Animals and brain slice preparation

Experiments were performed on brain slices from 12- to 15-week-old genetically marked (with ZsGreen) Glp1r- and Lepr-expressing POMC neurons using POMC^Dre Lepr^Cre ROSA26lSlrSrZsGreen^+/− or POMC^Dre Glp1r^Cre ROSA26lSlrSrZsGreen^+/− male and female mice. Animals were kept under standard laboratory conditions, with tap water and chow available ad libitum, on a 12 h light/dark cycle. The animals were lightly anesthetized with isoflurane (B506; AbbVie) and decapitated. Coronal slices (270–300 µm) containing the ARC were cut with a vibration microtome (HM-650 V; Thermo Scientific) under cold (4 °C), carbogenated (95% O₂ and 5% CO₂), glycerol-based modified artificial cerebrospinal fluid (GaCSF)⁵⁰. GaCSF contained (in mM): 244 glycerol, 2.5 KCl, 2 MgCl₂, 2 CaCl₂, 1.2 NaH₂PO₄, 10 HEPES, 21 NaHCO₃ and 5 glucose, adjusted to pH 7.2 with NaOH. If not mentioned otherwise, the brain slices were continuously superfused with carbogenated aCSF at a flow rate of ~2.5 ml min⁻¹. aCSF contained (in mM): 125 NaCl, 2.5 KCl, 2 MgCl₂, 2 CaCl₂, 1.2 NaH₂PO₄, 21 NaHCO₃, 10 HEPES and 5 glucose, adjusted to pH 7.2 with NaOH. To block GABAergic and glutamatergic synaptic input, in all recordings, the aCSF 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 suppress action-potential-dependent synaptic release, blocked voltage-dependent Na⁺ channels were blocked by 10⁻⁶ M TTX (T-550, Alomone).