03 mM Alexa Fluor 594 or Alexa Fluor check details 350 hydrazide dye (pH 7.3). K-glucoate was replaced by equimolar Cs-gluconate for some experiments. Epifluorescence was briefly used to target fluorescent cells, at which time the light source was switched to infrared differential interference contrast imaging to obtain the whole-cell recording (Zeiss Axioskop FS2 Plus equipped

with a fixed stage and a QuantEM:512SC electron-multiplying charge-coupled device camera). Electrophysiological signals were recorded using an Axopatch 700B amplifier (Molecular Devices), low-pass filtered at 2–5 kHz, and analyzed offline on a PC with pCLAMP programs (Molecular Devices). Recording electrodes had resistances of 2.5–5 MΩ when filled with the K-gluconate internal solution. Input resistance was assessed by measuring voltage SAHA HDAC cell line deflection at the end of the response to a hyperpolarizing rectangular current pulse steps (500 ms of −10 to −50 pA). Membrane potential values were compensated to account for junction potential (−8 mV). mCPP (4 μM, Aldrich) and leptin (100 nM; provided by A.F. Parlow, through the National Hormone and Peptide Program) were added to

the ACSF for specific experiments. Solutions containing mCPP or leptin were typically perfused for 2–4 min. A drug effect was required to be associated temporally with peptide application, and the response had to be stable within a few minutes. A neuron was considered depolarized very or hyperpolarized if a change in membrane potential

was at least 2 mV in amplitude. After recording, slices were fixed with 4% formalin in PBS at 4°C overnight. After washing in PBS, slices were mounted onto slides, covered in Vectashield (Vector Laboratories), and coverslipped to reduce photo-oxidation during visualization with fluorescent light. Cells were then visualized with ApoTome imaging system (Imager Z1; Zeiss) to identify post hoc the anatomical location of the recorded neuron. TTX, SKF96365, 2-APB, baclofen, and CGP54626 were obtained from Tocris. U73122 was obtained from Calbiochem. All solutions were made according to manufacturer’s specifications. Stock solutions of SKF96365, 2-APB, CGP54626, U73122 were made by dissolution in DMSO (Sigma). The concentration of DMSO in the external solution was <0.1%. Stock solutions of leptin were made by dissolution in D-PBS (GIBCO). Stock solutions of mCPP, TTX, and baclofen were made by dissolution in deionized water. Statistical data are expressed as mean ± SEM, where n represents the number of cells studied. The significance of differences between was evaluated using unpaired two-tailed Student’s t test with a confidence level of p < 0.05(∗) or p < 0.01 (∗∗). We thank Dr. Jefferey Friedman (Rockefeller University) for kindly providing us with the LepR-cre mice. This work was supported by grants to J.-W.S. (American Diabetes Association), Y.X.