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).
3h 8 oh dpat
Manufactured by PerkinElmer
Sourced in United States
[3H]8-OH-DPAT is a radiolabeled compound that acts as a selective agonist for the 5-HT1A receptor. It is commonly used as a research tool in various experimental studies.
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Lab products found in correlation
3h 5 ht, by PerkinElmer (2 mentions)
35s gtpγs, by PerkinElmer (2 mentions)
Way100635, by Merck Group (2 mentions)
3h prazosin, by PerkinElmer (2 mentions)
3h ketanserin, by PerkinElmer (2 mentions)
125i rti 55, by PerkinElmer (1 mentions)
Ip 1 elisa kit, by PerkinElmer (1 mentions)
25i nboh, by Cayman Chemical (1 mentions)
Gtpγs, by Merck Group (1 mentions)
Prazosin, by Merck Group (1 mentions)
8 oh dpat, by Merck Group (1 mentions)
Citalopram, by Lipomed (1 mentions)
Mazindol, by Lipomed (1 mentions)
Tramadol, by Lipomed (1 mentions)
Venlafaxine, by Lipomed (1 mentions)
Fluoxetine, by Lipomed (1 mentions)
Nisoxetine, by Merck Group (1 mentions)
3h mesulergine, by PerkinElmer (1 mentions)
Pindolol, by Merck Group (1 mentions)
Spiperone, by Merck Group (1 mentions)
14 protocols using 3h 8 oh dpat
1
Pharmacological Characterization of NBOMes
2
Radioligand Binding Assay Protocol
3
Radioligand Binding Assay Protocol
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Buprenorphine , citalopram, codeine , dextromethorphan , dihydrocodeine , fentanyl, heroin (diacetylmorphine, diamorphine) , 6‐acetylmorphine (6‐mono‐acetylmorphine), hydrocodone , hydromorphone, mazindol, MDMA, methadone , morphine, oxycodone , oxymorphone , pethidine (meperidine), tramadol, O‐desmethyl‐cis‐tramadol, tapentadol, venlafaxine and fluoxetine were purchased from Lipomed (Arlesheim, Switzerland). Mianserin, nisoxetine, pargyline, pindolol and spiperone were supplied by Sigma‐Aldrich (Buchs, Switzerland). D(S)‐methadone and l(R)‐methadone were obtained from Alsachim (Illkirch Graffenstaden, France). The HPLC purity of all of the substances was >98%. [3H]‐8‐OH‐DPAT, [3H]‐ketanserin and [3H]‐mesulergine were supplied by Perkin‐Elmer.
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4
Radioligand Binding Assay for 5-HT1A
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[3H]-8-OH-DPAT (135.2 Ci/mmol) was purchased from PerkinElmer and (R)-(+)-8-OH-DPAT, 5-HT and ZnCl2 were obtained from Sigma-Aldrich.
5
Radioligand Binding Assay for 5-HT1A Receptors
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Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser, and was then centrifuged at 32000 g for 10 min. The resulting pellet was then resuspended in the same buffer, incubated for 10 min at 37 °C, and centrifuged at 32000 g for 10 min. The final pellet was resuspended in Tris-HCl buffer containing 10 µM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.5 nM [3H]8-OH-DPAT (187.4 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris–HCl buffer containing 10 µM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H] 8-OH-DPAT, 50 µL of 10 µM serotonin. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]8-OH-DPAT, 50 µL of 50 µM new compounds. The tubes were incubated at 37 °C for 30 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
6
Autoradiographic Binding Assays of 5-HT1A, SERT, NET
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The autoradiographic binding assays for 5-HT1AR, SERT and norepinephrine transporter were performed using the following radioligands: (a) [3H]-8-OH-DPAT (233 Ci mmol−1), (b) [3H]-citalopram (70 Ci mmol−1) and (c) [3H]-nisoxetine (85 Ci mmol−1), respectively (Perkin-Elmer, Madrid, Spain) as described previously.17 The experimental conditions are summarized in Supplementary Table S2 . For 5-HT1AR-stimulated [35S]GTPγS autoradiography, coronal dorsal raphe nucleus (DR) sections were labeled with 0.04 nM [35S]GTPγS.17 Details are shown in Supplementary Information .
7
Quantification of 5-HT1A Receptor Autoradiography
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Brain slices were hydrated in 50 mM Tris-HCl buffer for 30 min at RT and then incubated for 1 h at RT in 50 mM Tris-HCl containing 4 mM CaCl2, 0.1% ascorbic acid and 2 nM [3H]8-OH-DPAT (Perkin Elmer, USA). To determine nonspecific radioligand binding, parallel brain slices were incubated for 1 h at RT in the same buffer enriched with 10 μM serotonin (Sigma-Aldrich, Germany). The [3H]8-OH-DPAT concentration corresponded to the Kd value [41 (link)]. Radioligand incubation was terminated by three washes in ice-cold 50 mM Tris-HCl buffer for 5 min. Tissue slices were then briefly immersed in distilled water, air-dried, and exposed to Fuji Imaging Plates (Fujifilm, Japan) with autoradiographic microscales (GE Healthcare, Germany) for 7 days. Developed autoradiograms were quantified using ImageGauge software (Fujifilm, Japan).
