cyano n  (Tocris)

Journal: bioRxiv

Article Title: mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder

doi: 10.1101/2022.01.28.478021

Figure Lengend Snippet: ( A ) Co-IP of cortical synaptosomal fraction (P2) from P56 mice by using anti-Homer1bc. IgG: control lane in the absence of antibodies. Immunoprecipitates and inputs were analyzed by immunoblotting for mGluR5 and Homer1bc. ( B ) Bar graphs showing Co-IP quantitation expressed as optical density (O.D.). ( C ) Confocal microscopy images showing mGluR5 + (green) and PSD-95 + (red) immunopuncta in layers II/III of S1 cortex (scale bar: 5 μm). ( D ) Bar graphs displaying the density of mGluR5 + puncta. Student T test *p < 0.05 (Co-IP: n = 8 IFL: n = 4).

Article Snippet: In pharmacological rescue experiments, animals were treated with the selective positive allosteric modulator (PAM) of mGluR5 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB; Tocris, UK) that was diluted in saline solution containing 5-10% final concentration of DMSO and polyethylene glycol 400 (DMSO: PEG 400 = 1:9) 5,23.

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Quantitation Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder

doi: 10.1101/2022.01.28.478021

Figure Lengend Snippet: ( A, C ) Representative confocal images showing Homer1bc + and mGluR5 + puncta in layer II-III of S1 cortex from either vehicle- or CDPPB-treated mice (scale bar: 5 μm). ( B, D ) Bar graphs showing both Homer1bc + ( B ) and mGluR5 + ( D ) immunopuncta density in layers II-III and V of both S1 and V1 cortices in either vehicle- or CDPPB-treated mice. ( E ) Confocal images of ARC immunostaining on coronal sections of the V1 cortex from mice treated with vehicle or CDPPB (scale bar: 25 μm), and relative ARC + cells density quantitation ( F ) throughout the cortical layers. Two-way ANOVA followed by Fisher’s multiple comparison test, *p < 0.05, ** p < 0.01, *** p < 0.001; (n = 6 animals for each genotype).

Article Snippet: In pharmacological rescue experiments, animals were treated with the selective positive allosteric modulator (PAM) of mGluR5 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB; Tocris, UK) that was diluted in saline solution containing 5-10% final concentration of DMSO and polyethylene glycol 400 (DMSO: PEG 400 = 1:9) 5,23.

Techniques: Immunostaining, Quantitation Assay, Comparison

Journal: bioRxiv

Article Title: mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder

doi: 10.1101/2022.01.28.478021

Figure Lengend Snippet: ( A) Illustrative confocal images taken from layers II-III of the BA17 cortex. ( A ) PSD-95 + (red), Homer1bc + (green), VGluT1 + (green) immunofluorescence puncta. Note the virtually complete overlapping of PSD-95 and Homer1bc immunofluorescence (scale bar: 5 μm). ( B, C, D ) Bar graphs showing the analysis of puncta density in layers II-III of BA17 cortices. ( E ) Western blotting showing the expression of PSD95, Homer1bc and mGluR5 in lysates from BA17 cortices. ( F-H ) Bar graphs displaying the optical density (O.D.) analysis of PSD95 ( F ), Homer1bc ( G ) and mGluR5 ( H ) expression. Student’s t-test, * p < 0.05, ** p < 0.01 (C1 = F, 4 years old; P1 = F, 5.7 years old; C2 = F, 29 years old; P2 = F, 30 years old).

Article Snippet: In pharmacological rescue experiments, animals were treated with the selective positive allosteric modulator (PAM) of mGluR5 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB; Tocris, UK) that was diluted in saline solution containing 5-10% final concentration of DMSO and polyethylene glycol 400 (DMSO: PEG 400 = 1:9) 5,23.

Techniques: Immunofluorescence, Western Blot, Expressing

Journal: Annals of neurology

Article Title: Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity

doi: 10.1002/ana.24708

Figure Lengend Snippet: Direct current stimulation-induced long-term depression (DCS-LTD) depends on metabotropic glutamate receptor 5 (mGluR5) activation in mouse M1 slices. (A, B) DCS-LTD effect can be elicited by cathodal DCS with treatment by either 50μM D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5; slopes were decreased to 84.3 ± 0.7% of baseline; n = 5 mice, 7 slices; p < 0.001) or 25μM bicuculline (BIC; 79.6 ± 0.7% of baseline; n = 5 mice, 6 slices; p < 0.001), antagonists of N-methyl-D-aspartate receptor and γ-aminobutyric acid type A receptor, respectively. (C) Bath application of mGluR5 negative allosteric modulator (2-chloro-4-[(2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]-1H-imidazol-4-yl)ethynyl]pyridine [CTEP], 10μM) completely abolished DCS-LTD in M1 slices (98.3 ± 0.7% of baseline; n = 7 mice, 10 slices; p > 0.05). Black bars under the traces in A–C indicate timing of drug exposure. (D) Statistical analysis of DCS-LTD changes on specific drug conditions (F7,56 = 423.2, p < 0.001); 1-way analysis of variance (ANOVA) post-test between each treatment and its baseline, ### = p < 0.001 and ns = p > 0.05; 1-way ANOVA post-test between 2 means as indicated in the graph, ***p < 0.001. fEPSP = field excitatory postsynaptic potential.

