Cdppb

Manufactured by Bio-Techne

Sourced in Macao, United Kingdom

CDPPB is a laboratory reagent produced by Bio-Techne. It functions as a selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5).

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3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB,

12 protocols using cdppb

CDPPB Administration and Signaling Pathways

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With reference to the dosages used in other studies^{24 (link),40 (link)}, CDPPB (Tocris) was dissolved in DMSO to a concentration of 10 mg/ml, and mixed with PBS containing 10% Tween-80 in a 1:9 ratio for intraperitoneal injection. PBS containing 10% Tween-80 was given as vehicle. Mice were given a single intraperitoneal injection at 10 mg/kg/day from P16 to 22. The whole little was weaned at P35.
For rescue experiments performed in adult mice, only males were employed. CDPPB (10 mg/kg) was injected 30 min before the open field test and three-chamber test. Cortices were collected within 10 min after the open field test. Protein samples were prepared in IP buffer supplemented with inhibitors including sodium orthovanadate. Samples were probed with antibodies against phospho-ERK (Cell Signaling, cat. 9101), total ERK (Cell Signaling, cat. 9102), phospho-JNK (Cell Signaling, cat. 9251), and total JNK (Cell Signaling, cat. 9252).

Chong C.H., Li Q., Mak P.H., Ng C.C., Leung E.H., Tan V.H., Chan A.K., McAlonan G, & Chan S.Y. (2019). Lrrc7 mutant mice model developmental emotional dysregulation that can be alleviated by mGluR5 allosteric modulation. Translational Psychiatry, 9, 244.

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Pharmacological induction of NMDAR and mGluR-LTD

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NMDAR-dependent LTD was induced by applying NMDA (20 µM) for 3 min, and mGluR-LTD was induced by applying R,S-DHPG (50 µM) for 5 min. In pharmacological pre-treatment experiments, slices were pre-incubated with the respective drug for 40 min before the beginning of baseline recordings, and then kept in bath throughout the entire experiment (except Fig. S5 and Fig. 5D). NMDA, MK-801 (40 µM) were purchased from Sigma. R,S-DHPG, D-AP5 (50 µM), 7-CK (100 µM), U0126 (20 µM) were purchased from Tocris Biosciences. Rapamycin (20 nM) was purchased from LC labs. Fresh bottles of DHPG were prepared as a 100× stock in H₂O, divided into aliquots and stored at −20 °C. Fresh stocks were made once a week. CHX (60 µM) and CDPPB (3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; 10 µM) were purchased from Tocris Biosciences. CDPPB was made up weekly in DMSO; CHX was fresh each experimental day.

Thomazeau A., Bosch M., Essayan-Perez S., Barnes S.A., De Jesus-Cortes H, & Bear M.F. (2020). Dissociation of functional and structural plasticity of dendritic spines during NMDAR and mGluR-dependent long-term synaptic depression in wild-type and fragile X model mice. Molecular Psychiatry, 26(9), 4652-4669.

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Estradiol and Cocaine Reinforcement in Ovariectomized Rats

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Estradiol (17β-estradiol; E2758, Sigma-Aldrich, St. Louis, MO) was dissolved in cottonseed oil to a final concentration of 2 μg/0.1 ml and was injected s.c. at a volume of 0.1 ml. The mGluR5 antagonist MPEP (1212, Tocris, Minneapolis, MN) was dissolved in sterile saline (1 mg/ml/kg) and injected i.p. This dose of MPEP has been shown to block estradiol-induced changes in dendritic spines within the NAc (Peterson et al., 2014 (link)), as well as estradiol enhancement of behavioral sensitization (Sircar and Kim, 1999 (link); Martinez et al., 2014 (link)), in OVX female rats. The mGluR5-positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB; 3235, Tocris) was dissolved in 10% Tween-80 (low CDPPB, 10 mg/ml/kg; high CDPPB, 25 mg/2 ml/kg) and injected i.p. The 10 mg/ml/kg dose of CDPPB mimics the effects of estradiol on dendritic spine density in the NAc in OVX females (Gross et al., 2016 (link)). Cocaine (cocaine hydrochloride; 0406-1520-53, Mallinckrodt, St. Louis, MO) was dissolved in sterile PBS (9.3 mg/ml) and infused i.v. (1.5 mg/kg/infusion). Previous work has shown that under extended access conditions, estradiol treatment enhances cocaine intake in OVX females self-administering at this dose (Ramôa et al., 2013 (link)).

