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125i rti 55

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[125I]RTI-55 is a radiotracer that can be used to label and detect dopamine transporters in research studies. It is a selective and potent ligand for the dopamine transporter.

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Lab products found in correlation

Nomifensine, by Merck Group (3 mentions) Fluoxetine, by Bio-Techne (2 mentions) Standard 125i microscale slides, by American Radiolabeled Chemicals (2 mentions) 3h 8 oh dpat, by PerkinElmer (1 mentions) 3h 5 ht, by PerkinElmer (1 mentions) Ip 1 elisa kit, by PerkinElmer (1 mentions) 25i nboh, by Cayman Chemical (1 mentions) 35s gtpγs, by PerkinElmer (1 mentions) Cryostat, by Leica (1 mentions) Biomax mr, by Kodak (1 mentions) Superfrost plus slides, by Avantor (1 mentions) Cryocut 1800, by Leica (1 mentions) Mr film, by Kodak (1 mentions) Perfection v750 pro, by Epson (1 mentions) Scion image for windows, by Techcomp Instruments (1 mentions) Fluoxetine hydrochloride, by Eli Lilly (1 mentions) Nisoxetine, by Bio-Techne (1 mentions) 3h dopamine, by PerkinElmer (1 mentions) 3h serotonin, by PerkinElmer (1 mentions) Fluvoxamine, by Bio-Techne (1 mentions)

Spelling variants of this product

Iodinated 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([125I]RTI-55; 2200 Ci/mmol) (2 citations)

11 protocols using 125i rti 55

1

Pharmacological Characterization of NBOMes

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25D-NBOMe, 25E-NBOMe, 25H-NBOMe, 25I-NBOH and 25N-NBOMe were purchased from Cayman Chemicals (Ann Arbor, MI). (+)LSD(-)tartrate, (-)DOM, (-)cocaine, and S(+)METH were provided by the National Institute on Drug Abuse Drug Supply Program (Rockville, MD). [3H]8-OH-DPAT, [125I]2,5-dimethoxy-4-iodoamphetamine (DOI), [125I]RTI-55, [3H]DA, [3H]5-HT, [3H]NE and [35S]GTPγS were purchased from Perkin Elmer Life and Analytical Sciences (Boston, MA). The IP-1 Elisa kit was purchased from Cisbio (Bedford, MA). Other reagents were purchased from Sigma (St. Louis, MO).
Eshleman A.J., Wolfrum K.M., Reed J.F., Kim S.O., Johnson R.A, & Janowsky A. (2018). Neurochemical pharmacology of psychoactive substituted N-benzylphenethylamines: high potency agonists at 5-HT2A receptors. Biochemical pharmacology, 158, 27-34.
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2

Quantifying Dopamine Transporter Density

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On postnatal day 62, brains from all rats were collected via rapid decapitation. Once removed from the skull, the brain was hemidissected, and the left hemisphere was frozen in powdered dry ice and stored at −80° C until further processing. The left hemisphere was sliced on a Leica cryostat into a series of 16 μm thick sagittal sections. [125I]-RTI-55 (Perkin-Elmer Life Sciences, Boston, MA, USA) was used to detect dopamine transporter sites and remaining autoradiography was performed as described by van Bregt et al., (2012) (link). The optical density of regions within the medial striatum and frontal cortex, as shown in Figure 2, and roughly analogous to plate # 81 of Paxinos and Watson (1986) , was quantified using ImageJ software (v. 2.0). Raters were blind to the treatment status of each brain. Mean uncalibrated optical density was calculated for each individual region from a series of nine contiguous sections. Non-specific binding was determined by measuring the optical density of a similarly sized area located within the corpus callosum just posterior to the sampled striatal sites (Figure 2). This value was subtracted from all density measures, and the product was used for statistical analyses.
Bardgett M.E., Downnen T., Crane C., Baltes Thompson E.C., Muncie B., Steffen S.A., Yates J.R, & Pauly J.R. (2020). Chronic risperidone administration leads to greater amphetamine-induced conditioned place preference.. Neuropharmacology, 179, 108276.
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3

