Plasmid β-loop-TMD-Dendra2 consists of the cytoplasmic M3-M4 loop of mouse GlyRβ (residues N334–A454 excluding signal peptide, UniProt ID P48168) fused to a single transmembrane domain and extracellular Dendra2 (in analogy to βLwt-TMD-pHluorin; Specht et al., 2011). The fusion constructs were cloned in a eukaryotic expression vector derived from pEGFP-N1 (Clontech) with a partial deletion of the cytomegalovirus promoter. Spinal cord dissociated neuron cultures were prepared from Sprague-Dawley rats (at E14) and from homozygous mRFP-gephyrin KI mice (at E13) as described elsewhere (Calamai et al., 2009), in accordance with the guidelines of the French Ministry of Agriculture

and the Direction départamentale des services vétérinaires de Paris (Ecole Normale Supérieure, Animalerie des Rongeurs, license GSK1349572 in vitro B 75-05-20). Neurons were plated at a density of 6 × 104/cm2 on 18 mm coverslips (thickness, 0.13–0.16 mm); cultured in neurobasal medium containing B-27, 2 mM Crizotinib datasheet glutamine, 5 U/ml penicillin, and 5 μg/ml streptomycin at 36°C and 5% CO2; transfected with 0.5 μg plasmid DNA per coverslip using Lipofectamine 2000; and used for experiments on the following day (at 12–24 days in vitro [DIV]). COS-7 cells were grown on coverslips in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, and cotransfected with β-loop-TMD-Dendra2 and mRFP-gephyrin in a stoichiometry of 1:4 on the day

prior to the experiments using FuGENE 6. Cell cultures were fixed for 10 min in 0.1 M sodium phosphate, pH 7.4, containing 4% paraformaldehyde (PFA) and 1% sucrose, rinsed, and imaged in PBS (pH crotamiton 7.4) (PALM and fluorophore counting). For PALM and STORM imaging, fiducial markers (TetraSpeck microspheres, 100 nm diameter, Invitrogen T7279) were attached to the coverslips after fixation. For immunolabeling, fixed neurons were permeabilized with 0.25% Triton X-100 where necessary and labeled in PBS containing 3% bovine serum albumin with antibodies against extracellular epitopes of GlyRα1 (Synaptic Systems, mAb2b, 146111, 1:200–400 dilution) and GABAARα2 (Synaptic Systems, 224103, 1:400),

the phosphorylated C domain of gephyrin (Synaptic Systems, mAb7a, 147011, 1:500; Kuhse et al., 2012), or the N terminus of bassoon (sap7f, 1:500; tom Dieck et al., 1998), followed by Alexa Fluor 647- or 488-tagged secondary antibodies (Invitrogen, 1:250–500). dSTORM was conducted in PBS (pH 7.4), containing 10% glucose, 50 mM β-mercaptoethylamine, 0.5 mg/ml glucose oxidase, and 40 μg/ml catalase, degassed with N2 (Izeddin et al., 2011). Spinal cord and cerebral cortex sections were prepared from mRFP-gephyrin KI mice. Male animals of 1 week to 6 months of age were perfused intracardially with 4% PFA and 0.1% glutaraldehyde in PBS (pH 7.4). Spinal cords (thoracic dorsal horn) and cortices (nonsuperficial layers of the frontal lobe) were dissected, postfixed with 4% PFA in PBS, cut into 1 mm segments, and incubated overnight in 2.3 M sucrose in PBS at 4°C.