Martinez L.A., Gross K.S., Himmler B.T., Emmitt N.L., Peterson B.M., Zlebnik N.E., Foster Olive M., Carroll M.E., Meisel R.L, & Mermelstein P.G. (2016). Estradiol Facilitation of Cocaine Self-Administration in Female Rats Requires Activation of mGluR5. eNeuro, 3(5), ENEURO.0140-16.2016.

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CDPPB Behavioral Assay in Mice

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CDPPB (Tocris) was dissolved in DMSO and polyethylene glycol 400
(DMSO:PEG 400 = 1:9) for the in vivo experiments and in DMSO only for the
in vitro experiments. Wild-type and Shank3Δ11-/- mice
received an intraperitoneal injection of the CDPPB (3 mg/kg)-containing solution
or the same volume of a DSMO:PEG400 mixture 70 min before each behavioral
test.

Vicidomini C., Ponzoni L., Lim D., Schmeisser M., Reim D., Morello N., Orelanna D., Tozzi A., Durante V., Scalmani P., Mantegazza M., Genazzani A.A., Giustetto M., Sala M., Calabresi P., Boeckers T.M., Sala C, & Verpelli C. (2016). Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice. Molecular psychiatry, 22(5), 689-702.

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Induction of NMDAR-LTD and mGluR-LTD

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NMDAR-dependent LTD was induced by applying NMDA (20 µM) for 3 min, and mGluR-LTD was induced by applying R,S-DHPG (50 µM) for 5 min. In pharmacological pretreatment experiments, slices were pre-incubated with the respective drug for 40 min before the beginning of baseline recordings, and then kept in bath throughout the entire experiment (except Supplementary Fig. S5 and Fig. 5d). NMDA, MK-801 (40 µM) were purchased from Sigma. R,S-DHPG, D-AP5 (50 µM), 7-CK (100 µM), U0126 (20 µM) were purchased from Tocris Biosciences. Rapamycin (20 nM) was purchased from LC labs. Fresh bottles of DHPG were prepared as a 100× stock in H₂O, divided into aliquots and stored at −20 °C. Fresh stocks were made once a week. CHX (60 µM) and CDPPB (3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; 10 µM) were purchased from Tocris Biosciences. CDPPB was made up weekly in DMSO; CHX was fresh each experimental day.

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Hippocampal Neuron Culture and Analysis

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Littermatte embryos derived from heterozygous parents were collected at day 16.5 of gestation. The hippocampus was dissected and dissociated in 0.0125% trypsin. Dissociated cells were plated at a density of 10,000 cells/cm² on poly-d-lysine-coated coverslips in a 24-well plate, and cultured with neurobasal medium containing 1% B27, 2 mM GlutaMax, 25 µM l-glutamate and 1% penicillin/streptomycin for 24 h [0 day in vitro (DIV)]. Half of the medium was replaced with medium without l-glutamate on the next day.
Neurons were transfected with pEGFP (Clontech) on 5 DIV using calcium phosphate precipitation. Photomicrographs were taken on 7 DIV and analyzed blinded to genotype with the Imaris imaging software (Bitplane).
The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) (Abcam) was dissolved in PBS at 5 mM and added to culture on 3 DIV at a final concentration of 10 µM. Hippocampal neurons were transfected with pEGFP and neurite analysis was performed as described above. In rescue experiment, CDPPB (Tocris) was dissolved in DMSO (27.4 µM) and added to culture to reach a final concentration of 50 nM on 3 DIV.

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ACSF and Internal Solutions Preparation

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The chemicals used for the ACSF and internal solutions were purchased from Merck and Sigma. DL-APV, CNQX, picrotoxin, DHPG, and CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide] were purchased from Tocris. All of the drugs were prepared in a high-concentration stock that was kept in an -80°C freezer. The chemicals were added to an ACSF bath prior to being applied.