Dopamine Transporter Binding Assay

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Frozen brains were cryostat cut in 12 μm sections and mounted on SuperFrost microscope slides (Gerhard Menzel GmbH, Braunschweig, Germany) for DAT binding by autoradiography. Sections were preincubated in 50 mM Tris–HCl/120 mM NaCl (pH 7.5) for 20 min, and then incubated in binding buffer with 50 pM [125I] RTI-55 (Perkin-Elmer Life Sciences, Boston, United States) with 1 μM fluoxetine (Tocris Bioscience, Bristol, United Kingdom) for 60 min. 100 μM nomifensine (Sigma-Aldrich) was added to the assay to determine non-specific binding. The slides were washed in ice-cold binding buffer (2 × 10 s) and in deionized water, dried, and exposed to Kodak Biomax MR film (Sigma-Aldrich). Autoradiograms from ligand binding autoradiography were digitized using a Dia-Scanner (Epson Perfection 4870 PHOTO). Optical density values were measured using Image J (1.52 h, Wayne Rasband National Institutes of Health, United States).
Zhang X., Mantas I., Fridjonsdottir E., Andrén P.E., Chergui K, & Svenningsson P. (2020). Deficits in Motor Performance, Neurotransmitters and Synaptic Plasticity in Elderly and Experimental Parkinsonian Mice Lacking GPR37. Frontiers in Aging Neuroscience, 12, 84.
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4

Quantifying DAT Binding in METH-Pretreated Rats

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Upon completion of FSCV recording, animals were sacrificed, brains were rapidly removed and frozen in 2-methyl butane on dry ice, and tissue samples were stored at −80 °C until processing for DAT binding. Brains were sectioned coronally (12 μm) using a cryostat (Cryocut 1800; Leica, Wetzlar, Germany), and sections were thaw-mounted onto Superfrost Plus slides (VWR, Aurora, CO) and then stored at −20 °C until needed. Striatal DAT levels were determined by [125I]RTI-55 (PerkinElmer, Waltham, MA) binding, as reported previously (Boja et al. 1992 (link); O’Dell et al. 2011 (link); Pastuzyn et al. 2012 (link); Son et al. 2013 (link)). The slides were apposed to film (Biomax MR; Eastman Kodak, Rochester, NY, USA) for 24 h and subsequently developed. Images were digitized and average gray values of the DM and DL striata were determined using ImageJ software (NIH, Bethesda, MD, USA). This densitometric analysis was performed in one hemisphere of striatal sections +1.6 and +0.7 mm from bregma. DAT binding was normalized across slides by subtracting the average gray value of the corpus callosum from the values for DM and DL striatum (background subtraction) for each section. DAT binding in METH-pretreated rats was then converted to percent of average value per brain region in saline-pretreated rats.
Robinson J.D., Howard C.D., Pastuzyn E.D., Byers D.L., Keefe K.A, & Garris P.A. (2014). METHAMPHETAMINE-INDUCED NEUROTOXICITY DISRUPTS PHARMACOLOGICALLY EVOKED DOPAMINE TRANSIENTS IN THE DORSOMEDIAL AND DORSOLATERAL STRIATUM. Neurotoxicity research, 26(2), 152-167.
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5

