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Supporting InformationHasbi et al. 10.1073/pnas.0903676106SI TextDrugs. Dopaminergic agonists and antagonists SKF 83959, SKF83822, dopamine, SCH 22390, and raclopride were purchasedfrom Tocris Bioscience. TPG, an inhibitor of intracellular cal-cium mobilization, by depleting intracellular stores (1), waspurchased from Sigma. 2-APB, which inhibits IP3 receptor-dependent calcium stores (2), was purchased from CaymanChemical. SQ, an inhibitor of AC (3), was purchased fromSigma. 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 dis-sected striatal tissue of adult rats (500 �g) were incubated(overnight at 4 °C) with 5 �g of rabbit anti-D2 (Chemicon) or ratanti-D1 (Sigma). After 1-h incubation with protein G/A-agarosesolution (Thermo Scientific), the immunoprecipitates werewashed three times with RIPA buffer, and 50 �L of SDS bufferwas added for 30 min at room temperature. The proteins wereresolved by electrophoresis and then transferred onto PVDFmembranes for Western blot analysis. Immunoblots were probedby using a monoclonal D1 receptor antibody (Sigma) or D2antibody (Chemicon), diluted to 1:1,000. The appropriate HRP-conjugated secondary antibodies were used at 1:1,000 to revealthe bands.
Immunocytochemistry. The primary antibodies used were: mouseanti-MAP2 (Abcam; 1:500), rat anti-D1 (Sigma; 1:400), rabbitanti-D2 (Chemicon; 1:400), mouse anti-Gq/11 (Santa Cruz;1:400), and mouse anti-BDNF (Chemicon; 1: 400). The second-ary antibodies conjugated to fluophores (Alexa Fluor 488; AlexaFluor 350, Alexa Fluor 568, Alexa Fluor 647) were all purchasedfrom Molecular Probes and used at 1: 500.
Paraformaldehyde-fixed neurons or floating brain sectionswere incubated with the primary antibodies overnight at 4 °C.After three washes with PBS-Tween20, the samples were incu-bated with the appropriate secondary antibody for 2–4 h at roomtemperature. After three washes, the slides were mounted byusing a mounting solution (Dako), and the images were acquiredby using a confocal Fluoview Olympus microscope (FV 1000).All images were acquired in sequential mode to minimize anybleed-through.
Neuronal Cultures. Neonatal rat striata (1 day of age) weretrypsinized in Hanks’ balanced salt solution (HBSS) with 0.25%trypsin and 0.05% DNase (Sigma) at 37 °C, and cells werewashed three times in HBSS with 12 mM MgSO4. Cells weredissociated in DMEM with 2 mM glutamine and 10% FBS andplated at 2 � 105 cells per poly-L-lysine-coated well (Sigma; 50�g/mL). The next day, media were changed to Neurobasalmedium with 50X B27 Supplement and 2 mM glutamine (In-vitrogen). On day 3 of culture, 5 �M cytosine arabinoside wasadded to inhibit glial cell proliferation. Half of the medium waschanged every 3 days.
Transfection Procedure. Neonatal striatal neurons in culture for7–21 days were transfected by using a combination of twoproducts: Effectene (Qiagen) and Exgen 500 (Fermentas). Usedindividually, these products gave restricted transfection effi-ciency (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, Effectenereagent was added and mixed. Ten minutes later, 6 mL of culturemedia was added to the mixture, which was split into 24 wells byremoving 250 �L of medium from each well and replacing it with250 �L of the transfection mixture.
Animals. Sprague–Dawley rats (Charles River), weighing 200–250 g, were housed paired in polyethylene cages in a colony roommaintained on a 12-h light–dark cycle with free access to foodand water. All treatments were performed during the light phaseof the day–night cycle. Animals were housed and tested incompliance with the guidelines described in the Guide to theCare and Use of Experimental Animals (Canadian Council onAnimal Care, 1993). SKF 83959 and SKF 83822 (Tocris Bio-science) were dissolved in physiological saline containing 5%DMSO.
