Supporting Information

Transcription

1 Supporting Information Hasbi et al /pnas SI Text Drugs. Dopaminergic agonists and antagonists SKF 83959, SKF 83822, dopamine, SCH 22390, and raclopride were purchased from Tocris Bioscience. TPG, an inhibitor of intracellular calcium mobilization, by depleting intracellular stores (1), was purchased from Sigma. 2-APB, which inhibits IP 3 receptordependent calcium stores (2), was purchased from Cayman Chemical. SQ, an inhibitor of AC (3), was purchased from Sigma. U73122, a PLC inhibitor (4), was purchased from Tocris. YM, a Gq-specific inhibitor (5), was a kind gift from Astellas. Coimmunoprecipitation. Solubilized striatal membranes from dissected striatal tissue of adult rats (500 g) were incubated (overnight at 4 C) with 5 g of rabbit anti-d2 (Chemicon) or rat anti-d1 (Sigma). After 1-h incubation with protein G/A-agarose solution (Thermo Scientific), the immunoprecipitates were washed three times with RIPA buffer, and 50 L of SDS buffer was added for 30 min at room temperature. The proteins were resolved by electrophoresis and then transferred onto PVDF membranes for Western blot analysis. Immunoblots were probed by using a monoclonal D1 receptor antibody (Sigma) or D2 antibody (Chemicon), diluted to 1:1,000. The appropriate HRPconjugated secondary antibodies were used at 1:1,000 to reveal the bands. Immunocytochemistry. The primary antibodies used were: mouse anti-map2 (Abcam; 1:500), rat anti-d1 (Sigma; 1:400), rabbit anti-d2 (Chemicon; 1:400), mouse anti-gq/11 (Santa Cruz; 1:400), and mouse anti-bdnf (Chemicon; 1: 400). The secondary antibodies conjugated to fluophores (Alexa Fluor 488; Alexa Fluor 350, Alexa Fluor 568, Alexa Fluor 647) were all purchased from Molecular Probes and used at 1: 500. Paraformaldehyde-fixed neurons or floating brain sections were incubated with the primary antibodies overnight at 4 C. After three washes with PBS-Tween20, the samples were incubated with the appropriate secondary antibody for 2 4 h at room temperature. After three washes, the slides were mounted by using a mounting solution (Dako), and the images were acquired by using a confocal Fluoview Olympus microscope (FV 1000). All images were acquired in sequential mode to minimize any bleed-through. Neuronal Cultures. Neonatal rat striata (1 day of age) were trypsinized in Hanks balanced salt solution (HBSS) with 0.25% trypsin and 0.05% DNase (Sigma) at 37 C, and cells were washed three times in HBSS with 12 mm MgSO 4. Cells were dissociated in DMEM with 2 mm glutamine and 10% FBS and plated at cells per poly-l-lysine-coated well (Sigma; 50 g/ml). The next day, media were changed to Neurobasal medium with 50X B27 Supplement and 2 mm glutamine (Invitrogen). On day 3 of culture, 5 M cytosine arabinoside was added to inhibit glial cell proliferation. Half of the medium was changed every 3 days. Transfection Procedure. Neonatal striatal neurons in culture for 7 21 days were transfected by using a combination of two products: Effectene (Qiagen) and Exgen 500 (Fermentas). Used individually, these products gave restricted transfection efficiency (10 20%). In combination, the efficiency of transfection rose to 40 70%. Briefly, 2.7 g of cameleon YC.6.1 cdna (5) was mixed in the following order with buffer EC (Effectene kit), 150 mm NaCl, Exgen reagent, and enhancer (Effectene kit). After vortexing and a 5-min wait at room temperature, Effectene reagent was added and mixed. Ten minutes later, 6 ml of culture media was added to the mixture, which was split into 24 wells by removing 250 L of medium from each well and replacing it with 250 L of the transfection mixture. Animals. Sprague Dawley rats (Charles River), weighing g, were housed paired in polyethylene cages in a colony room maintained on a 12-h light dark cycle with free access to food and water. All treatments were performed during the light phase of the day night cycle. Animals were housed and tested in compliance with the guidelines described in the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, 1993). SKF and SKF (Tocris Bioscience) were dissolved in physiological saline containing 5% DMSO. Rats were injected s.c. (1.0 ml/kg) once daily for 3 days with saline, SKF 83959, or SKF (0.4 mg/kg). One hour after the final injection rats were anesthetized with pentobarbital and perfused with ice-cold 4% paraformaldehyde. Brains were removed, fixed in 10% sucrose for 2 h followed by 20% sucrose in 0.1 M PBS for 5 days, frozen in Isopentane ( 60 C), and stored at 80 C until cryostat sectioning. Serial coronal sections through the striatum (bregma 1.5 mm) were cut at 10- or 30- m thickness. Free-floating sections were prepared with the Elite ABC kit (Vector Laboratories) and incubated with primary antibody for BDNF (1:500; Chemicon) for 24 h at room temperature. Images were obtained with an Axioplan2 microscope (Zeiss). Cells were counted within similar 400- m 2 areas within the caudate putamen, nucleus accumbens core, and shell regions. Confocal Microscopy FRET and Data Processing. Dopamine D1 D2 receptor interaction was assessed previously by coimmuprecipitation studies performed from rat brain (6). To further characterize this interaction we used FRET methodology. Paraformaldehyde-fixed striatal neurons or floating sections (10 m) from rat brain were incubated for 24 h at 4 C with primary antibodies raised against D1 and D2 receptors (6) and the species-specific secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 350 dyes, respectively. The primary antibodies have been shown to be highly specific to D1 or D2 receptors using HEK cells expressing individual D1, D2, D3, D4 or D5 receptors (6). They were further validated by immunohistochemistry showing lack of reactivity in slices from D1 / and D2 / mice. The D2-Alexa Fluor 350 was used as a donor dipole, while D1-Alexa Fluor 488 was used as acceptor dipole. The images were acquired with an Olympus Fluoview FV 1000 laser scanning confocal microscope with a 60 /1.4 NA objective. The donor was excited with a krypton laser at 405 nm, while the acceptor was excited with an argon laser at 488 nm. The emissions were collected at 430/20-nm and 530-nm LP filter. Other FRET pairs ( and ) were tested and showed comparable results. Eleven images were acquired for each FRET analysis (Table S1 and Table S2) in accordance with the algorithm described by Chen et al. (7). 1of10

