Millicell cm filters

The organotypic slice culture was performed as previously described^{17 (link)}. Hypothalamic slices were obtained using a previously reported method with slight modifications^{8 (link)}. C57BL/6 pups at 8–11 days old were decapitated and the brains were quickly removed. Hypothalamic tissues were sectioned at a depth of 250 μm on a vibratome (Leica VT1200S) in chilled Gey’s balanced salt solution enriched with glucose (0.5%) and KCl (30 mM). Coronal slices containing the arcuate nucleus (ARC) were then placed on Millicell-CM filters (Millipore, pore size 0.4 μm, diameter 30 mm) and maintained at an air-medium interface in minimum essential media supplemented with heat-inactivated horse serum (25%), glucose (32 mM), and GlutaMAX (2 mM). Cultures were typically maintained for 8–10 days in standard medium, which was replaced 2 or 3 times a week. Healthy slices typically showed a slight reduction in the thickness of the hypothalamus after 10 days of incubation, while marked decreases in unhealthy slices, which mostly lost the hypothalamic structure. After 10 days, the selected healthy slices were then randomly divided into experimental and control groups.

Mouse pups between 8 and 11 day old were decapitated, and the NG were quickly removed and cultured in chilled Gey's Balanced Salt Solution (Invitrogen) enriched with glucose (0.5%) and KCl (30 mM). The NG were then placed on Millicell-CM filters (Millipore; pore size 0.4 μm) and then maintained at the air-media interface in minimum essential medium (Invitrogen) supplemented with heat-inactivated horse serum (25%, Invitrogen), glucose (32 mM), and GlutaMAX (2 mM, Invitrogen). Cultures were typically maintained for 10 days in standard medium, which was replaced three times a week. After an overnight incubation in low serum, (1.5%) MEM supplemented with GlutaMAX (2 mM), slices were stimulated with vehicle, 5 μM GW3965 for 4 hr. RNA was harvested using Acturus PicoPure RNA Extraction kit (Applied Biosystems).