Normal aCSF (35 °C) and normal pipette solution were used in electrophysiological recording (Ibrahim et al., 2003 (link)) (Supplemental Methods ). When the cationic blocker La3+ was added to the bath, a HEPES-buffered CSF solution was utilized (Zhang et al., 2008 (link)). In some experiments, low Na+ (5 mM) HEPES-buffered CSF solution and Ca2+ free extracellular CSF solution were used, where N-methyl-D-glucamine (NMDG) replaced Na+, and Mg2+ replaced Ca2+, respectively. For experiments measuring the ramp current-voltages (I–Vs) , K+-gluconate in the normal internal solution was replaced with Cs+-gluconate (pH 7.35 with CsOH), and the extracellular solution contained Na+, K+, Ih (HCN), Ca2+ and GABAA channel blockers (in mM: NaCl, 126; 4-aminopyridine, 5; KCl, 2.5; MgCl2, 1.2; CsCl, 2; CaCl2, 1.4; CoCl2, 1; nifedipine, 0.01; HEPES, 20; NaOH, 8; glucose, 10; tetrodotoxin, 0.001; picrotoxin, 0.1). Ramp I-Vs were also recorded in a solution that both Na+ and K+ were replaced with same concentration of Cs+(pH 7.35 with CsOH).
All drugs were purchased from Calbiochem (La Jolla, CA) unless otherwise specified. Leptin was provided by Dr. Parlow (Harbor-UCLA Medical Center, Torrance, California, USA) through the National Hormone and Peptide Program. The Jak2 inhibitor (E)-N-benzyl-2-cyano-3-(3,4-dihydroxyphenyl) acrylamide a-Cyano-(3,4-dihydroxy)-N-benzylcinnamide Tyrphostin B42 (AG 490), the PLC inhibitor U73122, the less active analog U73343 and the PI3 kinase inhibitor wortmannin (Alomone Laboratories) were dissolved in dimethylsulfoxide (DMSO) as stock solutions. The selective PLCγ inhibitor 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholline (ET-18-OCH3) was dissolved in H2O. 1, 2-Bis-(o-aminophenoxyethane)-N, N, N', N'-tetra-acetic acid (BAPTA) tetrasodium salt was dissolved in the internal solution at a 10 mM concentration. The ion channel blockers/activators used were as follows: 1-[β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365); 2-aminoethyl diphenylborinate (2-APB); flufenamic acid (FFA) and 1-(cis-9-Octadecenoyl)-2-acetyl-sn-glycerol, 2-Acetyl-1-oleoyl-sn-glycerol (OAG), all were dissolved in DMSO. LaCl3 was dissolved in H2O.
All drugs were purchased from Calbiochem (La Jolla, CA) unless otherwise specified. Leptin was provided by Dr. Parlow (Harbor-UCLA Medical Center, Torrance, California, USA) through the National Hormone and Peptide Program. The Jak2 inhibitor (E)-N-benzyl-2-cyano-3-(3,4-dihydroxyphenyl) acrylamide a-Cyano-(3,4-dihydroxy)-N-benzylcinnamide Tyrphostin B42 (AG 490), the PLC inhibitor U73122, the less active analog U73343 and the PI3 kinase inhibitor wortmannin (Alomone Laboratories) were dissolved in dimethylsulfoxide (DMSO) as stock solutions. The selective PLCγ inhibitor 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholline (ET-18-OCH3) was dissolved in H2O. 1, 2-Bis-(o-aminophenoxyethane)-N, N, N', N'-tetra-acetic acid (BAPTA) tetrasodium salt was dissolved in the internal solution at a 10 mM concentration. The ion channel blockers/activators used were as follows: 1-[β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365); 2-aminoethyl diphenylborinate (2-APB); flufenamic acid (FFA) and 1-(cis-9-Octadecenoyl)-2-acetyl-sn-glycerol, 2-Acetyl-1-oleoyl-sn-glycerol (OAG), all were dissolved in DMSO. LaCl3 was dissolved in H2O.
