9% phosphate buffered saline (PBS) followed by 10% neural buffered formalin. Brains were removed, stored in the same fixative for 4–6 hr at 4°C, transferred to a 20% sucrose DEPC-treated PBS, pH 7 at 4°C overnight, and cut into 30 μm coronal sections on a microtome. Brain slices were prepared from young adult male mice (5–7-week-old) as previously described (Dhillon et al., 2006 and Vong et al., 2011). Briefly, 300 μM PLX-4720 thick coronal sections were cut with a Leica VT1000S vibratome and then incubated in carbogen-saturated (95% O2/5% CO2) aCSF at room temperature for at least 1 hr before recording. Slices were transferred to the recording chamber perfused with aCSF

(in mM: 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 21.4 NaHCO3, 10 glucose) at a flow rate of ∼2 ml/min. The slices were allowed to equilibrate for 10–20 min before performing whole-cell recordings. All electrophysiology recordings were performed at room temperature. To verify the deletion of NMDARs in AgRP neurons or POMC neurons, we performed whole-cell, voltage-clamp recordings in the presence of low Mg2+ (MgCl2 in aCSF Ion Channel Ligand Library concentration was reduced from 1.2 mM to 0.1 mM) to avoid Mg2+-block of NMDARs, and 100 μM picrotoxin (PTX) to block GABAA receptor-mediated IPSCs. A stimulating electrode was placed near the VMH 300–500 μm from the recording electrode. Excitatory postsynaptic currents were evoked by 0.1 Hz stimulation. The stimulation strength

chosen for evoking AMPAR- and NMDAR-mediated EPSCs in each case was to produce half maximal EPSC amplitudes within the linear region of the stimulation strength-peak amplitude curve. The evoked NMDAR- or AMPAR-mediated currents were constructed by averaging 12 EPSCs elicited at −60 mV. NMDA currents were calculated by subtracting the average response in the presence of Fossariinae 50 μm D-APV from that recorded in its absence. AMPA current was then calculated by subtracting the background

currents (recorded in the presence of 50 μm D-APV and 30 μm CNQX) from that recorded in the presence of D-APV only. EPSCs were measured in whole-cell voltage-clamp mode with a holding potential of −60 mV. The internal recording solution contained (in mM): CsCH3SO3 125; CsCl 10; NaCl 5; MgCl2 2; EGTA 1; HEPES 10; (Mg)ATP 5; (Na)GTP 0.3 (pH 7.35 with NaOH). Currents were amplified, filtered at 1 kHz, and digitized at 20 kHz. EPSCs were measured in the presence of 100 μM picrotoxin (PTX). Miniature EPSCs were recorded with 1 μm tetrodotoxin in aCSF recording solution. Frequency and peak amplitude were measured by using the Mini Analysis program (Synaptosoft). Membrane potential and firing rate were measured by whole-cell current clamp recordings from AgRP neurons in brain slices. Recording electrodes had resistances of 2.5–4 MΩ when filled with the K-gluconate internal solution (128 mM K-gluconate, 10 mM HEPES, 1 mM EGTA, 10 mM KCl, 1 mM MgCl2, 0.