2 1. Ghosh TK, Bian J, Short AD, Rybak SL, Gill DL (1991) Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth. J Biol Chem 266: Peppiatt CM, et al. (2003) 2-Aminoethoxydiphenyl borate (2-APB) antagonizes inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps, and has a usedependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium 34: Harris DN, Asaad MM, Phillips MB, Goldenberg HJ, Antonaccio MJ (1979) Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives. J Cyclic Nucleotide Res 5: Smith RJ, et al. (1990) Receptor-coupled signal transduction in human polymorphonuclear neutrophils: Effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Ther 253: Takasaki J, et al. (2004) A novel G q/11-selective inhibitor. J Biol Chem 279: Lee SP, et al. (2004) Dopamine D1 and D2 receptor coactivation generates a novel phospholipase C-mediated calcium signal. J Biol Chem 279: Chen Y, Elangovan M, Periasamy A (2005) FRET data analysis: The algorithm. Molecular Imaging: FRET Microscopy and Spectroscopy, eds Periasamy A, Day RN (Oxford Univ Press, New York), pp of10

3 a D1 (Alexa 488) D2 (Alexa 568) Merge b D1(Alexa 568) D2(Alexa 647) FRET Distance ROI Average FRET Efficiency Average Distance between donor and acceptor c A B CPu NAc Str Str Mock Str Mock IgG IgG WB: anti-d1r anti-d2r CPu: Caudate Putamen NAc: Nucleus Accumbens Str :Striatum IP: anti-d2r anti-d1r anti-d2r WB: anti-d1r anti-d2r Str :Striatum Fig. S1. (a) Representative immunocytochemistry-based colocalization of endogenous dopamine D1 and D2 receptors in cultured neonatal rat striatal neurons. D1 receptors were detected by using a specific anti-d1 antibody and a secondary antibody conjugated to Alexa Fluor 488. D2 receptors were detected by using a specific anti-d2 antibody and a secondary antibody conjugated to Alexa Fluor 568. Similar results were obtained with other flurophore pairs (Alexa Fluor 350-Alexa Fluor 488; Alexa Fluor 568-Alexa Fluor 647). Note the colocalization at the cell surface and proximal dendrites and the localization of D2 receptors in cytosol. (b) Example of colocalization and confocal FRET analysis of the interaction between D1 and D2 receptors in rat striatal neurons. Anti-D1-Alexa Fluor 568 was used as the donor dipole, anti-d2-alexa Fluor 647 was used as the acceptor dipole. Eleven images were taken to generate images of pfret, and calculation of distances separating the pair, using an algorithm as described (7). ROIs within neurons coexpressing both D1 and D2 receptors were analyzed, to calculate FRET efficiency (E), and distance between donor and acceptor. A distance of 10 nm (100 Å) indicates no FRET and no interaction. (c)(a) Western blots of D1R and D2R from rat striatum are shown. (B) Coimmunoprecipitation of D1 with D2-specific antibody and coimmunoprecipitation of D2 with D2-specific antibody from the rat striatum are shown. A nonspecific band of kda appearing in the immunoprecipitation samples represents IgG. 3of10

