Sensory deprivation drives cell-wide synaptic enhancement that globally sensitizes a neuron. Experiments were conducted according to National Institutes of Health guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Cold Spring Harbor Laboratory and University of California, San Diego. SEP-GluR1, SEP-GluR1(S831A,S845A), SEP-GluR2(R586Q), untagged-GluR2(edited), and SEP-GluR3 from rat were PCR amplified click here and subcloned into an expression vector with a ubiquitous promoter CAG, pCALNL. pCALNL-DsRed and pCAG-ERT2CreERT2 were obtained from Addgene. All the DNA plasmids were amplified with the endotoxin-free Maxiprep kit (QIAGEN).

For the formation of homomeric GluR2, SEP-GluR2(R586Q) was expressed. Heteromeric AMPA receptors were formed by coexpressing untagged-GluR2(edited) with either

SEP-GluR1 or SEP-GluR3 at a 1:1 molar ratio. L2/3 progenitor cells were transfected by in utero electroporation. E15 time pregnant C57BL/6J mice (Charles River) were anesthetized with an isoflurane-oxygen mixture (Lei Medical). Approximately 0.5 μl of DNA solution containing fast green was pressure injected through a pulled-glass capillary tube by mouth into the right lateral ventricle of each embryo. The head of each embryo was placed PLX-4720 in vivo between tweezers electrodes with the anode contacting the right hemisphere. Electroporation was achieved with five square pulses (duration = 50 ms, frequency = 1 Hz, voltage = 25V; Harvard Apparatus). 4-OHT (Sigma-Aldrich) was dissolved in ethanol at a concentration of 20 mg/ml and diluted with 9 vol of corn oil (Sigma-Aldrich). Diluted 4-OHT (2 mg/ml) was i.p. injected into each mouse at P11 (100 μl per animal) or P34–P35 (300–450 μl per animal). For sensory deprivation all the major whiskers were trimmed daily from P11. Whisker-intact animals were handled similarly to whisker-trimmed animals. Acute coronal brain slices (350 μm thick) from in utero electroporated mice at P13 or P36–P37 were prepared. Slices were cut

in gassed (95% O2 and 5% CO2) ice-cold solution containing 25 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 0.5 mM CaCl2, 7 mM MgCl2, Idoxuridine 25 mM D-glucose, 110 mM choline chloride, 11.4 mM sodium ascorbate, and 3.1 mM sodium pyruvate. Slices were then incubated in artificial cerebrospinal fluid (ACSF) containing 118 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.2 mM NaH2PO4, 11 mM D-glucose, 4 mM MgCl2, and 4 mM CaCl2 at 35°C for 30 min and then at room temperature until used. All experiments were performed at 30°C. We used a two-photon laser-scanning microscope (Prairie) to image L2/3 pyramidal cells of the mouse barrel cortex (40× 0.8 NA objective lens and 1.4 NA oil condenser; Olympus) in a perfusion chamber containing ACSF. SEP and DsRed were excited at 910 nm with a Ti:sapphire laser (Coherent). Green and red fluorescence signals were separated by a set of dichroic mirrors and filters (Chroma).