To first confirm that Ro 25-6981 showed an antidepressant-like effect in the FST of non-mutants, mice were intraperitoneally injected (10 mL/kg body weight) with 10 mg/kg Ro 25-6981 (Tocris, Ellisville Missouri) or 0.9% saline vehicle 30 minutes prior to the FST. This dose was chosen based on previous studies in the FST [7 (link), 20 (link), 23 (link)]. Using the same procedure, GluA1KO, GluN2AKO, GluN1INTER-KO, and PSD-95 mutants were tested in the FST after injection of Ro 25-6981 or vehicle. One week later, treatment assignments were reversed for each mouse and mice were tested in the open field test 30 minutes after injection of Ro 25-6981 or vehicle. Mice were place in a 40 × 40 × 35 cm square arena (60 lux) constructed of white Plexiglas for 30 minutes, as previously described [47 (link)]. Total distance traveled was measured by the Ethovision videotracking system (Noldus Information Technology Inc., Leesburg, VA) and expressed in meters.
Ro25 6981
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
Ro25-6981 is a selective and potent antagonist of the NR2B subunit of the NMDA receptor. It acts by inhibiting the function of the NR2B subunit, which is involved in synaptic plasticity and neuronal excitability.
Automatically generated - may contain errors
Lab products found in correlation
Nvp aam077, by Merck Group (3 mentions)
Picrotoxin, by Merck Group (3 mentions)
Ifenprodil, by Bio-Techne (3 mentions)
Ro 25 6981, by LC Laboratories (2 mentions)
Baclofen, by Bio-Techne (2 mentions)
Cgp35348, by Bio-Techne (2 mentions)
Picrotoxin, by Bio-Techne (2 mentions)
Tcn201, by Merck Group (2 mentions)
Ethovision video tracking system, by Noldus (1 mentions)
Rapamycin, by Bio-Techne (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
R 5000, by LC Laboratories (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Vitexin, by Merck Group (1 mentions)
Isovitexin, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Cytarabine, by Merck Group (1 mentions)
Strychnine, by Merck Group (1 mentions)
Bicuculline methochloride, by Merck Group (1 mentions)
26 protocols using ro25 6981
1
Behavioral Effects of Ro 25-6981 in Mutant Mice
2
Ro25-6981 Dose for Associative Recognition
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Ro25-6981 (Tocris, Abingdon, UK) was dissolved in sterile saline at a final concentration of 5 mg/mL. Mice were administered a single 10 mg/kg dose of Ro25-6981 or vehicle IP, 30 minutes before the start of the associative recognition OiP task. This dose was selected based on previously published behavioral work (e.g., Mikics et al., 2017 (link)).
3
Pharmacological Modulation of Synaptic Plasticity
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For behavioral and in vivo experiments, animals received intraperitoneal (i.p.) injections of Ro-25-6981 (10 mg/kg, Tocris, 1594) with or without CGP35348 (100 mg/kg; Tocris, 1245), or 0.9% physiological saline vehicle (0.01 ml/g, LabChem). Animals in the RNA immunoprecipitation (RIP)-Seq experiment received either Ro-25-6981 (10 mg/kg) with or without rapamycin (6 mg/kg; LC Laboratories, #R-5000) or 0.9% physiological saline (200 μl). For in vitro experiments, Ro-25-6981 (10 µM), rapamycin (200 nM), and baclofen (50 µM; Tocris, #0796) were used. Vehicle controls cells were treated with water (vehicle for Ro-25-6981 and baclofen) and/or 0.1% DMSO (vehicle for rapamycin).
