The study has been performed according to the ARRIVE Guidelines to improve the reporting of bioscience research using laboratory animals. Experiments were performed in compliance with the European Union animal welfare guidelines [European Communities Council Directive of September 22, 2010 (2010/63/EU)] and the Italian Decree n.26/2014, authorization n. 152/2020-PR, Ministry of Health, Italy. Adult (16-wk-old) male C57BL/6j mice were purchased from Charles River Laboratories (Sulzfeld, Germany); male mice with spontaneous nonsense mutation of the ob gene for leptin (ob/ob, JAX mouse strain) B6.Cg-Lepob/J and WT ob gene expressing homozygous siblings of different ages were obtained from breeding ob gene heterozygotes and genotyped with PCR. Since orexin levels exhibit a diurnal fluctuation (concentrations increase during the dark period or active phase (i.e., ZT13- 24) and decrease during the light period or rest phase), the animals were maintained under a 12 h light:12 h dark cycle, light on at 8:00 PM, i.e., ZT0, for at least 4 weeks before euthanizing at ZT20-22. For the same reason, all the pharmacological treatments were performed at ZT20-22. Adult (16-wk-old) male B6.Cg-Tg(TH-GFP)21-31 (C57BL/6JJcl) mice were used for the electron microscopy analysis.

All mice were housed in controlled temperature and humidity conditions and fed ad libitum to exclude the effects of fasting on endocannabinoid-mediated plasticity in the VTA (Godfrey and Borgland, 2020). Wt and ob/ob mice were injected i.p. with several treatments as following: OX-A (Tocris, 40 μg/kg, 2 h), Leptin (Sigma Aldrich, 5 mg/kg, 2 h), SB-334867 (Tocris, 30 mg/kg for wt and 60 mg/kg for ob/ob mice, 3 h alone or 1 h before OX- A injection), AM251 (Tocris, 10 mg/kg, 3 h alone or 1 h before OX-A injection), O-7460 (Cayman, 12 mg/kg, 1 h alone or 30 min before OX-A injection), L741 (Tocris, 1.5 mg/kg, 30 min).

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