4 Fig. S2. (a) Dose-dependent effect of quinpirole on intracellular calcium mobilization, measured by using cameleon Y6.1 FRET and live cell confocal microscopy in rat striatal neurons. Results are mean SEM (n 21). (b) Effects of 100 nm SKF alone (n 20) or in combination with 100 nm quinpirole (n 33) on intracellular calcium increase. Results are mean SEM. (c) Comparison of the effects of SKF (n 43) and SKF (n 20) on intracellular calcium mobilization in rat striatal neurons. Results are mean SEM. (d) Intracellular calcium measurements after treatment with SKF (100 nm, n 23 cells) in D5 / mice-derived striatal neurons. Results are mean SEM. (e) Intracellular calcium measurements after treatment with dopamine (100 nm) in WT and D1 / mice-derived striatal neurons. Results are mean SEM. (f) Intracellular calcium measurements after treatment with dopamine (1 M) in striatal neurons preincubated without (Dop; n 24) or with thapsigargine (Dop TPG; n 26). Results are mean SEM. 4of10

5 a % of total fluorescence Plasma Membrane Cytosol Control 2 min 5 min Dopamine (100 nm) b plasma membrane Gq fluorescence (% over basal) Control 2 min 5 min 100 nm Dopamine (Time) Control 2 min 5 min Cytosolic Gq fluorescence (% beyond basal) Fig. S3. (A) Quantification of Gq/11 immunofluorescence at plasma membrane and cytosol after treatment with vehicle (control, 0 min) or SKF (100 nm) for 2 and 5 min. (B) Quantification of endogenous Gq immunofluorescence at the plasma membrane (Upper) and cytosolic (Lower) in rat striatal neurons treated with vehicle (control) or with 100 nm dopamine for 2 or 5 minutes. Results are mean SEM of three independent experiments. 5of10

6 a Control 2 min 5 min b c Control SKF (2 hr) Racl + SKF 89359: 5 min SKF89359: 5 min pcamkii d BDNF in WT (Control) BDNF in WT (SKF 83959, 2 hrs) BDNF in D-/- (SKF 83959, 2 hrs) Fig. S4. (A) Representative example of CaMKII activation in D5 / mice-derived striatal neurons treated with vehicle (control) or 100 nm SKF for 2 (Center) and 5 minutes (Right). Confocal microscopy immunofluorescence of activated-phosphorylated CaMKII (p CaMKII ) was performed on fixed neurons. Note the activation in the cytosolic and nuclear compartments. (B) Confocal images showing that D2-antagonist raclopride (10 M) inhibited BDNF expression induced by 100 nm SKF (C) Confocal microscopy immunofluorescence of BDNF expression levels in D5 / mice-derived striatal neurons treated with vehicle (control) or 100 nm SKF for 5 minutes (Upper Left) and 2 hours (Lower Left). DAPI staining of nuclei is shown in the Right frames. (D) Confocal microscopy immunofluorescence of BDNF expression levels in D1 / mice-derived striatal neurons treated with 100 nm SKF for 2 hours. 6of10

7 Fig. S5. (a) Confocal microscopy immunofluorescence of MAP2 in rat striatal neurons treated intermittently, from postnatal days 4 10, with vehicle (control) (Left), 10 nm SKF (Center) or 500 nm dopamine (Right). The nuclei were stained with DAPI (blue), and MAP2 was detected by using a specific anti-map2 antibody and a secondary antibody conjugated to Alexa Fluor 488. (b) Effect of 10 M raclopride on SKF triggered MAP2 production and striatal neuron growth. (c) Immunofluorescence of MAP2 in D5 / mice-derived striatal neurons treated with 10 nm SKF as described in a. (d) Immunofluorescence of MAP2 in D1 / mice-derived striatal neurons treated with 10 nm SKF as described in a. 7of10

8 Fig. S6. Examples of FRET analysis methodology to measure the energy transfer and the distance separating endogenous dopamine D1 and D2 receptors in situ using sections from rat nucleus accembens core. Anti-D2-Alexa Fluor 350 was used as the donor dipole, and anti-d1-alexa Fluor 488 was used as the acceptor dipole. Eleven images were taken to generate images of pfret, and distances separating the receptor pair, using an algorithm (7). ROIs within neurons coexpressing both D1 and D2 receptors were analyzed, and the data, representing average pfret, FRET efficiency (E), and distance between donor and acceptor are shown. 8 of 10

9 Table S1. Control for FRET data analysis (images a g) Symbol Fluorophore or sample Excitation wavelength Emission wavelength a Donor only Donor (405) Donor (405) b Donor only Donor (405) Acceptor (488) c Acceptor only Donor (405) Acceptor (488) d Acceptor only Acceptor (488) Acceptor (488) e Donor and acceptor Donor (405) Donor (405) f Donor and acceptor Donor (405) Acceptor (488) g Donor and acceptor Acceptor (488) Acceptor (488) 9of10

10 Table S2. Four more images (h k) are required to implement the back-bleed-through correction (total: 11 images) Excitation/sample Donor emission Acceptor emission Donor/donor a B Acceptor/donor h I Donor/acceptor j C Acceptor/acceptor d Donor/double e f Acceptor/double k g 10 of 10