3h sch23390

Manufactured by PerkinElmer

Sourced in United States, Germany, France

[3H]SCH23390 is a high-affinity and selective dopamine D1 receptor radioligand. It is commonly used in receptor binding assays to study the distribution and pharmacology of dopamine D1 receptors in the central nervous system.

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Lab products found in correlation

3h raclopride, by PerkinElmer (4 mentions) 3h spiperone, by PerkinElmer (3 mentions) Gtpγs, by Merck Group (3 mentions) Butaclamol, by Merck Group (3 mentions) Hygromycin, by Merck Group (2 mentions) Fluphenazine, by Merck Group (2 mentions) Tetracycline, by Merck Group (2 mentions) Blasticidin, by Merck Group (2 mentions) 3h methylspiperone, by PerkinElmer (2 mentions) 35s gtpγs, by PerkinElmer (2 mentions) Cell culture reagents, by Thermo Fisher Scientific (2 mentions) Ascorbic acid, by Merck Group (2 mentions) 3h ym 09151 2, by PerkinElmer (2 mentions) 3h mazindol, by PerkinElmer (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Isobutylmethylxanthine, by Merck Group (1 mentions) 3h camp, by PerkinElmer (1 mentions) Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions) Pramipexole, by Bio-Techne (1 mentions) Pd128907, by Bio-Techne (1 mentions)

18 protocols using 3h sch23390

Dopamine Receptor Ligand Binding Protocol

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ATR was purchased from Chem Service (West Chester, PA, USA). Reagents for western blotting were obtained from BioRad (Hercules, CA, USA). [³H]-SCH23390 (dopamine D1-like receptor antagonist) and [³H]-Spiperone (dopamine D2-like receptor antagonist) were purchased from Perkin-Elmer (Boston, MA, USA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.

Márquez-Ramos J.A., Hernández-Plata I., Díaz-Muñoz M, & Rodríguez V.M. (2017). The Hypoactivity Associated with the Repeated Exposure to Atrazine Is Related to Decreases in the Specific Binding to D1-DA Receptors in the Striatum of Rats. Journal of Toxicology, 2017, 2169212.

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Receptor Binding Density Quantification

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The receptor binding density for D1 and D2 receptors was adapted from the protocols described in Sommer et al. (31 (link)) and Behuet et al.’s study (32 (link)). The tritiated ligands [³H]-SCH23390 and [³H]-Raclopride were purchased from PerkinElmer (Germany). SCH23390 is a potent D1 and D5 receptor antagonist. Raclopride is a D2 and D3 selective receptor antagonist. Both ligands bind to the receptors localized on the cell surface, without penetrating inside the cell membrane. In the first step, the pre-incubation, endogenous ligands were washed off. In the following main incubation, the tritiated ligands were incubated both in the presence of a competitor, in order to determine the unspecific binding, and without it, in order to assess the total binding. Finally, the slices were rinsed. The slices incubated with [³H]-Raclopride were additionally dried with a cold air stream. A detailed description of the protocols used is reported in Table 1.

Chhabra S., Nardi L., Leukel P., Sommer C.J, & Schmeisser M.J. (2023). Striatal increase of dopamine receptor 2 density in idiopathic and syndromic mouse models of autism spectrum disorder. Frontiers in Psychiatry, 14, 1110525.

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Radioligand binding assay for dopamine receptors

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[³H]Methylspiperone (85.5 Ci/mmol), [³H]SCH23390 (73.1 Ci/mmol), [³H]cAMP (25 Ci/mmol) and [³⁵S]GTPγS (1250 Ci/mmol) were purchased from PerkinElmer (Boston, MA). Tetracycline, hygromycin, blasticidin, DA, (+)-butaclamol, fluphenazine, phenylmethylsulfonyl fluoride (PMSF), GDP, GTPγS, cAMP, isobutylmethylxanthine and ascorbic acid were obtained from Sigma-Aldrich (St. Louis, MO). G418 was obtained from Gemini Bio-Products (West Sacramento, CA), while cell culture reagents were obtained from Invitrogen (Carlsbad, CA). (−)-L-SPD was generously provided by Dr. David Y.W. Lee from the Bio-Organic and Natural Products Laboratory at McLean Hospital (Harvard Medical School, Belmont, MA).

Hicks C., Huang P., Ramos L., Nayak S.U., Caro Y., Reitz A.B., Smith G.R., Lee D.Y., Rawls S.M, & Liu-Chen L.Y. (2018). Dopamine D1-like receptor agonist and D2-like receptor antagonist (−)-Stepholidine reduces reinstatement of drug-seeking behavior for 3,4-methylenedioxypyrovalerone (MDPV) in rats. ACS chemical neuroscience, 9(6), 1327-1337.

