPCTs were passaged on a type I collagen (637-00653, Nitta gelatin, Osaka, Japan)-coated glass base dish (3910-035, Iwaki, Tokyo, Japan) and incubated at 37 °C. After 3 d, 5 × 10³ BMVECs were added to it and grown in a Complete Serum-Free Medium Kit With RocketFuel™ (SF-4Z0-500, Cell Systems, WA, USA) with vehicle, 50 µM serotonin hydrochloride (H9523, Sigma-Aldrich, MO, USA) and 10 µM 5-HT1A antagonist WAY-100635 maleate (ab120550, abcam, Cambridge, UK), for an additional 4 days, being subjected to immunostaining.