2.3. Ex Vivo Studies

Twenty-four male adult Sprague–Dawley rats (200–250 g) were housed in Plexiglass cages (40 × 25 × 15 cm), two rats per cage, in climatized colony rooms (22 ± 1 °C; 60% humidity), on a 12 h/12 h light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food, 24 h/day throughout the study, with no fasting periods. Rats were fed a standard laboratory diet (3.5% fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value; 13.39 kJ/g). Housing conditions and experimentation procedures were strictly in accordance with the European Union ethical regulations on the care of animals for scientific research. The experimental paradigm was approved by the Local Ethical Committee (University “G. d’Annunzio” of Chieti-Pescara, Chieti-Pescara, Italy) and the Italian Health Ministry (Authorization N. F4738.N.XTQ, delivered on 11 Novembre 2018). Specifically, rats were sacrificed by CO₂ inhalation (100% CO₂ at a flow rate of 20% of the chamber volume per min) and hypothalami were immediately collected and maintained in humidified incubator with 5% CO₂ at 37 °C for 4 h (incubation period), in DMEM enriched with CBD (1000 nM) or CBG (1 nM). Afterwards, the hypothalamic samples were subjected to analytical procedure for gene expression and biogenic amine level assessment, as described in the following paragraphs. The samples (n = 12) for the determination of biogenic amines were dissected in 1 mL of a perchloric acid solution (50 mM) and filtered (PTFE 0.45 µm), whereas the hypothalami (n = 12) intended to be used for gene expression analysis were dissected and stored in RNAlater solution (Ambion, Austin, TX, USA) at −20 °C.