Dopamine Transporter Density Quantification

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DAT density was assessed via 125I-RTI-55 binding to striatal core slices as previously described (O'Dell et al., 2012 ). Briefly, slides were thawed on a slide warmer (5-10 min) and pre-incubated in buffer-sucrose (10 mM sodium phosphate, 120 mM sodium chloride, 320 mM sucrose, pH 7.4) containing 100 nM fluoxetine at room temperature for 5 min. Slides were then incubated for 2 h in buffer-sucrose containing 25 pM 125I-RTI-55 (2200 Ci/mmol, PerkinElmer, Watham, MA). In a previous study conducted in the same laboratory, nonspecific binding was demonstrated by slides incubated in buffer-sucrose containing 25 pM 125I-RTI-55, 100 nM fluoxetine, and 100 μM nomifensine (Vieira-Brock et al., 2015 ). Slides were rinsed twice in ice-cold buffer and distilled water for 2 min and air-dried. Sample slides and standard 125I microscale slides (American Radiolabeled Chemicals, St. Louis, MO) were exposed to Kodak MR film (Easterman Kodak Co., Rochester, NY, USA) for 24 hours.
Baladi M.G., Nielsen S.M., McIntosh J.M., Hanson G.R, & Fleckenstein A.E. (2016). Prior nicotine self-administration attenuates subsequent dopaminergic deficits of methamphetamine in rats: Role of nicotinic acetylcholine receptors. Behavioural pharmacology, 27(5), 422-430.
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6

Serotonin Transporter Density Quantification

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SERT density was assessed via [125I]RTI-55 binding to dorsal hippocampus and PRh slices as previously described (O’Dell et al., 2012 (link)). Briefly, slides were thawed on a slide warmer (5–10 minutes) and incubated in sucrose buffer (10mM sodium phosphate, 120mM sodium chloride, 320mM sucrose, pH 7.4) containing 21 pM 125I-RTI-55 (2200 Ci/mmol, PerkinElmer, Waltham, MA). Nonspecific binding was determined by slides incubated in sucrose buffer containing 21 pM [125I]RTI-55 and 100nM fluoxetine. Slides were rinsed twice in ice-cold buffer and distilled water for 2 minutes and air-dried. Sample slides and standard 125I microscale slides (American Radiolabeled Chemicals, St. Louis, MO) were placed on 1 cassette and exposed to same Kodak MR film (Eastman Kodak Co., Rochester, NY) for 24 hours to keep variables constant.
Vieira-Brock P.L., McFadden L.M., Nielsen S.M., Smith M.D., Hanson G.R, & Fleckenstein A.E. (2015). Nicotine Administration Attenuates Methamphetamine-Induced Novel Object Recognition Deficits. International Journal of Neuropsychopharmacology, 18(12), pyv073.
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7

Autoradiographic Detection of DAT

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Fresh frozen sections mounted on Superfrost slides were used for autoradiographic detection of DAT. Sections were pre-incubated in binding buffer (50 mM Tris–HCl/120 mM NaCl, pH 7.5) for 20 min. They were incubated in binding buffer with 50 pM [125I] RTI-55 (Perkin-Elmer Life Sciences) and 1 μM fluoxetine (Tocris Bioscience) for 60 min. For non-specific binding, 100 μM nomifensine (Sigma-Aldrich) was added to the assay. Lastly, sections were washed in an ice-cold binding buffer for 2 × 10 s and rapidly dipped in deionized water. When dried, sections were exposed to Kodak Biomax MR Film (Sigma-Aldrich) in a dark room. After 24 h, autoradiograms were digitized using a high-resolution scanner (Epson Perfection V750 PRO). Optical densitometry was done with Image J in grayscale in the same way as aforementioned.
He Y., Kaya I., Shariatgorji R., Lundkvist J., Wahlberg L.U., Nilsson A., Mamula D., Kehr J., Zareba-Paslawska J., Biverstål H., Chergui K., Zhang X., Andren P.E, & Svenningsson P. (2023). Prosaposin maintains lipid homeostasis in dopamine neurons and counteracts experimental parkinsonism in rodents. Nature Communications, 14, 5804.
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8

