Plants were removed with a trowel, individually placed in bags, returned to the laboratory and kept at 4°C until further processing.
At the beginning of a trial mice were individually placed onto the central platform facing an open arm followed by 5 min free exploration.
Each forewing (approximately 1 mg) was tightly folded into a 5×× 9 mm tin capsule (ThermoQuest, Milan, Italy), individually placed in a 96-well plate and assigned a specific number.
Thirty minutes after the i.p. treatment with the methanol extract of H. tiliaceus (3, 10 and 30 mg/kg) or nortriptyline HCl (Pamelor®, 2 mg/kg), a reference antidepressant drug, or saline, mice were individually placed in an open cylindrical container (35 cm height × 24 cm diameter) containing 13.5 cm of water at 22 25°C for 6 min.
To habituate each mouse to the testing environment, they were individually placed in an empty testing chamber (a 51 cm×51 cm×39 cm opaque Plexiglas box) for 5 minutes prior to the beginning of testing.
Mice were injected with a saline solution and individually placed in the activity box for 30 min during 3 days consecutively for habituation before all locomotor experiments.
Rats were individually placed in a cage with a wire-mesh bottom.
Thirty minutes later, the treatment with the extract (50, 100, and 200 mg/kg, p.o). or diazepam (1 mg/kg, i.p ., a standard drug, or distilled water (0.1 mL/mouse, p.o)., mice were individually placed in an open cylindrical container (45 cm height × 20 cm diameter) containing 17 cm of water at 25 °C for 5 min.
Then, every pup was weighed, excretion was stimulated by a cotton swab, then the pup was reweighed and individually placed in a plastic beaker containing 2 ml of high-fat milk (UHT long life cream, 10% fat, Tnuva dairy, Israel) sweetened by the addition of sucrose to yield a 10% solution, warmed to 38°C and spread uniformly over tissue paper cut to fit the bottom of the beaker.
After injection, the three mice were individually placed in a dose calibrator which measured an average of 102.5 ± 0.5 μCi decay corrected, in a reasonable agreement with the requested amount.