Rats were injected s.c. (1.0 mL/kg) once daily for 3 days withsaline, SKF 83959, or SKF 83822 (0.4 mg/kg). One hour after thefinal injection rats were anesthetized with pentobarbital andperfused with ice-cold 4% paraformaldehyde. Brains were re-moved, fixed in 10% sucrose for 2 h followed by 20% sucrose in0.1 M PBS for 5 days, frozen in Isopentane (�60 °C), and storedat �80 °C until cryostat sectioning. Serial coronal sectionsthrough the striatum (bregma 1.5 mm) were cut at 10- or 30-�mthickness. Free-floating sections were prepared with the EliteABC kit (Vector Laboratories) and incubated with primaryantibody for BDNF (1:500; Chemicon) for 24 h at room tem-perature. Images were obtained with an Axioplan2 microscope(Zeiss). Cells were counted within similar 400-�m2 areas withinthe caudate putamen, nucleus accumbens core, and shell re-gions.
Confocal Microscopy FRET and Data Processing. Dopamine D1–D2receptor interaction was assessed previously by coimmuprecipi-tation studies performed from rat brain (6). To further charac-terize this interaction we used FRET methodology. Paraform-aldehyde-fixed striatal neurons or floating sections (10 �m) fromrat brain were incubated for 24 h at 4 °C with primary antibodiesraised against D1 and D2 receptors (6) and the species-specificsecondary antibodies conjugated to Alexa Fluor 488 and AlexaFluor 350 dyes, respectively. The primary antibodies have beenshown to be highly specific to D1 or D2 receptors using HEKcells expressing individual D1, D2, D3, D4 or D5 receptors (6).They were further validated by immunohistochemistry showinglack of reactivity in slices from D1�/� and D2�/� mice. TheD2-Alexa Fluor 350 was used as a donor dipole, while D1-AlexaFluor 488 was used as acceptor dipole. The images were acquiredwith an Olympus Fluoview FV 1000 laser scanning confocalmicroscope with a 60�/1.4 NA objective. The donor was excitedwith a krypton laser at 405 nm, while the acceptor was excitedwith an argon laser at 488 nm. The emissions were collected at430/20-nm and 530-nm LP filter. Other FRET pairs (488–568and 568–647) were tested and showed comparable results.Eleven images were acquired for each FRET analysis (Table S1and Table S2) in accordance with the algorithm described byChen et al. (7).
Hasbi et al. www.pnas.org/cgi/content/short/0903676106 1 of 10
1. Ghosh TK, Bian J, Short AD, Rybak SL, Gill DL (1991) Persistent intracellular calcium pooldepletion by thapsigargin and its influence on cell growth. J Biol Chem 266:24690–24697.
2. Peppiatt CM, et al. (2003) 2-Aminoethoxydiphenyl borate (2-APB) antagonizes inositol1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps, and has a use-dependent and slowly reversible action on store-operated calcium entry channels. CellCalcium 34:97–108.
3. Harris DN, Asaad MM, Phillips MB, Goldenberg HJ, Antonaccio MJ (1979) Inhibition ofadenylate cyclase in human blood platelets by 9-substituted adenine derivatives. JCyclic Nucleotide Res 5:125–134.
4. Smith RJ, et al. (1990) Receptor-coupled signal transduction in human polymorpho-nuclear neutrophils: Effects of a novel inhibitor of phospholipase C-dependent pro-cesses on cell responsiveness. J Pharmacol Ther 253:688–697.
5. Takasaki J, et al. (2004) A novel G�q/11-selective inhibitor. J Biol Chem 279:47438–47445.
6. Lee SP, et al. (2004) Dopamine D1 and D2 receptor coactivation generates a novelphospholipase C-mediated calcium signal. J Biol Chem 279:35671–35678.
7. Chen Y, Elangovan M, Periasamy A (2005) FRET data analysis: The algorithm. MolecularImaging: FRET Microscopy and Spectroscopy, eds Periasamy A, Day RN (Oxford UnivPress, New York), pp 126–145.
