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 (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, , a second round of glutathione treatment was applied to cleave SCH23390, NMDA 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 . 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 (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. Slices currents. The data were evaluated with paired t-tests. were transferred into a submerged recording chamber at ap- proximately 35–36 °C. For current clamp, the recording pipettes Open Field Test. The experimental apparatus consisted of four were filled with intracellular solution containing 120 mM K- Plexiglas Digiscan automated open fields (Accuscan) (42 ϫ 42 ϫ gluconate, 6 mM KCl, 0.5 mM CaCl2, 0.2 mM EGTA, 4 mM 30 cm), evenly illuminated by overhead red lighting (9 Ϯ 1 lux) ATP-Mg, 10 mM HEPES, and 0.3% biocytin, with a final pH of and equipped with photobeam sensors to quantify locomotor 7.25. The resistances of patch pipettes were 5–10 M⍀. To record activity. Before testing and between animals, we cleaned the spontaneous inhibitory postsynaptic currents (sIPSC), a differ- apparatus with 95% ethanol. Activity was recorded during the ϩ ent Cs -based intracellular solution (134 mM CsCl2,2mM first exposure to the open field arena for 1 h.

Ji et al. www.pnas.org/cgi/content/short/0904289106 2of8 A B dys +/+Endocytosis time (min) dys -/- Endocytosis time (min) 0 30 60 0 30 60 GTH cleavage - +++ GTH cleavage - +++ Biotin D2R Biotin D2R

Total D2R Total D2R Biotin Cadherin Biotin Cadherin Biotin TfR Biotin TfR

Total Tubulin Total Tubulin

C 100 100 100 dys +/+

80 80 dys -/- 80 60 60 60

40 40 40

20 20 20 % TfR internalized % D2R internalized

0 % N-Cad internalized 0 0 30 60 30 60min 30 60 30 60 min 30 60 30 60 min

Fig. S1. Spontaneous endocytosis of D2. (A and B) Biotinylation assay of receptor endocytosis in cortical neurons. Surface proteins were labeled with cleavable biotin and neurons were incubated at 37 °C for various times (internalization times). The residual extracellular biotin was removed by treatment with glutathione (GTH). Representative immunoblots show internalization of D2, N-Cad, TfR in wild-type (A) and dysϪ/Ϫ (B) cultures. (C) Quantitative analysis of D2 basal internalization. Receptors internalized at 30 and 60 min at 37 °C were normalized to total surface receptors to quantify their internalization.

Ji et al. www.pnas.org/cgi/content/short/0904289106 3of8 A B dys +/+Endocytosis time (min) dys -/- Endocytosis time (min) 0 30 60 0 30 60 GTH cleavage - +++ GTH cleavage - +++ Biotin D2R Biotin D2R

Total D2R Total D2R Biotin Cadherin Biotin Cadherin Biotin TfR Biotin TfR

Total Tubulin Total Tubulin

100 100 100 C dys +/+

80 80 dys -/- 80

60 60 60

40 40 40

20 20 20 % TfR internalized % D2R internalized 0 % N-Cad internalized 0 0 30 60 30 60min 30 60 30 60 min 30 60 30 60 min

Fig. S2. Biochemical analysis of dopamine-induced D2 endocytosis. (A and B) Biotinylation assay of receptor endocytosis in cortical neurons. Surface proteins were labeled with cleavable biotin, treated with dopamine (10 ␮M) at 37 °C for various times (internalization times). The residual extracellular biotin was removed by GTH treatment. Representative immunoblots show internalization of D2, N-Cad, TfR in wild-type (A) and dysϪ/Ϫ (B) cultures. Immunoblots of total D2 show no change in overall D2 protein levels. (C) Quantitative analysis of dopamine-induced internalization of D2 (left), N-Cad (middle), and TfR (right). Receptors internalized after dopamine treatment at 30 and 60 min were normalized to total surface receptors to quantify their internalization.

Ji et al. www.pnas.org/cgi/content/short/0904289106 4of8 A B Control DA + Strip

DA- induced Endocytosis GFP

Strip dys -/- dys +/+

Secondary antibody

Flag tagged D2R Flag-D2 M1 antibody Secondary antibody surface intracellular

C GFP 100

80

60

40

20 % Internalized D2R

0 Flag-D2 dys +/+ dys -/- surface intracellular

Figure S3 Ji et al.

