Morphine sulfate pentahydrate

To reduce handling stress, fish were acclimatized for a week before the treatment. Fish were then assigned randomly to acute morphine treatment or control; no particular randomization method was performed to allocate the subjects in groups in this study. Fish were individually treated by immersion in a tank [sized 361 mm (L) × 218 mm (W) × 256 mm (D)] with water containing 2 mg/L morphine sulfate pentahydrate (Lipomed AG, Switzerland) for 20 min. The morphine treatment procedure was chosen based on a protocol previously reported (Stewart et al., 2011 (link)). As morphine is an analgesic, it does not cause any form of pain or suffering to the fish, and the doses used are those utilized in previous studies that do not cause toxicity (Magalhães et al., 2017 (link); Chatigny et al., 2018 (link)). Therefore, no major effects from the treatments on fish were expected and only normal healthy fish were used in this study. After the treatment, the fish were anesthetized by immersion in water containing benzocaine (0.1 g benzocaine/200 mL, Sigma) and the brain was dissected for in situ hybridization and real-time PCR analysis. The same treatment protocol was employed for control samples, but they were immersed 20 min in water without morphine. In both the morphine and control group, the treatments were carried out simultaneously on separate immersion tanks from 1,400 to 1,600 h.

HBMECs were seeded in a 96-wells plate onto collagen Type I, Rat Tail at a seeding density of 10,000 cells/well. After 24 h incubation at 37 °C, 5% CO₂, medium with serum was changed for medium without serum. Morphine sulfate pentahydrate was purchased from Lipomed AG (Switzerland). Morphine treatment was performed at three different concentrations: 1 µM, 10 µM and 100 µM (n = 6) for 24 h and 48 h. Medium and morphine were renewed daily. Distilled water was used as negative control and lysis buffer as positive control. Cell proliferation was assessed with CellTiter 96 AQ_euous One Solution Cell Proliferation Assay (MTS) (Promega). Cytotoxicity was assessed with Pierce LDH Cytotoxicity Assay Kit (ThermoScientific) according to manufacturer’s instructions. Absorbance was measured on FilterMax F3 (Molecular Devices) with SofMax Pro 7 (Version 7.0.3, Molecular Devices).

To examine the effect of morphine on fear conditioning, fish were exposed with morphine (Groups 3 and 4, Fig. 4B) during the conditioning and post-conditioning phase (Fig. 4A). Briefly, on the Day-2, after 5-min of conditioning with AS and 60-min of recovery (consolidation), fish was immersed in a 1 L container containing 800 ml of morphine (2 mg/l, morphine sulfate pentahydrate, Lipomed AG, Switzerland) for 30 min and transferred to the housing tank or proceed for Kiss1 administration. Next day (Day-3), prior to post-conditioning assessment, the fish was again immersed in morphine solution for 30 min. This was because the protective effect of morphine on fear memory and responses has been demonstrated to be most effective 24 h or 48 h after the traumatic event^{6 (link), 49 (link)}. The dose for morphine was chosen based on a previous study in adult zebrafish^{66 (link)}.