Wire Hanging Test

The wire hanging test was conducted following the previous report (Niimi et al., 2009). The animal was placed on a stainless steel bar (50 cm in length, 2 mm diameter, 37 cm above the floor) at a point midway between the supports and observed for 30 s. The score was evaluated according to the following scheme: 0, fell off; 1, hung onto the wire by two forepaws; 2, hung onto the wire by two forepaws, but also attempted to climb onto the wire; 3, hung onto the wire by two forepaws plus one or both hindpaws around the wire; 4, hung onto the wire by all four paws plus tail wrapped; 5, escaped. Latency to falling off and the score were also recorded, and the cutoff latency was set at 30 s. Each mouse had three opportunities, and the inter-trial intervals were 30 min. Thirty minutes after saline or (pyr)apelin-13 treatment, formalin was i.pl. injected. After 15 min, the wire hanging test was started. The mice were divided into four groups: saline and (pyr)apelin-13 (2, 10, and 20 mg/kg, n = 10 per group).