Electrophysiological Recordings of ARC Neurons

The electrophysiological experiments were carried out essentially as described^{18 (link),52 (link)}. Perforated patch-clamp recordings were performed from genetically marked AgRP and POMC neurons in coronal slices (270 µm) containing the ARC from adult POMC-Gi or AgRP-Gq;POMC-Gi male and female mice. Neurons were identified by their anatomical location in the ARC and by their ZsGreen or tdTomato fluorescence. Unless otherwise stated, the artificial cerebrospinal fluid contained 10⁻⁴ M picrotoxin (P1675, Sigma-Aldrich), 5 × 10⁻⁶ M CGP (CGP-54626 hydrochloride, BN0597, Biotrend), 5 × 10⁻⁵ M DL-AP5 (dl-2-amino-5-phosphonopentanoic acid; BN0086, Biotrend) and 10⁻⁵ M CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; C127, Sigma-Aldrich) to block GABAergic and glutamatergic synaptic input. Perforated patch-clamp experiments were conducted using protocols modified from previous studies. The used DMSO concentration (0.1–0.3%) had no noticeable effect on the investigated neurons. CNO was bath-applied at a flow rate of ~2.5 ml min⁻¹ at a concentration of 3 µM for 5 min.