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Supporting Information Supporting Information Ji et al. 10.1073/pnas.0904289106 SI Materials and Methods were then returned to serum-free Neurobasal media containing Reagents and Antibodies. N-terminal FLAG-D2 human cDNA glutathione (50 mM, 37 °C) for various times to allow internal- was a gift from Dr. Sibley’s laboratory, NINDS. Dysbindin-GFP ized receptors to recycle before neurons were cooled to 4 °C and was from GeneCopeia. Quinpirole, dopamine, haloperidol, a second round of glutathione treatment was applied to cleave SCH23390, NMDA receptor antagonist D-(Ϫ)-2-amino-5- phos- any newly appearing surface biotin. The disappearance of bio- phono-pentanoic acid (D-APV), and AMPA receptor antago- tinylated proteins as a function of time represents the rate of nist 6-Cyano-7-nitroquinoxaline-2, 3-dione (CNQX) were pur- recycling. Residual biotinylated receptors were isolated by chased from Sigma. EZ-Link sulfo-NHS-LC-biotin, sulfo-NHS- streptavidin precipitation and detected by immunoblotting. SS-Biotin, and Streptavidin agarose resins were from Pierce. Commercial antibodies were used against D2 (Abcam), amino- Receptor Recycling Experiments. Cells were incubated with M1 terminal D2 (Chemicon), D1 (Chemicon), TfR (Zymed labora- anti-FLAG mAb to selectively label FLAG-tagged receptors tories), LAMP1 (Developmental studies hybridoma bank), N- presented on the plasma membrane at the beginning of the Cad (Abcam), Tubulin (Abcam), M1, and M2 anti-FLAG experiment. Then cells were incubated at 37 °C for 60 min in the antibodies (Sigma). HRP-conjugated secondary antibodies were presence of 10 ␮M dopamine to drive internalization. At the end from Pierce. Alex-546 and Alex-488 conjugated secondary an- of this incubation, cells were quickly washed three times in PBS ϩ ϩ tibodies were from Molecular Probes. containing 0.04% EDTA and lacking Ca2 /Mg2 to dissociated FLAG antibody bound to residual surface receptors remaining Subjects. All procedures were approved by the National Institutes on the plasma membrane, thereby leaving antibody bound only of Health (NIH) Animal Care and Use Committee and followed to the internalized pool of receptors. Stripped cells were then the NIH Guidelines ‘‘Using Animals in Intramural Research.’’ incubated at 37 °C for 60 min in the presence of either D2 Sandy mice carry an autosomal recessive coat mutation occurred receptor antagonist haloperidol or D1 receptor antagonist spontaneously in the inbred DBA/2J strain in 1983 at the Jackson SCH23390 to prevent subsequent receptor activation and to Laboratory. In the mice that we used, the mutation was trans- allow recycling to occur, and then cells were fixed with 4% ferred to the C57BL/6J genetic background by more than ten paraformaldehyde under nonpermeable conditions, and incu- generation backcrossing. The Sandy mice express no dysbindin bated with Alex-546 conjugated secondary antibody to detect protein owing to a deletion in the gene dystrobrevin-binding recycled, antibody-labeled receptors. Neurons were then per- protein 1 (Dtnbp1; encoding dysbindin). Dysbindin null mutant meabilized and stained with Alex-488 conjugated secondary mice (dysϪ/Ϫ), and wild-type (ϩ/ϩ) littermates were bred by antibodies to visualize prelabeled internalized receptors. In each heterozygous (dys-/ϩ) mating. Mice were identified by PCR experiment, and for each receptor construct examined, two analysis of tail DNA. Mice were group-housed (2–4/cage) in a control coverslips were included, one in which cells were fixed climate-controlled animal facility (22 Ϯ 2 °C) and maintained on after a 60 min incubation in the absence of agonist and without a 12-hr light/dark cycle, with free access to food and water, unless a stripping step (100% surface receptor control) and the other specified in particular experiments. in which cells were fixed immediately after the stripping step (0% recycled control) (Fig. S2B). The efficiency of strip was Ͼ95%. Biotinylation Assay of Receptor Endocytosis and Recycling with Cleav- Red fluorescence intensities indicative of recycling were divided able Biotin Reagent. Biotinylation assay of receptor endocytosis by total (red ϩ green) fluorescence intensities. The percentage and recycling was performed as previously described (15). of receptors recycled in an individual cell was then determined Cultured cortical neurons grown in 100 mm dishes were incu- from the control conditions according to the following formula: bated with lysosomal protease inhibitor Leupeptin (100 ␮g/mL, (E-Z)/(C-Z) ϫ 100, where E ϭ the mean ratio for the experi- 1 h, 37 °C) to block the activity of lysosomes. Leupeptin was mental coverslip, Z ϭ the mean ratio for the zero surface present throughout all steps except the biotinylation reaction. control, and C ϭ the mean ratio for the 100% surface control. For internalization assays, neuron surface was biotinylated with Units of internalization were normalized to untreated controls. cleavable sulfo-NHS-S-S biotin (30 mg/mL, 30 min, RT). Unre- acted biotin was quenched and removed by