Article Snippet: Drugs NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA A R) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Activation Assay

Journal: Annals of neurology

Article Title: Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity

doi: 10.1002/ana.24708

Figure Lengend Snippet: Metabotropic glutamate receptor 5 (mGluR5) positive allosteric modulator (PAM) promotes direct current stimulation-induced long-term depression (DCS-LTD) in mouse M1 slices. (A) Short-term depression induced by 10-minute 400μA cathodal DCS (99.6 ± 0.7% of baseline 1 hour after DCS; n = 5 mice, 5 slices; p > 0.05) was enhanced to DCS-LTD (83.3 ± 0.4% of baseline 1 hour after DCS; n = 4 mice, 9 slices; p < 0.001) by exposure of M1 slices to an mGluR5 PAM (3-cyano-N-[1,3-diphenyl-1H-pyrazol-5-yl]benzamide [CDPPB], 10μM). (B) Statistic analysis of the aftereffects induced by cathodal DCS (400μA, 10 minutes) with and without CDPPB compared to normal DCS-LTD (F5,42 = 1317, p < 0.001); 1-way analysis of variance (ANOVA) post-test between each treatment and its respective baseline, ### = p < 0.001 and ns = p > 0.05; 1-way ANOVA post-test between 2 means as indicated in the graph, ***p < 0.001. fEPSP = field excitatory postsynaptic potential.

Article Snippet: Drugs NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA A R) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques:

Journal: Annals of neurology

Article Title: Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity

doi: 10.1002/ana.24708

Figure Lengend Snippet: Cathodal direct current stimulation (DCS) increases phosphorylated (p) ribosomal protein S6 via metabotropic glutamate receptor 5 and mechanistic target of rapamycin activation in mouse neocortex. (A) p-S6 in mouse M1 slices was measured 0, 15, 30, and 60 minutes after cathodal DCS (400μA, 25 minutes) by immunoblot, and a time-dependent increase of p-S6 was found. Actin was used as loading reference. Control slices (CT) were collected without DCS treatment. Total S6, normalized to actin, was unchanged (F4,15 = 0.85, p = 0.52 by 1-way analysis of variance [ANOVA]). (B) Statistical analysis of normalized p-S6:S6 ratio from M1 slices collected at different time points (F4,15 = 3.35, p < 0.05 by 1-way ANOVA). The normalized p-S6:S6 ratio is significantly increased 60 minutes after DCS (213.0 ± 26.9% of control; n = 4 mice, 4 slices; **p < 0.01 by 1-way ANOVA post-test). (C, D) p-S6 and S6 levels in M1 collected 1 hour after in vivo cathodal transcranial DCS (tDCS; 1mA, 25 minutes) with or without pretreatment with rapamycin (Rapa; C) or 2-chloro-4-([2,5-dimethyl-1-(4-[trifluoromethoxy]phenyl)-1H-imidazol-4-yl]ethynyl)pyridine (CTEP; D). p-S6 increases in the vehicle (Veh) + tDCS condition, and is otherwise stable. Total S6, normalized to actin, was unchanged (F5,24 = 1.29, p = 0.30 by 1-way ANOVA). (E) Statistical analysis of normalized p-S6:S6 ratio from M1 tissues of in vivo tDCS work (F5,40 = 4.76, p < 0.01). Normalized p-S6:S6 is increased (227.8 ± 32.0% of control) after cathodal tDCS (control [±vehicle]: n = 9 mice, tDCS [±vehicle]: n = 9 mice; ***p < 0.001). This increase is abolished by either rapamycin (73.7 ± 21.2% of control; n = 6 mice; p > 0.05 as compared to the control; ***p < 0.001 as compared to tDCS group) or CTEP pretreatment (135.8 ± 21.7% of control; n = 6 mice; p > 0.05 as compared to the control; *p < 0.05 as compared to tDCS group). Rapamycin (48.7 ± 5.4% of control; n = 3 mice; p > 0.05) or CTEP (122.0 ± 58.6% of control; n = 3 mice; p > 0.05) exposure without tDCS did not significantly affect the p-S6:S6 relative to the control group. ***p < 0.001 and *p < 0.05 by 1-way ANOVA post-test.