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FRETD1(Alexa 568) Distance
6.6136.6130.2030.20321219.5269.5260.0220.02220209.9989.9980019191010001818
5.8145.8140.2650.26517176.5056.5050.2030.20316166.516.510.2030.20315159.2079.2070.0370.03714149.4329.4320.0290.02913139.7189.7180.0120.01212129.0949.0940.0510.05111119.1129.1120.0410.04110107.1847.1840.1530.153997.8777.8770.120.12887.0587.0580.1790.179776.0286.0280.2560.256666.2656.2650.2230.223555.9945.9940.2410.241447.0137.0130.1790.179335.9625.9620.2360.236226.885 6.885 0.1950.19511
Distance between donor and Distance between donor and acceptoracceptorFRET EfficiencyFRET Efficiency
AverageAverageAverageAverageROIROI
6.6136.6130.2030.20321219.5269.5260.0220.02220209.9989.9980019191010001818
5.8145.8140.2650.26517176.5056.5050.2030.20316166.516.510.2030.20315159.2079.2070.0370.03714149.4329.4320.0290.02913139.7189.7180.0120.01212129.0949.0940.0510.05111119.1129.1120.0410.04110107.1847.1840.1530.153997.8777.8770.120.12887.0587.0580.1790.179776.0286.0280.2560.256666.2656.2650.2230.223555.9945.9940.2410.241447.0137.0130.1790.179335.9625.9620.2360.236226.885 6.885 0.1950.19511
Distance between donor and Distance between donor and acceptoracceptorFRET EfficiencyFRET Efficiency
AverageAverageAverageAverageROIROI
D1 (Alexa 488) D2 (Alexa 568) Merge
D2(Alexa 647)
a
b
c A BCPu NAc
43
95
72
130
55
170
95
72
4355
43
95
72
55
Str
43
9572
34
55
Mock
Str
Mock
Str
WB: anti-D1R anti-D2R
CPu: Caudate PutamenNAc: Nucleus AccumbensStr :Striatum
IP: anti-D2R anti-D1RWB: anti-D1R anti-D2R
Str :Striatum
CPu NAc
43
95
72
130
55
170
95
72
4355
43
95
72
55
Str
43
9572
34
55
Mock
Str
Mock
Str
WB: anti-D1R anti-D2R
CPu: Caudate PutamenNAc: Nucleus AccumbensStr :Striatum
IP: anti-D2R anti-D1RWB: anti-D1R anti-D2R
Str :Striatum
CPu NAc
43
95
72
130
55
170
95
72
4355
43
95
72
55
Str
43
9572
34
55
Mock
Str
Mock
Str
WB: anti-D1R anti-D2R
CPu: Caudate PutamenNAc: Nucleus AccumbensStr :Striatum
IP: anti-D2R anti-D1RWB: anti-D1R anti-D2R
Str :Striatum
CPu NAcCPu NAc
43
95
72
130
55
170
43
95
72
130
55
170
95
72
4355
43
95
72
55
43
95
72
130
55
170
Str
43
9572
34
55
Mock
Str
Mock
Str
WB: anti-D1R anti-D2R
CPu: Caudate PutamenNAc: Nucleus AccumbensStr :Striatum
IP: anti-D2R anti-D2RWB: anti-D1R anti-D2R
Str :Striatum
IgGIgG
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 usinga specific anti-D2 antibody and a secondary antibody conjugated to Alexa Fluor 568. Similar results were obtained with other flurophore pairs (Alexa Fluor350-Alexa Fluor 488; Alexa Fluor 568-Alexa Fluor 647). Note the colocalization at the cell surface and proximal dendrites and the localization of D2 receptorsin cytosol. (b) Example of colocalization and confocal FRET analysis of the interaction between D1 and D2 receptors in rat striatal neurons. Anti-D1-Alexa Fluor568 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, andcalculation of distances separating the pair, using an algorithm as described (7). ROIs within neurons coexpressing both D1 and D2 receptors were analyzed, tocalculate 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 blotsof D1R and D2R from rat striatum are shown. (B) Coimmunoprecipitation of D1 with D2-specific antibody and coimmunoprecipitation of D2 with D2-specificantibody from the rat striatum are shown. A nonspecific band of 95–100 kDa appearing in the immunoprecipitation samples represents IgG.