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Radioligand Binding Assays for Dopamine Receptors

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[³H]Methyl spiperone (85.5 Ci/mmol), [³H]SCH23390 (73.1 Ci/mmol), and [³⁵S]GTPγS (1250 Ci/mmol) were purchased from PerkinElmer (Boston, MA). Tetracycline, hygromycin, blasticidin, dopamine (DA), (+) butaclamol, fluphenazine, phenylmethylsulfonyl fluoride (PMSF), GDP, GTPγS, and ascorbic acid were obtained from Sigma-Aldrich (St. Louis, MO). G418 was obtained from Gemini Bio-Products (West Sacramento, CA); cell culture reagents were obtained from Invitrogen (Carlsbad, CA); and protease inhibitor cocktail, phosphatase inhibitor cocktail, and lysis buffer were obtained from Thermo Scientific (Rockford, IL). All test molecules (Table 1) were either synthesized in-house as described below or purchased from vendors. Asinex Corp. (Winston-Salem, NC): SK603, SK609, SK610, SK611; Enamine, Ltd. (Kiev, Ukraine): Compounds described in Lichtenberger patent²⁸ L1 and L2; Asiba Pharmatech, Inc. (Milltown, NJ): R- and S-enantiomers of SK609. DA and D2R/D3R agonists PD128907 and pramipexole were purchased from Tocris Bioscience (Bio-Techne, Inc., CA).

Xu W., Wang X., Tocker A.M., Huang P., Reith M.E., Liu-Chen L.Y., Smith AB I.I.I, & Kortagere S. (2016). Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists. ACS chemical neuroscience, 8(3), 486-500.

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Quantifying Dopamine Receptor Changes in Amphetamine-Treated Rats

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Animals (N = 12 AMPH pre-treated, N = 12 saline pre-treated) were sedated with isoflurane and euthanized by cervical dislocation and the brains were rapidly removed, snap frozen and stored at -80°C. Frontal brain sections (14 μm) were cut and DAT and DRD1 autoradiography was performed on the CPu and NAcc. [³H]WIN35428 (Perkin-Elmer^®, France; specific radioactivity = 3.034 MBq/nmol; 5 concentrations from 0.55 to 15.0 nM) was used for the DAT binding experiments according to protocols described before by Hebert [24 (link)]. Non-specific binding was determined by incubation of adjacent brain slices in the presence of 10 μM nomifensine. For the DRD1 binding experiments, [³H]SCH-23,390 (Perkin-Elmer^®, France; specific radioactivity = 3.119 MBq/nmol; 5 concentrations from 0.10 to 8.1 nM) was used and performed according to the Savasta protocol [25 (link)]. Non-specific binding was determined by incubation of adjacent brain slices in the same conditions and in the presence of 10 μM SKF38393. Brain sections were exposed to tritium-sensitive phosphor imaging plates (Perkin-Elmer^®) before acquisition of images (Cyclone^®, Perkin-Elmer^®). Specific binding was calculated as the difference between total and non-specific binding and K_d and B_max values were derived from raw data using nonlinear fitting procedures (Prism^®).

Schrantee A., Tremoleda J.L., Wylezinska-Arridge M., Bouet V., Hesseling P., Meerhoff G.F., de Bruin K.M., Koeleman J., Freret T., Boulouard M., Desfosses E., Galineau L., Gozzi A., Dauphin F., Gsell W., Booij J., Lucassen P.J, & Reneman L. (2017). Repeated dexamphetamine treatment alters the dopaminergic system and increases the phMRI response to methylphenidate. PLoS ONE, 12(2), e0172776.

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Radioligand Binding Assay Protocol

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Dopamine hydrochloride and L-(-)-norepinephrine (+)-bitartrate salt monohydrate were purchased from Sigma. (-) quinpirole hydrochloride, clonidine hydrochloride, 7-OH-PIPAT maleate, RO-105824 dihydrochloride, RX821002 and yohimbine hydrochloride were purchased from Tocris. [³H]RX821002 (63.9 Ci/mmol), [³H]SCH 23390 (81.9 Ci/mmol) and [³H]YM-09151-2 (84.4 Ci/mmol) were from Perkin-Elmer. Pertussis toxin was purchased from Sigma.

Sánchez-Soto M., Casadó-Anguera V., Yano H., Bender B.J., Cai N.S., Moreno E., Canela E.I., Cortés A., Meiler J., Casadó V, & Ferré S. (2018). α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands. Molecular neurobiology, 55(11), 8438-8454.