Article Snippet: Drugs NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA A R) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Control, In Vivo

Journal: eLife

Article Title: Sex-dependent effects of in utero cannabinoid exposure on cortical function

doi: 10.7554/elife.36234

Figure Lengend Snippet: Figure 8. Prenatal cannabinoid treatment affects expression of endocannabinoid system components in a sex-dependent fashion. CB1, TRPV1, mGlu5, and DAGLa mRNA levels in medial prefrontal cortex at PND90 were determined by real-time PCR in male (A, C, E, and G) and female (B, D, F, and H) rats using Taqman probes and a QuantStudio7 thermocycler. Treatment of dams with WIN from GD5 to GD20 decreased TRPV1, mGlu5, and DAGLa mRNA in female offspring. However, the same treatment decreased only mGlu5 in male offspring. *p<0.05. Error bars represent SEM. Figure 8 continued on next page

Article Snippet: The CB1 cannabinoid receptor antagonist SR141716A [5-(4-chloro-phenyl) 1-(2,4-dichlorophenyl) 4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (National Institute of Mental Health, USA), the positive allosteric modulator of mGlu5 receptors CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide) (National Institute of Mental Health, USA), the vanilloid TRPV1 antagonist capsazepine (N-[2-(4-Chlorophenyl)ethyl] 1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) (Tocris) and the FAAH inhibitor URB597 (Cyclohexylcarbamic acid 3’-(Aminocarbonyl)-[1,1’biphenyl] 3-yl ester) (Tocris) were dissolved in 5% Tween 80/5% polyethylene glycol/saline and given intraperitoneally (i.p.).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Sex-dependent effects of in utero cannabinoid exposure on cortical function

doi: 10.7554/elife.36234

Figure Lengend Snippet: Figure 9. Positive allosteric modulation of mGlu5 restores LTD and normalizes social interaction in male rats prenatally exposed to WIN. (A) Average time-courses of mean field EPSPs showing that, in WIN-exposed males (WIN, n = 17, green circle), LTD can be restored with the mGlu5positive allosteric modulator CDPPB (10 mM, n = 4, blue navy circle). (B) Peak amplitude measurements before (baseline) and after (LTD) stimulation protocol from individual experiments in absence or presence of CDPPB. After 45 min CDPPB preincubation: 0.190 ± 0.004 mV before and 0.147 ± 0.012 after LTD Figure 9 continued on next page

Article Snippet: The CB1 cannabinoid receptor antagonist SR141716A [5-(4-chloro-phenyl) 1-(2,4-dichlorophenyl) 4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (National Institute of Mental Health, USA), the positive allosteric modulator of mGlu5 receptors CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide) (National Institute of Mental Health, USA), the vanilloid TRPV1 antagonist capsazepine (N-[2-(4-Chlorophenyl)ethyl] 1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) (Tocris) and the FAAH inhibitor URB597 (Cyclohexylcarbamic acid 3’-(Aminocarbonyl)-[1,1’biphenyl] 3-yl ester) (Tocris) were dissolved in 5% Tween 80/5% polyethylene glycol/saline and given intraperitoneally (i.p.).

Techniques:

Journal: Neuropharmacology

Article Title: Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: implications for optimizing preclinical neuroscience drug discovery

doi: 10.1016/j.neuropharm.2016.07.001

Figure Lengend Snippet: Affinity and cooperativity estimates for allosteric modulation of orthosteric agonist-mediated iCa 2+ mobilization and IP 1 accumulation in HEK293A-mGlu 5 -low and cortical neurons. Data are mean ± SEM of 3–9 independent experiments performed in duplicate.

Article Snippet: N -(1,3-Diphenyl-1 H -pyrazolo-5-yl)-4-nitrobenzamide (VU29), 3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and 7-(Hydroxyimino)cyclopropa[ b ]chromen-1a-carboxylate ethyl ester (CPCCOEt) were purchased from Tocris Bioscience (Melbourne, Australia).

Techniques:

Journal: Neuropharmacology

Article Title: Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: implications for optimizing preclinical neuroscience drug discovery

doi: 10.1016/j.neuropharm.2016.07.001

Figure Lengend Snippet: DHPG concentration-response curves for iCa2+ mobilization in the absence and presence of indicated concentrations of allosteric ligands. Interaction studies for DPFE, VU0409551, VU0424465, and VU0403602 were performed using simultaneous addition of both ligands, to minimize allosteric ligand-induced acute desensitization due to intrinsic agonist activity. VU29, VU0405398, CDPPB and VU0360172 were added 1 min prior to addition of glutamate. Data sets were globally fitted to an operational model of allosterism to estimate affinity and cooperativity. Curves represent the best fit of the data. Data are mean + SEM of n=3–10 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Article Snippet: N -(1,3-Diphenyl-1 H -pyrazolo-5-yl)-4-nitrobenzamide (VU29), 3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and 7-(Hydroxyimino)cyclopropa[ b ]chromen-1a-carboxylate ethyl ester (CPCCOEt) were purchased from Tocris Bioscience (Melbourne, Australia).

Techniques: Concentration Assay, Activity Assay