Assessment of depression-like behavior in rats
Depression-like behavior was assessed in the FST, originally proposed as a model of stress-induced depression-like behavior (Porsolt, Le Pichon, & Jalfre, 1977). FST is broadly used for the early screening of novel molecules with putative antidepressant-like (AD) effects since immobility is commonly qualified as “despair” and considered to reflect depression-like states. Remarkably, the immobility response in the FST can be prevented by various types of AD treatments, including tricyclic antidepressants, monoamine oxidase inhibitors, SSRIs, and NA reuptake inhibitors (Planchez et al., 2019).
Behavioral experiments were performed between 09:00 and 14:00 hours. Animals were adapted to handling and were taken into the experimental room (80-90 lux) for at least 1 hour to reach habituation and assigned randomly to the experimental groups. Peptides were freshly prepared and intranasal treatments were administered 24 hours before the test phase (20 μl total volume). Doses for Galanin receptor 2 agonist (M1145), Y1R receptor agonist [Leu31, Pro34]NPY, GALR2 Antagonist M871 and ANA-12 were chosen based on previous published protocols (Borroto-Escuela et al., 2022; Ribeiro et al., 2020; Serova et al., 2017). We performed dose-response curves to determine effective doses. Separate group of rats were randomly allocated into six experimental groups: (1) Control: distilled water; (2) M1145- treated group (132 µg); (3) Y1R agonist-treated group receiving the Y1R agonist [Leu31- Pro34]NPY (132 µg); (4) M1145+Y1R: group administered with both substances; (5) M1145+Y1R+M871: group treated with M1145, [Leu31- Pro34]NPY and the GALR2 antagonist (M871; 132 µg); (6) M1145+Y1R+ANA-12: group treated with M1145, [Leu31- Pro34]NPY and the TrkB antagonist (ANA-12, 5.06304, Sigma, St. Louis, MO, USA; 0.5 mg/kg, i.p.) (N=6 in each group).
Swimming sessions were conducted by placing individually the rats in cylinders containing water (25±0.2°C), 30 cm deep. Two sessions were conducted: an initial 15 min pretest followed 48 h later by a 5-min test. The water in the cylinders was changed after every trial. The FST was performed according to previously reported methods (Borroto-Escuela et al., 2021; Koike & Chaki, 2014). The total duration of floating (immobility) and swimming periods were scored during the 5 min test and analyzed using the Raton Time 1.0 software (Fixma S.L., Valencia, Spain). Floating in the water without struggling and only making movements necessary to keep its head above the water was regarded as immobility. Swimming was scored when they actively swam around the cylinder. Following swimming sessions, the rats were removed from the tank, carefully dried in heated cages, and then returned to their home cages. Behavioral experiments were carried out by observers blinded to all experimental conditions.