Hasbi et al. www.pnas.org/cgi/content/short/0903676106 3 of 10
Fig. S2. (a) Dose-dependent effect of quinpirole on intracellular calcium mobilization, measured by using cameleon Y6.1 FRET and live cell confocal microscopyin rat striatal neurons. Results are mean � SEM (n � 21). (b) Effects of 100 nM SKF 83959 alone (n � 20) or in combination with 100 nM quinpirole (n � 33) onintracellular calcium increase. Results are mean � SEM. (c) Comparison of the effects of SKF 83822 (n � 43) and SKF 83959 (n � 20) on intracellular calciummobilization in rat striatal neurons. Results are mean � SEM. (d) Intracellular calcium measurements after treatment with SKF 83959 (100 nM, n � 23 cells) inD5�/� 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 neuronspreincubated without (Dop; n � 24) or with thapsigargine (Dop � TPG; n � 26). Results are mean � SEM.
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0
10
20
30
40
50
60
70
80
Control 2 min 5 min
-60
-50
-40
-30
-20
-10
0
100 nM Dopamine (Time)
plasm
a mem
brane
Gq f
luores
cenc
e (%
over
basa
l)Cy
tosoli
c Gq f
luores
cenc
e (%
beyo
nd ba
sal)
5 min2 minControl
Control 2 min 5 min0
25
50
75
100Plasma MembraneCytosol
Dopamine (100 nM)
% o
f to
tal
flu
ore
scen
cea
b
Fig. S3. (A) Quantification of Gq/11 immunofluorescence at plasma membrane and cytosol after treatment with vehicle (control, 0 min) or SKF 83959 (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 treatedwith vehicle (control) or with 100 nM dopamine for 2 or 5 minutes. Results are mean �SEM of three independent experiments.
Hasbi et al. www.pnas.org/cgi/content/short/0903676106 5 of 10
Control 5 min2 min
Control
SKF 83959 (2 hr)
a
b
d
BDNF in WT (SKF 83959, 2 hrs)
BDNF in D-/- (SKF 83959, 2 hrs)
BDNF in WT (Control)
pCaMKII5 minRacl + SKF 89359: 5 min SKF89359:
c
Fig. S4. (A) Representative example of CaMKII� activation in D5�/� mice-derived striatal neurons treated with vehicle (control) or 100 nM SKF 83959 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 expressioninduced by 100 nM SKF 83959. (C) Confocal microscopy immunofluorescence of BDNF expression levels in D5�/� mice-derived striatal neurons treated with vehicle(control) or 100 nM SKF 83959 for 5 minutes (Upper Left) and 2 hours (Lower Left). DAPI staining of nuclei is shown in the Right frames. (D) Confocal microscopyimmunofluorescence of BDNF expression levels in D1�/� mice-derived striatal neurons treated with 100 nM SKF 83959 for 2 hours.
Hasbi et al. www.pnas.org/cgi/content/short/0903676106 6 of 10
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 83959 (Center) or 500 nM dopamine (Right). The nuclei were stained with DAPI (blue), and MAP2 was detected by using a specific anti-MAP2antibody and a secondary antibody conjugated to Alexa Fluor 488. (b) Effect of 10 �M raclopride on SKF 83959-triggered MAP2 production and striatal neurongrowth. (c) Immunofluorescence of MAP2 in D5�/� mice-derived striatal neurons treated with 10 nM SKF 83959 as described in a. (d) Immunofluorescence ofMAP2 in D1�/� mice-derived striatal neurons treated with 10 nM SKF 83959 as described in a.
Hasbi et al. www.pnas.org/cgi/content/short/0903676106 7 of 10
Fig. S6. Examples of FRET analysis methodology to measure the energy transfer and the distance separating endogenous dopamine D1 and D2 receptors insitu 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 acceptordipole. Eleven images were taken to generate images of pFRET, and distances separating the receptor pair, using an algorithm (7). ROIs within neuronscoexpressing both D1 and D2 receptors were analyzed, and the data, representing average pFRET, FRET efficiency (E), and distance between donor and acceptorare shown.
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Table S1. Control for FRET data analysis (images a–g)
SymbolFluorophore
or sampleExcitation
wavelengthEmission
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)
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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 BAcceptor/donor h IDonor/acceptor j CAcceptor/acceptor dDonor/double e fAcceptor/double k g
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