P1675

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.

The MOR agonist DAMGO (H-2535, Bachem), MOR antagonist CTAP (H-3698, Bachem), delta opioid receptor agonist [D-Pen²,D-Pen⁵]-enkephalin (DPDPE; H-2905m, Bachem), α7 nicotinic acetylcholine receptor antagonist methyllycaconitine citrate (MLA; 1029, Tocris), AMPA antagonist NBQX (0373, Tocris), tetrodotoxin citrate (ARCD-0640, ARC Inc.), EtOH (E7148, Sigma-Aldrich), and GABA_A receptor antagonist picrotoxin (P1675, Sigma-Aldrich) were used.

The quinolinium salt-based halide-sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE; ab145418, Abcam) was used as a measure of chloride ion influx activity13 (link). Following cell culture, the PRNCs were incubated with 5 mM MQAE for 2 h at RT in the dark, and subsequently washed twice with NbActive 4 (BrainBit). Cells were then treated with 50 μM GABA (A2129, Sigma-Aldrich) or 10 nM GABA analog THIP, δ-GABA_AR specific agonist8 (link) (T101, Sigma-Aldrich), then fluorescence intensity was consequently measured at 0, 5, 10, and 20 min at RT. For inhibition assay, the cells were pretreated with 1 μM flumazenil (F6300, Sigma-Aldrich) or 1 μM picrotoxin (P1675, Sigma-Aldrich) or PBS (control) for 45 min at RT, and then stimulated with 50 μM GABA for 10 min at RT. Intercellular MQAE is quenched by 10 μM tributyltin chloride (T50202, Sigma-Aldrich) and 10 μM nigericin sodium salt (N7143, Sigma-Aldrich). The fluorescence intensity was measured by the Gemini EX florescence plate reader (Ex/Em = 360/460; Molecular Devices). The kinetic analysis was performed by using GraphPad Prism 6^® software.