Bn0086

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.

Noradrenaline-bitartrate(10 nM - 100 μM; I9278, Sigma), the α₁-AR antagonist prazosine (5 µM; P7791, Sigma), the α₂-AR antagonist yohimbine (5 µM; Y3125, Sigma), the α_2A-AR antagonist BRL 44408 (10 μM, C5776, Sigma), the α_2B-AR antagonist ARC 239, the α_1A-AR antagonist WB 4101, the α_1C, D-AR antagonist CEC (1 nM – 1 µM, Q102, Sigma) and the α_1A-AR agonist A61603 (100 nM; A5861, Sigma) were added to the normal aCSF.
To reduce glutamatergic and GABAergic synaptic input to the recorded neurons, 10 µM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, C127, Sigma-Aldrich), 50 µM DL-AP5 (DL-2-amino-5-phosphonopentanoic acid, BN0086, Biotrend), and 100 µM PTX (picrotoxin, P1675, Sigma Aldrich) was added to the extracellular saline.