Quantification of Dopamine Transporter Binding

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After all the functional MRI scans were performed, animals were killed using CO2 and then decapitated, their brain dissected out and fresh frozen in isopentane chilled in dry ice. The brains were then sectioned (14 μm) with a cryostat. Slide mounted sections were preincubated in 50 mM Tris-HCl/120 mM NaCl (pH 7.5) for 20 min. Incubation in binding buffer (50 mM Tris–HCl/120 mM NaCl, pH 7.5/1 μM fluoxetine) was conducted with 50 pM [125I] RTI-55 (Perkin-Elmer Life Sciences, Boston, USA) for 60 min. For nonspecific binding, 100 μM nomifensine (Sigma Aldrich) was added to the assay. The slides were washed 2 × 10 s in ice-cold binding buffer, rapidly dipped in deionized water, dried, and exposed to autoradiographic films (BioMax MR, Merck Eurolab, Sweden) at − 20 °C for 7 days. The films were then developed (using Kodak D19 and Kodak Unifix; Kodak, Rochester, New York, USA). Autoradiograms were digitized using a Dia-Scanner (Epson Perfection 4870 PHOTO; Seiko Epson Corporation, Suwa, Japan), and optical density values were measured using Scion Image for Windows (version alfa 4.0.3.2; Scion Corporation).
Monnot C., Zhang X., Nikkhou-Aski S., Damberg P, & Svenningsson P. (2017). Asymmetric dopaminergic degeneration and levodopa alter functional corticostriatal connectivity bilaterally in experimental parkinsonism. Experimental Neurology, 292, 11-20.
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9

Quantifying Dopamine Transporter Binding

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DA transporter ligand binding was performed as reported by Burhans et al.,11 (link) and Andrews et al.,42 (link) [125I]-RTI-55 was purchased from Perkin Elmer (Boston, MA). The slides were incubated in a solution of [125I]-RTI-55 (1098.7 µCi/ml, 2200 Ci/mmol) and protease inhibitor cocktail (PIC) diluted in a phosphate buffer (50 mM NaH2PO4; 50 mM NaHPO4; pH 7.4) 1:10 for 90 min at 4°C. We added 10 µM fluoxetine hydrochloride (Eli Lilly, Indianapolis, IN) to block serotonin transporter binding. Thus the presence of GBR 12935 (1 μM) and fluoxetine hydrochloride (10 μM) were essential for non-specific binding. The slides were washed 3 times in ice-cold fresh phosphate buffer for 5 min each, after the incubation period. Following the final wash, the slides were quickly dipped once in ice-cold double distilled H2O to desalt thetissue and dried by a steady flow of air overnight at room temperature. DAT slides and an autoradiographic [125I] Microscale (Amersham Biosciences, Picataway, NJ) were exposed to Kodak BioMax MR-1 film (Amersham Biosciences) at 4°C for 24 hours.
, & Mohamed W. (2015). Possible alteration of catecholaminergic transporters in specific brain areas of iron deficit rats. Annals of Neurosciences, 22(1), 26-30.
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10

Molecular Modeling of Neurotransmitter Binding

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Molecular modeling was performed using MOE
v2010 and v2011.10 software from Chemical Computing Group (Montreal,
Quebec, CA). The radioligands [3H]serotonin (∼28
Ci/mmol), [3H]dopamine (∼26 Ci/mmol), [3H]norepinephrine (∼26 Ci/mmol), and [125I]RTI-55
(∼2200 Ci/mmol) were obtained from PerkinElmer Life and Analytical
Sciences (Foster City, CA). Nonradioactive citalopram, mazindol, nisoxetine,
and fluvoxamine were obtained from Tocris Bioscience (Ellisville,
MO). Virtual screening hit compounds were purchased from Ambinter
(Orleans, FR). C57BL/6J mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). Data analysis was performed using GraphPad Prism
5.0 (GraphPad Software, San Diego, CA).
Nolan T.L., Geffert L.M., Kolber B.J., Madura J.D, & Surratt C.K. (2014). Discovery of Novel-Scaffold Monoamine Transporter Ligands via in Silico Screening with the S1 Pocket of the Serotonin Transporter. ACS Chemical Neuroscience, 5(9), 784-792.
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