Cells were fixed in 4% paraformaldehyde for 20 min, blocked in 5% goat serum with 0.1% Triton X-100 in PBS for 1 hr, and stained overnight at 4°C for PSD-95 (1:200) or synapsin (1:100) in blocking solution. Appropriate secondary antibodies conjugated to AlexaFluor 488 and 567 (Molecular Probes) were incubated with samples for 1 hr at room temperature after three washes with TBST. Slides were prepared by using Fluormount G (Southern Biotech) and images were taken with a Zeiss 510 Meta confocal microscope at 63 ×. Analysis of PSD-95 clusters was performed as described (Mukai et al., 2008). Briefly, the particle measurement

function of ImageJ was used to count discrete fluorescent puncta in a proximal 20 μM section of the largest one Kinase Inhibitor Library solubility dmso or two dendrites per neuron. Settings of minimal punctum size and threshold were maintained constant across all treatment conditions. Pearson’s coefficients were calculated by using the colocalization threshold plugin for ImageJ. Hippocampal neurons were treated with and without 20 μM 2-bromopalmitate for 8 hr. For live staining, neurons

(DIV 15) were incubated with 50 mg/ml anti-GluR2 N-terminal selleck chemicals antibody (Millipore) in conditioned medium for 15 min at 37°C. Neurons were fixed with 4% PFA for 10 min on ice, blocked in 5 mg/ml bovine serum albumin in PBS for 10 min, and stained with secondary antibody under unpermeabilized conditions. Neurons were then permeabilized to detect the antigen. Images were taken with a confocal laser microscopy system (Carl Zeiss LSM 510; Carl Zeiss). The number of GluR2 fluorescent puncta, which are merged with VGLUT1, was calculated as surface GluR2 by using ImageJ. To quantitate changes in clustering, we chose twelve fields from two independent neuronal cultures and analyzed the largest proximal dendrite (30 mm long). Proteins from brain homogenate were extracted with modified RIPA buffer and incubated with anti-NR2B antibody Org 27569 (Invitrogen) overnight followed by a 2 hr incubation with protein A/G agarose-conjugated beads (Calbiochem). Beads were washed three times in modified

RIPA, aspirated to dryness with a syringe, eluted with 2 × LDS, and analyzed by SDS-PAGE. We thank Michael Koldobskiy for helpful discussions and Masoumeh Saleh for maintaining and genotyping the nNOS knockout mice. This work was supported by US Public Health Service Grant MH18501 and Research Scientist Award DA-00074 (to S.H.S.), National Institutes of Health grant MH67068 (to J.A.G.), a Simons Foundation grant (to J.A.G.), and a NARSAD Young Investigator Award (to J.M.). “
“Leptin is secreted by adipocytes in proportion to fat stores providing feedback on the status of lipid reserves (Friedman, 2009). Leptin circulates and binds its receptor (LEPR) in the brain where it decreases food intake and promotes energy expenditure (Myers et al., 2010).