Amylin

On P21–28, rats were perfused with a 4% sucrose solution, and neurons were dissociated from VMN punches, as previously described (28 (link),29 (link)). Neurons were cultured in growth media (Neurobasal plus 2.5 mmol/L glucose) for 5 days and exposed twice daily to 10 µmol/L amylin (Bachem) or PBS (n = 9 rats/group). On day 5, media were collected and kept at −80°C for cytokine assays. Neurons were exposed to 120 µL of lysis buffer (Ambion) from which mRNA was extracted and gene expression assayed by QPCR (Applied Biosystems) (28 (link)).

Sprague-Dawley male rats were killed on postnatal days (P) 21–28, and 350-µm sections of the VMH (from bregma −2.30 to −3.60 mm [27 ]) were cut with a vibratome in oxygenated slushed artificial cerebrospinal fluid (containing 118 mmol/L NaCl, 3 mmol/L KCl, 1 mmol/L MgCl₂, 2.5 mmol/L NaHCO₃, 1.5 mmol/L CaCl², 1.2 mmol/L NaH₂PO₄, 5 mmol/L HEPES, 2.5 mmol/L glucose, 15 mmol/L sucrose [pH 7.4]). Explant slices were transferred to individual wells and maintained in Neurobasal (Invitrogen, Grand Island, NY) containing 10% FBS, 5 mmol/L glucose, 10 µg/mL gentamicin, and 10,000 U/mL penicillin/streptomycin at 37°C for 5 days. They were exposed twice daily to 10 µmol/L amylin (Bachem, Torrance, CA) or PBS control (n = 9 rats/group). On day 5, media were collected and stored at −80°C for cytokine assays. Slices were placed in RNA Later (Ambion, Grand Island, NY), the VMH was punched under microscopic guidance, and mRNA expression was assayed by quantitative reverse transcriptase PCR (QPCR; Applied Biosystems, Grand Island, NY) (28 (link),29 (link)).

Male IL-6 KO and WT mice (9 weeks old) were fed mouse chow ad libitum throughout the experiment. After 1 week of acclimation, mice were randomized by body weight into four groups then anesthetized and implanted with subcutaneous intrascapular minipumps (Alzet Model 1002; Durect Corp.). Pumps contained amylin (Bachem) in 0.9% saline (WT-A and IL-6 KO-A infused at 1 mg/kg/day) or 0.9% saline vehicle (WT-S and IL-6 KO-S). Body weight and food intake were monitored biweekly for 2 weeks. Terminally, food was removed 2 h before lights off. Mice were injected intraperitoneally with murine leptin (5 mg/kg in PBS; NHPP) at lights off, anesthetized (ketamine/xylazine) 45 min later, and rapidly perfused with 0.9% saline followed by 2% PFA in PBS (32 (link)). Brains were removed, postfixed overnight in potassium phosphate-buffered saline containing 20% sucrose, and frozen with 2-methylbutane.