three successive Agonist-Induced Receptor Internalization Assay. Endocytic traffick- washes of biotinylated cells in PBS containing 10 mM Glycine. ing of receptors was visualized after specific labeling surface Biotinylated neurons were incubated with either control solution proteins. Briefly, transfected live neurons expressing FLAG-D2 or dopamine (10 ␮M, 37 °C) for various times as indicated in Fig. constructs were fed with M1 anti-FLAG monoclonal antibody 2. Assays were stopped by chilling neurons on ice. Neurons were (2.0 ␮g/␮L) at 37 °C for 25 min in the absence of agonist. either immediately scraped into lysis buffer or incubated with Neurons were then incubated with agonist (10 ␮M dopamine, 60 glutathione strip buffer at 4 °C (50 mM glutathione, 75 mM min, 37 °C) or left untreated. Untreated neurons were fixed with NaCl, 75 mM NaOH, 10% FBS in H2O) to cleave remaining 4% formaldehyde in PBS, and stained with Alexa-546 conju- surface biotin. Excess glutathione was then quenched at 4 °C in gated secondary antibody without permeabilization to label total iodoacetamide buffer (50 mM iodoacetamide, 1% BSA in PBS) surface D2 receptors. Residual surface receptors (those not for 30 min. Neurons were then scraped and lysed in lysis buffer internalized by agonist) in neurons treated with agonist were and remaining biotinylated receptors were isolated by strepta- stripped of antibody by washing in PBS containing 0.04% EDTA vidin precipitation and detected by immunoblotting. For recy- and lacking Ca2ϩ/Mg2ϩ (the M1 interaction is Ca2ϩ sensitive). cling assays, neuron surface was biotinylated with cleavable Neurons were fixed and then permeabilized, and stained with biotin, and neurons were transferred to 37 °C for 60 min to allow Alexa-546 conjugated secondary antibody to label internalized endocytosis in the presence of dopamine (10 ␮M). Neurons were D2 receptors. cooled to 4 °C and the remaining surface biotin was removed by one round of glutathione treatment. The internalized and bio- Image Analysis and Quantification. Fluorescence images were ac- tinylated proteins were spared from the first stripping. Cultures quired with Zeiss LSM510 confocal microscope. The confocal Ji et al. www.pnas.org/cgi/content/short/0904289106 1of8 microscope settings were kept the same for all scans. All MgCl2,2mMNa2-ATP, 0.5 mM Na2GTP,5mMNa2- measurements were performed using MetaMorph software. phosphocreatine, 1 mM EGTA, 10 mM HEPES, and 0.3% Single transfected neuron was selected and manually traced for biocytin, pH 7.25) was used. The sIPSCs were recorded at Ϫ70 maximum accuracy. Image analysis was based on measuring the mV in the presence of CNQX (50 ␮M) and D-APV (50 ␮M). area of labeled puncta rather than counting the number of The signals were amplified and filtered at 2 kHz with Multi- labeled puncta. The average intensity of surface fluorescence Clamp 700A amplifiers or at 5 KHz with Axopatch 200B (Axon staining was measured in the traced region and the background Instruments) and acquired at sampling intervals of 20–100 ␮s staining was subtracted. Intensity was expressed in arbitrary through a DigiData 1322 interface with program pCLAMP 8.2 units of fluorescence per square area. The number of neurons (Axon Instrument). The membrane potentials were not cor- used for quantification for each experimental condition ranged rected for liquid junction potential. The access resistance was from 12–15 from at least four independent experiments. monitored during recordings, and the data were excluded from analysis if the series resistance changed more than 20% from Physiological Recording and Data Analysis. Wild-type and dysϪ/Ϫ control levels (10–25 M⍀). mice (15–35 days old) were used in this study. Mice were killed All drugs were applied to the bath through gravity. Stock with a guillotine after being administered with an overdose of solutions of all tested chemicals were stored at Ϫ70 °C and were sodium pentobarbital (i.p., 100 mg/kg). Brains were quickly diluted to proper concentration before each experiment. removed, and 300-␮m thick coronal slices containing frontal The spontaneous excitatory and inhibitory postsynaptic cur- cortex were cut on a vibratome (Vibratome Co.). Slice cuttings rents recorded in voltage-clamp mode were analyzed with were maintained in oxygenated ice-cold Naϩ-free sucrose solu- Clampfit 9.2 (Axon Instrument). A typical EPSC and IPSC were tion containing 2.5 mM KCl, 1.25 mM NaH2PO4,26mM selected to create a sample template for event detection within NaHCO3, 0.5 mM CaCl2, 7.0 mM MgSO4, and 213 mM sucrose. a data period. The frequency (event number) and amplitude of Slices were initially incubated at 35 °C in an Ringer solution individual events were examined with Clampfit. The input (ACSF) containing 124 mM NaCl, 2.5 mM KCl, 1.25 mM resistances of the tested neurons were calculated offline from the NaH2PO4, 2 mM CaCl2, 1 mM MgSO4, 26 mM NaHCO3 and 10 voltage produced by negative current injection before the step mM dextrose, pH 7.4, and then kept at room temperature.
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