Fig. S3. Imaging analysis of dopamine-induced endocytosis of FLAG-D2. (A) Schematic of internalization assay. Neurons expressing FLAG-D2 were incubated at 37 °C with M1 antibody (Ca2ϩ sensitive) for 20 min, followed by dopamine for 60 min. The surface M1 antibody was removed by stripping (Fig. S4A). Neurons were then fixed, permeabilized, and stained with Alexa-546 conjugated secondary antibody to label internalized D2. (B) Representative images showing cell surface (left, control, step 1) and endocytosed (right, after dopamine treatment and stripping, step 2) FLAG-D2 (red) in transfected neurons (indicated by GFP fluorescence). (Scale bar, 20 ␮m.) (C) Quantification of D2 endocytosis. Intracellular D2 immunofluorescnece was normalized to cell surface immunofluorescence before DA treatment.

Ji et al. www.pnas.org/cgi/content/short/0904289106 5of8 A GFP Flag-D2

B dys +/+ dys -/- Surface Intracellular Surface Intracellular Step 1 Step Step 2 Step

Fig. S4. Surface and intracellular D2 before recycling. (A) Experiment controls of Fig. S3. A neuron transfected with eGFP and FLAG-D2 showed no surface staining by M1 antibody after stripping with EDTA and Ca2ϩ/Mg2ϩ free PBS (Right). (B) Experiment controls of Fig. 3. (Upper) Sister cultures of Fig. 5B were incubated at 37 °C with M1 antibody (Ca2ϩ sensitive) for 60 min in the absence of dopamine. Cultures were fixed without a prior stripping step (Step 1), and stained with Alexa-546 conjugated secondary antibody (red) to label surface D2 under nonpermeable conditions and Alexa-488 conjugated secondary antibody (green) to label intracellular D2 under permeable conditions. (Lower) Similar cultures as above, except that cells were fixed immediately after the stripping step (Step 2), and stained with Alexa-546 conjugated secondary antibody (red) to label surface D2 under nonpermeable conditions and Alexa-488 conjugated secondary antibody (green) to label intracellular D2 under permeable conditions.

Ji et al. www.pnas.org/cgi/content/short/0904289106 6of8 ABCD FS

fAHP half-width

-64 mV

Non-FS

2 mV 300 ms

-64 mV

100 pA 40 mV 40 mV

40 mV 15 ms 1 ms

300 ms

EFFS (19 mice, 29 slices, 29 cells) dys +/+ (10 mice, 16 slices, 16 cells) Non-FS (59 mice, 158 slices, 158 cells) dys -/- (9 mice, 13 slices, 13 cells)

-25 ** 2 ** 1 -25

-20 0.8 -20 1.5 -15 0.6 -15 1 -10 0.4 -10 AHP (mV) AHP 0.5 (mV) AHP -5 0.2 -5 Half-width of APs (ms) Half-width of Half-width of APs (ms) Half-width of 0 0 0 0

Fig. S5. Identification of prefrontal layer V interneurons. (A–D) Samples of FS and Non-FS interneurons. In upper panel, FS cell was identified by its high-frequency firing of action potentials without adaptation, short half-width, large fAHP, and spontaneous postsynaptic potentials with high frequency and large amplitude. In lower panel, Non-FS neuron had adaptive firing, wider action potentials, smaller fAHP, and relatively less spontaneous postsynaptic potentials. (E) Quantification of half-width and fAHP of action potentials. FS cells exhibited remarkably shorter half-width larger fAHP than those of Non-FS neurons. (F) Comparatively, no difference was observed between wild-type and dysϪ/Ϫ FS interneurons in both half-width and fAHP of action potentials.

Ji et al. www.pnas.org/cgi/content/short/0904289106 7of8 A 1900 dys +/+

dys -/- 1575 (cm) 1250

Distance 925

600 5 1015202530354045505560 Minutes

Fig. S6. Higher locomotor activity in dysϪ/Ϫ mice. (A) Ambulatory distance in 5-min intervals displayed by dysϩ/ϩ, and dysϪ/Ϫ littermates during the first 1-h exposure to the open field arena. n ϭ 13–14/group. Values represent mean Ϯ SEM.

Ji et al. www.pnas.org/cgi/content/short/0904289106 8of8