8
Radioligand Binding Assay for 5-HT1A
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For the 5-HT1A assay, ten concentrations equally spaced on a log scale (10−14 M–10−5 M) of each compound were incubated in duplicate with 1 nM [3H]8-OH-DPAT (specific activity: 200 Ci/mmol, Perkin Elmer, MA, USA) for 60 min. at 36 °C in a 50 mM Tris-HCl (pH 7.4) buffer, supplemented with 0.1% ascorbate, 5 mM MgCl2 and 80 µg of hippocampal membrane suspension. Non-specific binding was determined with 10 μM serotonin. The final DMSO concentration in the assay was 5%. After incubation, the reaction mixture was deposited with the FilterMate-96 Harvester (Perkin Elmer, MA, USA) onto Unifilter® GF/C plates (Perkin Elmer, MA, USA) presoaked in 0.4% PEI for 1 h. Each well was washed with 2 mL of 50 mM Tris-HCl (pH 7.4) buffer to separate bound ligands from free ones. Plates were left to dry overnight. Then, 35 µL of Microscint-20 scintillation fluid (Perkin Elmer, MA, USA) was added to each filter well and left to equilibrate for 2 h. Filter-bound radioactivity was counted in a MicroBeta2 LumiJet scintillation counter (Perkin Elmer, MA, USA). Binding curves were fitted with one site non-linear regression. Binding affinity (pKi and Ki) for each compound was calculated from the EC50 values with the Cheng–Prusoff equation from two separate experiments.
9
Synthesis and Characterization of 5-MeO-Tryptamines
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5-MeO-tryptamines were synthesized as hydrochloride salts (Supplementary Fig. 1), identified and characterized as described in the Supplementary Material. WAY100635 maleate and ketanserin tartrate were obtained from Tocris (Bio-Techne R&D Systems, S.L.U., Madrid, Spain). Solutions for injection were freshly prepared daily in isotonic saline solution (0.9% NaCl, pH 7.4). The radioligands [ 3 H]5-HT (33.2 Ci/mmol), [ 3 H]ketanserin (22.8 Ci/ mmol), [ 3 H]8-OH-DPAT (200 Ci/mmol), [ 3 H]imipramine (40 Ci/mmol) and the membrane preparations expressing human 5-HT1A and 5-HT2A receptors (h5-HT1AR and h5-HT2AR) were purchased from Perkin Elmer, Inc. (Waltham, MA, USA). p-Chloroamphetamine (pCA) and paroxetine were purchased from Sigma Aldrich. All other reagents were of analytical grade and purchased from several commercial sources. See the Supplementary Material for buffers and solutions composition.
10
Radioligand Binding Assay for 5-HT1A Receptor
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HEK cells expressing the human 5-HT1A receptor (HEK-h5HT1a) were used. Cells were grown to confluence on 150 mm plates in DMEM containing 10% FCS (HyClone), 0.05% penicillin/streptomycin, and 300 µg/mL G418. Cells were collected via scraping into phosphate-buffered saline (PBS) and centrifuged at 270 x g for 10 min. The cell pellet was subsequently homogenized in 50 mM Tris-HCl (pH 7.7) with a polytron, and centrifuged at 27000 × g. The homogenization and centrifugation were repeated to wash any remaining serotonin (5-HT) from the growth media. The final pellet was resuspended at 0.5 mg protein/mL in assay buffer (25 mM TrisHCl, 100 µM ascorbic acid, 10 µM pargyline, pH 7.4). The assay was performed in triplicate in a 96-well plate. The reaction mixture contained BrMeI, 100 µL of cell homogenate (0.05 mg protein/well) and 100 µL of [3H]8-OH-DPAT (0.5 nM final concentration, 170 Ci/mmol, Perkin Elmer) in a final volume of 1 mL. Plates were incubated at room temperature for 60 min and then filtered through polyethylenimine-soaked (0.05%) filters on a Tomtec cell harvester. Filters were then washed with cold 50 mM Tris buffer (pH 7.7) for 6 sec, dried, spotted with scintillation cocktail, and counted for 2 min after a 4 hr delay on a Perkin Elmer Betaplate 1205 liquid scintillation counter (Perkin Elmer). Nonspecific binding was determined with 1.0 µM dihydroergotamine.
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