The brain was quickly removed and

placed in ice-cold slicing solution containing (in mM) 87 NaCl, 2.5 KCl, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 25 glucose, and 75 sucrose saturated with 95%/5% O2/CO2. The brain was blocked and mounted on a vibrating slicer (Leica, Nussloch, Germany) submerged in ice-cold slicing solution. Angled horizontal slices (250 μm) containing the DMH were cut and incubated in 32.5°C artificial cerebrospinal fluid (aCSF) containing (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 2.5 CaCl2, 1.5 MgCl2, AT13387 solubility dmso 1.25 NaH2PO4, and 10 glucose saturated with 95%/5% O2/CO2 for a minimum of 60 min. Hypothalamic slices were then submerged in a recording chamber and superfused with 32.5°C aCSF at a flow rate of 1 ml/min. Whole-cell recordings were obtained from DMH neurons visualized with an Olympus upright microscope (Olympus, Center Valley, PA) fitted with infrared differential interference contrast optics. Recordings were obtained using borosilicate glass microelectrodes (tip resistance 4.5–6.5 MΩ) filled with a solution containing (in mM) 108 K gluconate, 8 Na gluconate, 2 MgCl2, 8 KCl, 1 potassium EGTA, 4 potassium ATP, 0.3 sodium GTP, and 10 HEPES, and corrected to pH 7.2 with

KOH. In a subset of experiments, 10 mM BAPTA was included in the intracellular solution to chelate postsynaptic Ca2+. www.selleckchem.com/products/abt-199.html Recordings were accepted for analysis if changes in access resistance were <15%. Cells were voltage clamped at −80 mV and the perfusate always contained DNQX (10 μM; Tocris, Ellisville, MO) to block AMPA and kainate receptor-mediated glutamatergic transmission. GABAergic fibers were stimulated extracellularly with a patch pipette filled with aCSF and positioned within

the DMH. Oxalosuccinic acid IPSCs were evoked at a rate of 0.2 Hz and paired-pulse responses were obtained by applying a pair of synaptic stimuli 50 ms apart. For HFS, afferents were stimulated at 100 Hz for 4 s, repeated twice, 20 s apart, unless otherwise specified. Electrophysiological signals were amplified using the Multiclamp700 B amplifier (Molecular Devices, Union City, CA), low-pass-filtered at 1 kHz, digitized at 10 kHz using the Digidata 1322 (Molecular Devices), and stored for offline analysis. Evoked currents were analyzed using Clampfit 9 (Molecular Devices). The amplitude of the synaptic current was calculated from the baseline (current before evoked response) to the peak of each evoked. For clarity, the stimulus artifacts were removed digitally from the traces depicted. Spontaneous IPSCs were analyzed using the threshold detection criteria in Minianalysis (Synaptosoft). Results are expressed as means ± SEM. In most cases, significance was determined using a one-sample or paired Student’s t test comparing the means following HFS or drug treatment to baseline with significance level of p < 0.05.