Lin C.W., Chen C.Y., Cheng S.J., Hu H.T, & Hsueh Y.P. (2014). Sarm1 deficiency impairs synaptic function and leads to behavioral deficits, which can be ameliorated by an mGluR allosteric modulator. Frontiers in Cellular Neuroscience, 8, 87.

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Pharmacological Intervention in Mice

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Drugs were purchased from Sigma-Aldrich (MDL 100907 and MK801) and Tocris (CDPPB, Olanzapine, Sulpiride).
All agents were dissolved in a vehicle solution (80% saline, 10%DMSO, 10% Cremophor) and were administered i.p. on a daily basis at a volume of 10ml/kg.
At the age of 6-weeks-old, mice were randomly assigned to the respective treatment groups. For each treatment group, 12 mice were used to achieve a statistically significant power.

Mellios N., Papageorgiou G., Gorgievski V., Maxson G., Hernandez M., Otero M., Varangis M., Dell’Orco M., Perrone-Bizzozero N, & Tzavara E. (2024). Regulation of neuronal circHomer1 biogenesis by PKA/CREB/ERK-mediated pathways and effects of glutamate and dopamine receptor blockade. Research Square.

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Modulating mGluR5 in Adult Mice

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Mice received a daily intraperitoneal injection (at 9 am) from the postnatal day 56 (PnD56) to PnD91 during 5 weeks, see Fig 1. Negative allosteric modulator of mGluR5, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP), (2 mg/ml, 3 mg/kg animal, Tocris, Cat. No. 2921) and positive allosteric modulator of mGluR5, 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), (6.66 mg/ml, 10 mg/kg animal, Tocris, Cat. No. 3235) solutions were prepared with the same dissolving solution (accordingly to manufacturer’s recommendation). Drug preparation was made so that each animal received equivalent volume (1.5 μl/g animal). Equivalent volume of dissolving solution was used for the control treatment. Fresh solutions were prepared every 2–3 days and preserved at 4°C. The CDPPB and MTEP doses used in this study are known to be active at the behavioral and cellular level [5 (link), 40 (link)–43 (link)]. Since mGluR5 is known to play a function during the cerebral development and in the adult brain, the treatment was started in adulthood to exclude the effect of mGluR5 on development and, consequently, limiting the present results to the effect of mGluR5 in mature/adult brain.

Arsenault D., Coulombe K., Zhu A., Gong C., Kil K.E., Choi J.K., Poutiainen P, & Brownell A.L. (2015). Loss of Metabotropic Glutamate Receptor 5 Function on Peripheral Benzodiazepine Receptor in Mice Prenatally Exposed to LPS. PLoS ONE, 10(11), e0142093.

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Investigating Metabotropic Glutamate Receptor Signaling

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S-DHPG [(S)-3, 5-dihydroxyphenylglycine] and CDPPB (3-cyano-N-(1, 3-diphenyl-1H-pyrazol-5-yl) benzamide), were purchased from Tocris Bioscience (Ellisville, MO). Polyclonal antibodies against total-ERK 1/2 (Cat#9102) phospho-ERK1/2 (phosphorylated at Thr202 and Tyr204 of Erk1;Thr185 and Tyr187 of Erk2, Cat#9101), and phospho-PKC (Pan) (autophosphorylated at carboxy terminal Ser660 residue; Cat #9371) were from Cell Signaling (Beverly, Massachusetts, USA). Monoclonal antibody against mGluR1 was purchased from BD Transduction (Franklin Lakes, NJ). Polyclonal antibody for mGluR5 was obtained from Upstate Biotechnology (Lake Placid, NY). Antibody against the housekeeping protein β-actin was from Sigma (St. Louis, Missouri, USA). All other materials were purchased from commercial sources.

Bhardwaj S.K., Ryan R.T., Wong T.P, & Srivastava L.K. (2015). Loss of dysbindin-1, a risk gene for schizophrenia, leads to impaired group 1 metabotropic glutamate receptor function in mice. Frontiers in Behavioral Neuroscience, 9, 72.

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