4
In Vivo and In Vitro Pharmacological Assays
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For behavioral and in vivo experiments, animals received intraperitoneal (i.p.) injections of Ro-25-6981 (10 mg/kg, Tocris 1594) with or without CGP35348 (100 mg/kg; Tocris, 1245), or 0.9% physiological saline vehicle (0.01 ml/g, LabChem). Animals in the RIP-Seq experiment received either Ro-25-6981 (10 mg/kg) with or without rapamycin (6 mg/kg; LC Laboratories, #R-5000), or 0.9% physiological saline (200 μl). For in vitro experiments, Ro-25-6981 (10 µM), rapamycin (200 nM), and baclofen (50 µM; Tocris, #0796) were used. Vehicle controls cells were treated with water (vehicle for Ro-25-6981 and baclofen) and/or 0.1% DMSO (vehicle for rapamycin).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.08.25.266833 doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.08.25.266833 doi: bioRxiv preprint
5
Intrathecal Injection of Ro25-6981 for Mechanical Allodynia
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Intrathecal (i.t.) injection was performed as described previously (Minami et al., 1995 (link)). A 27-gauge stainless-steel needle (0.35 mm outer diameter) attached to a microsyringe was inserted between the L5 and L6 vertebrae of conscious mice, and Ro25-6981 (100 μg/mouse, Tocris Bioscience, Bristol, UK; Mihara et al., 2011 (link); Kim et al., 2012 (link)) in 1% dimethylsulfoxide/saline (5 μl) was injected i.t. 0.5 h before assessment of mechanical allodynia. Attenuation of the withdrawal threshold for mechanical allodynia by Ro25-6981, which is a specific antagonist of NMDAR containing GluN2B, was measured three times 30–90 min after the injection.
6
Extraction and Quantification of Bioactive Compounds
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Methanol (HPLC grade) was obtained from Merck (Darmstadt, Germany). Formic acid, ethanol, n-butanol, and Tween®-80 were obtained from Synth (Diadema, Brazil). Vitexin and isoVitexin standards (99.99%) were purchased from Sigma-Aldrich (São Paulo, Brazil). The 6-C-glycoside-diosmetin and vicenin-2 standards were generated in our laboratory according to the methods described by de Oliveira et al. (2014 (link)). Valium® (diazepam) was purchased from Roche (São Paulo, Brazil). Sintocalmy® (standardized extract of Passiflora incarnate L.—extract ACH 06) was obtained from Aché (Guarulhos, Brazil). Ro25-6981, picrotoxin and (S)-WAY100135 were purchased from Tocris Biosciences (Ellisville, MO, USA). NMDA was obtained from Sigma-Aldrich (São Paulo, Brazil). Buspirone hydrochloride was obtained from LIBBS Pharmaceutical Ltd (São Paulo, Brazil).
7
Pharmaceutical Compounds for Neuroscience Research
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N-Methyl-D-aspartate (NMDA), glycine, strychnine, bicuculline methochloride, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide (NBQX), NVP-AAM077, (+)-5-methyl-10,11-dihydro-5H-dibenzo(a, b)cyclohepten-5,10-imine maleate (MK-801), poly-l-lysine, and cytarabine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Leptin was purchased from Abcam (San Francisco, CA, USA). Ro25-6981 and tetrodotoxin were purchased from Tocris (Ellisville, MI, USA). All drugs were dissolved in water or saline.
8
Optogenetic Manipulation and Pharmacological Modulation
9
Modulation of Cortical Excitability Post-Stroke
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Ninety minutes after the start of photothrombosis, mice received a single injection (i.p.) of the GluN2B receptor antagonist Ro25-6981 (6 mg/kg, Tocris Bioscience), the α4β2 specific nAChR antagonist dihydro-β-erythroidine hydrobromide (3 mg/kg DHβE, Tocris Bioscience) or 0.9% saline (vehicle). An additional group of mice was injected with 3 mg/kg DHβE 180 min after stroke. Previous studies have shown that both drugs readily cross the blood brain barrier and alter cortical excitability (Liu et al., 2007 (link); Brown et al., 2012 (link)).
10
Transient Middle Cerebral Artery Occlusion
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Transient MCAO was adopted in our study, mainly according to the method of Rousselet et al. (2012 (link)). Briefly, 20–25 g male mice were anesthetized with isoflurane. During the whole surgery body temperature of the mice is maintained constant by a heating pad (Rousselet et al., 2012 (link)). A monofilament suture (about 9–10 mm is coated silicon, Doccol Corporation) was used to occlude the right MCA. Heart rate, rectal temperature, and blood gases were monitored during the MCAO surgery. All the mice were subjected a 1 h MCAO. For sham group, all procedures of operation were same except that the MCAO suture was not inserted into MCA. For drug treatment experiments, drugs were then performed via intraperitoneal injection 2 h after the onset of MCAO (GLYX-13, Tocris, 10 mg/kg, D-serine, Sigma, 50 mg/kg, Ro256981 Tocris 5 mg/kg, NVP-AAM077 Tocris 2.4 mg/kg).
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