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G9a Inhibitor Compound Evaluation

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UCL-2190^{41 (link)}, Ciproxifan⁴² and Pitolisant^{27 (link)} were from own laboratory stocks of which synthesis and analytics have been described previously. G9a-inhibitors A-366 and UNC-0642 as well as G9a enzyme, S-adenosylmethionine (SAM, (2S)-2-amino-4-[[(2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl-methylsulfonio]butanoate), biotinylated histone H3 (1–21) fragment and Dulbecco´s modified eagle medium (DMEM, article no. D5671) were purchased from Sigma-Aldrich, Taufkirchen, Germany. Fetal bovine serum albumin (FBS Good-Forte) and Dulbecco´s Phosphate Buffered Saline (DPBS) were provided by PAN biotech (Aidenbach, Germany). The radioligands [³H]N^α-methylhistamine, [³H]histamine, [³H]spiperone and [³H]SCH23390 were purchased from PerkinElmer (Rodgau, Germany) as well as AlphaLISA materials such as AlphaLISA H3K9me2 acceptor beads, streptavidin-coated donor beads, detection buffer (5x) and white 384-well microplates (OptiPlate). Human or animal blood/tissue/cell samples have not been used in this study.

Reiner D., Seifert L., Deck C., Schüle R., Jung M, & Stark H. (2020). Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader–Willi Syndrome. Scientific Reports, 10, 13558.

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Receptor Quantification in Substantia Nigra, VTA, and Striatum

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The DAT and D2 receptors (D2R) were quantified in the subtantia nigra pars compacta (SNc) and ventral tegmental area (VTA) (DAT: n = 6 per group; D2R: n = 5 and 6 for control and MPH rats, respectively) and in the striatum (DAT: n = 6 and 8; D2R: n = 5 and 8 for control and MPH rats, respectively) using [¹²⁵I]-PE2I (prepared according to Chalon et al., 1999 ) and [³H]-YM-09151-2 (PerkinElmer), and the D1 receptors (D1R) were quantified in the striatum (n = 5 and 8 for control and MPH rats) using [³H]-SCH-23390 (PerkinElmer) using the methodology previously detailed by Chalon et al. (1999) and Bouchez et al. (2008) (link). For more details, see the Supplementary Materials section.

Lepelletier F.X., Tauber C., Nicolas C., Solinas M., Castelnau P., Belzung C., Emond P., Cortese S., Faraone S.V., Chalon S, & Galineau L. (2015). Prenatal Exposure to Methylphenidate Affects the Dopamine System and the Reactivity to Natural Reward in Adulthood in Rats. International Journal of Neuropsychopharmacology, 18(4), pyu044.

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Quantifying Dopamine D1 Receptor Binding

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Autoradiography was performed as previously described to quantify dopamine type 1-like receptor (D1) binding [40 (link)–44 (link)]. Briefly, slides were pre-incubated for 60 min at room temperature in 50 mM Tris HCl buffer. Then, 2.5 nM of [3H] SCH 23390 (specific activity = 85 Ci/mmol) and 40 nM of ketanserin (both acquired from PerkinElmer, Waltham, MA, USA) were added to the re-incubation buffer, and the slides were incubated for an additional 60 min at room temperature. Non-specific binding was determined in the presence of 1 μM of flupenthixol (Sigma-Aldrich, St. Louis, MO, USA). Slides were washed 2× for 5 min in pre-incubation buffer, followed by a brief immersion in dH2O, all at 4 °C. This experiment was performed following a protocol adapted from [45 (link)].

Richardson B.J., Hamilton J., Roeder N., Thanos K.Z., Marion M, & Thanos P.K. (2023). Fatty acid-binding protein 5 differentially impacts dopamine signaling independent of sex and environment. Addiction neuroscience, 8, 100118.

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Radiolabeling D1 and D2 Receptors

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D1 and D2 receptors were radiolabeled as previously described (Hersi et al., 1995 (link)). Briefly, slides were incubated with ligands diluted in Tris buffer at room temperature for 60 minutes. For D1 receptor, total radioligand binding was determined using [³H]-SCH 23390 (1 nM; Perkin Elmer, Waltham, MA, USA). To measure the degree of non-specific binding, the contiguous sections were incubated in the same buffer, but supplemented with 1 µM unlabeled SCH23390 (Sigma-Aldrich, St. Louis, MO, USA). D2 receptor binding was determined using [³H]-raclopride (2.85 nM; Perkin Elmer). Non-specific binding of the D2 radioligand was assessed in adjacent sections that were incubated in the same buffer supplemented with 1 µM (+)-butaclamol (Sigma-Aldrich). Test cassettes containing slices from each age and genotype, each with its own [³H] standard, were used to ensure proper exposure time and signal strength. Representative autoradiograms are shown in Fig. 1.

Pokinko M., Grant A., Shahabi F., Dumont Y., Manitt C, & Flores C. (2017). Dcc Haploinsufficiency Regulates Dopamine Receptor Expression Across Postnatal Lifespan. Neuroscience, 346, 182-189.

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