All counts were performed blinded to the genotype of the animals. Golgi impregnated neurons were visualized in brightfield with a 40× objective and traced using Neurolucida software (MBF Bioscience). Neuronal tracings were subjected to Sholl analysis using Neuroexplorer software. The center of all concentric circles is defined as the center of the soma. The starting radius was 12.5 μm, and the click here ending radius was 200 μm from the center with an interval of 12.5 μm between radii. Mice were perfused with 4% PFA and postfixed overnight and vibratome sectioned at 70–100 μm. Sections were permeabilized and

blocked for 2 hr in PBS plus 0.1% Triton X-100, 10% serum, 0.2% gelatin. Sections were incubated 48 hr in primary antibodies: chicken anti-GFP (1:500, Aves Labs), rabbit anti-GABA (1:2,000, Sigma A2052,), rat anti-CTIP2 (1:500, Abcam ab18465), rabbit anti-SATB2 (1:1,000, Abcam ab34735), mouse anti-NeuN (1:500, Millipore MAB377), rabbit anti-RFP (1:500, MBL PM005), Protease Inhibitor Library solubility dmso and rabbit

anti-Shh (1:200, a kind gift from S. Scales, Genentech). Images were acquired using a Leica SP5 laser scanning confocal microscope. For synaptophysin-GFP puncta counts images were analyzed using Imaris (Bitplane). Only axon segments with a minimum length of 20 μm and a least one GFP puncta were included in the analysis. All counts were performed blind to the treatment. Corticospinal projections were retrogradely labeled with red fluorescent microspheres (Lumafluor) injected into the spinal cord at the C2–C3 level at P21–P28. Callosal projections were labeled by injecting fluorgold (Fluorochrome, LLC) into the contralateral sensorimotor cortex. Brains were collected for isothipendyl processing 24–48 hr after injections. Mutant mice and their wild-type littermates ages

P21–P28 were anesthetized with Avertin and decapitated. Brains were quickly dissected in ice-cold “sucrose-ACSF” buffer containing 252 mM sucrose, 126 mM NaCL, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 26 mM NaHCO3, and 10 mM D-glucose. Brains were vibratome sectioned in the same solution at 300 μm and transferred to ACSF without sucrose. Slices were recovered at 35°C for 30 min and then maintained at room temperature. Neurons were targeted for whole-cell patch clamp recording with borosilicate glass electrodes having a resistance of 2–6 MΩ. The electrode internal solutions was composed of 130 mM potassium gluconate, 10 mM KCl, 10 mM HEPES, 1 mM MgCl2, 16 mM sucrose, 5 mM EGTA, 4 mM Na2ATP, and 1 mM NaGTP, titrated to pH 7.3 with KOH for recording mEPSCs. Channelrhodopsin recordings were done with an internal solution composed of (in mM) 120 CsMeSO3, 15 CsCl, 8 NaCl, 0.5 EGTA, 10 HEPES, 5 QX-314, 10 TEA-Cl, 2 Mg-ATP, 0.3 Na-GTP; pH adjusted to 7.3 with CsOH, 290 mOsm. During collection of miniature EPSCs external solution was supplemented with 1 μM tetrodotoxin and 10 μM bicuculline.