60 adult zebrafish (Danio rerio) of wild-type short-fin strain from both sexes (50:50 ratio), n = 10 per group, were purchased from an authorized commercial dealer (Pet Product S.R.L., Bucharest, Romania). The animals were held under adequate conditions of acclimatization at least one week before the experiments. Fish were kept in the light–dark cycle (14/10 h) photoperiod (lights on at 8:00 am), fed twice a day with Norwin Norvitall flake (Norwin, Gadstrup, Denmark), and housed in 24 L housing tanks (30 × 30 × 30 cm) at 26 ± 1 °C, pH = 7.5, dissolved oxygen at 7.20 mg/L, ammonium concentration < 0.004 ppm, and a conductivity of 500 μS. All tanks were maintained under constant mechanical filtration to avoid the accumulation of organic toxins. All animals were divided into the following groups: the control group, the scopolamine group (Sco, 100 μM, Sigma–Aldrich, Darmstadt, Germany), and three rhoifolin treatment groups (Rho: 1, 3, and 5 μg/L), the imipramine group (IMP, 20 mg/L, Sigma–Aldrich, Darmstadt, Germany, as a positive control within an novel tank diving test (NTT)) and the galantamine group (GAL, 1 mg/L, Sigma–Aldrich, Darmstadt, Germany, as a positive control within Y-maze and novel object recognition (NOR) tests). The doses of Sco, Rho, IMP, and GAL were chosen following a previous report [17 (link)]. Rho (1, 3, and 5 μg/L) was individually delivered to fish through transferring into a 500 mL glass for 1 h, once daily, whereas the Sco (100 μM) treatment was administered once independently by moving into a 500 mL glass 30 min before the behavioral tests [18 (link)]. This study was previously approved by the local board of ethics for animal experimentation (No. 15309/2019) and fully complied with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals. Efforts were made to reduce animal suffering and the number of animals utilized.
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