Brain tissue and neuronal cultures were fixed in 4% PFA, and postfixed in ice-cold acetone-methanol (1:1) at –20°C for 10 min. The immunostainings with rabbit anti-Arc and anti-Notch1 antibodies were performed using the TSA fluorescence amplification kit (Perkin Elmer). ImageJ software (NIH) was used to quantify fluorescence intensity of immunostainings with NICD1 (Figure 2A), EGFP

(Figure S3B), and Notch1 (see legend for Figures 3C and 3D). Student’s t test was used to determine p values. Golgi-Cox staining (FD NeuroTechnologies) was performed according to the manufacturer’s instructions. Dendrite and spine lengths/widths were measured using Reconstruct software by the Neural Systems Laboratory (http://www.bu.edu/neural/Reconstruct.html). buy EPZ-6438 Spine length and width data were analyzed using the Kolmogorov-Smirnov selleck chemical statistical test. Transverse hippocampal slices (350 μm) were prepared from Notch1 cKO and control mice, and maintained in artificial cerebrospinal fluid at room temperature. Data were collected using an Axopatch 1D amplifier (Molecular Device); signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed using pCLAMP 8 software (Molecular Device). The authors thank Jason Shepherd, Richard Flannery, Marlin Dehoff, Vera Goh, and Keejung Yoon for technical and intellectual input during the course of this project. We also thank Ted Dawson and Jay Baraban for

critically reading the manuscript. Funding for this work came from the Institute for Cell Engineering

at Johns Hopkins University (N.G.), a NARSAD Young Investigator Award (N.G), the James S. McDonnell Foundation (N.G.), and the National Institute of Mental Health (P.F.W.). “
ent in each arm and number of entries in each arm using the StopWatch Plus software. The social interaction testing was carried out in three sessions using a three-chambered box with openings between the chambers. The Morris water maze Parvulin test was done according to a published protocol (Vorhees and Williams, 2006). Details for all behavioral tests are provided in the Supplemental Information. Neuronal cultures were prepared from the hippocampus of E17.5 embryos and plated on poly-L-lysine-coated 60 mm dishes or 18 mm glass coverslips. Neurons were exposed to pharmacological manipulations after 14 days in vitro (DIV). For Sindbis virus infection, the pSinRep5 vector (Invitrogen) was used to generate viruses expressing either full-length Arc or a nonfunctional form with residues 91–100 deleted (Chowdhury et al., 2006). Synaptosomal fractions were prepared as previously described (Blackstone et al., 1992). Standard western blot protocols were used. Details regarding fractionation, immunoprecipitation, and western blot protocols are provided in the Supplemental Information. Quantitation of individual protein bands was done using ImageJ software. Values were averaged between experiments, and Student’s t test was used to compare samples.