All procedures were approved by IACUC of Boston Children’s Hospital and conducted in the Mecp2 KO mouse line generated by A. Bird and colleagues (Guy et al., 2001). Double mutants for Mecp2 and NR2A were generated by crossing Mecp2 heterozygote females with NR2A KO males (originally generated by M. Mishina; Sakimura et al., 1995). fLox-STOP-Mecp2 (Guy et al., 2007) or PV-Cre x fLox-Mecp2 animals were used in some experiments (Hippenmeyer et al., 2005;
Jackson Laboratories). All control animals were WT age-matched littermates of the mutant mice. Electrophysiological recordings were performed using standard techniques (Fagiolini et al., 2003). Spontaneous and evoked single-unit responses were recorded with multichannel probes (A1x16-3mm-50-177-A16, Neuronexus Technologies) in response to high contrast (100%), low spatial frequency sine wave gratings (0.025 or 0.07 cpd; 2 Hz). Transient VEPs were recorded under nembutal/chlorprothixene anesthesia selleck products using standard techniques in mice (Fagiolini and Hensch, 2000). A tungsten electrode (1.5 MΩ, FST) was inserted into binocular V1. Receptive field was located within the visual field 20 degrees from the vertical meridian corresponding to the maximal VEP response. Behavioral threshold acuity was evaluated using the optomotor task (Prusky et al., 2004). Mice were tested
every 3–5 days starting after eye opening until adulthood (P60–P240). Experimenters were blind to genotype and the animal’s previously recorded thresholds. All animals were habituated before the Ipilimumab ic50 onset of testing by gentle handling and by placing them on the arena platform for a few minutes at a time. Mice were decapitated under brief isoflurane anesthesia and the brain processed as detailed in Supplemental Information. A stimulating
pulse Galactosylceramidase (1 ms) was delivered through an ACSF-filled patch pipette to the white matter in V1. The resultant change in emitted Di-4-ANEPPS fluorescence, corresponding to a change in membrane potential, was recorded with a MiCam Ultima (Brain Vision, SciMedia) camera (at 1 frame/ms). Changes in fluorescence were averaged across ten 512 ms trials. Regions of interest (125 × 125 μm2) in the upper (150 μm below the pia) and lower layers (300 μm above the white matter) “on beam” with the stimulating electrode were analyzed for maximum change in intensity normalized to the resting intensity (ΔF/F). Primary antibodies and dilutions are detailed in the Supplemental Information. Quantitative analyses of the binocular zone of visual cortex across all layers were performed blind to genotype and treatment. Mean pixel intensity (at 100×) of the PV signal in each field (1,024 × 1,024) was measured using MacBiophotonics ImageJ software. The number of perisomatic synapses (at 100x) was determined on triple-stained images (PV, GAD65, DAPI) using the “particle analysis” function (ImageJ).