glutamate site specific nmdar antagonist d  (Tocris)

Journal: Science Advances

Article Title: GluN2B-specific NMDAR positive allosteric modulation reverses cognitive and behavioral abnormalities in Mecp2 and Disc1 transgenic mice

doi: 10.1126/sciadv.ady3891

Figure Lengend Snippet: ( A ) Virtual high-throughput screening of drugs targeting three allosteric modulatory sites on the diheteromeric GluN1/GluN2B-NMDAR. ( B ) Structures of the lead GluN2B-NMDAR PAMs 170, 175, 182, and 189. ( C to H ) Functional characterization of lead compounds in HEK293 cells transiently expressing recombinant GluN1/GluN2B- or GluN1/GluN2A-NMDAR. NMDAR-gated currents are induced by a short glutamate exposure (100 μM for 2 s) with glycine (30 μM) present in the extracellular recording solution (ECS). Currents are recorded under the whole-cell voltage-clamp configuration at a holding membrane potential of –60 mV. (C) Representative current traces are recorded from HEK293 cells transiently expressing the GluN1 and GluN2B subunits. Lead compound 170, 175, 182, or 189 (1 μM) alone without glutamate (light blue) does not induce any noticeable currents but potentiates glutamate-evoked, GluN1/GluN2B-NMDAR-gated currents (blue). The black bars indicate the duration of glutamate application. (D) Bar graph showing the fold potentiation on glutamate-induced GluN1/GluN2B-NMDAR currents by lead modulators ( n = 5 to 7 cells for each compound). [(E) to (H)] Lead compounds (170, 175, 182, and 189) are more potent and/or efficacies at potentiating GluN1/GluN2B- (blue) or GluN1/GluN2A-NMDAR (red) currents (170: logEC 50 = –6.91 ± 0.14 versus −5.08 ± 0.21, *** P < 0.001; 175: logEC 50 = −7.36 ± 0.24 versus −5.61 ± 0.30, ** P < 0.01, top value: 4.01 ± 0.30 versus 2.00 ± 0.17, P = 0.05; 182: logEC 50 = –7.10 ± 0.20 versus −5.89 ± 0.24, *** P < 0.001; 189: logEC 50 = –7.19 ± 0.20 versus −7.47 ± 0.46, P = 0.69, top value: 5.31 ± 0.40 versus 1.63 ± 0.05, ** P < 0.01). All data are reported as mean ± SEM. Dose-response curves are fitted using a three-parameter Hill equation. LogEC 50 and top values are compared using an extra sum-of-squares F test.

Article Snippet: An NMDAR antagonist d -APV (100 μM; Hello Bio) was added to the cell culture medium to improve cell viability, and the transfected cells were cultured for an additional 18 to 30 hours before electrophysiology experiments.

Techniques: High Throughput Screening Assay, Functional Assay, Expressing, Recombinant, Membrane

Journal: Science Advances

Article Title: GluN2B-specific NMDAR positive allosteric modulation reverses cognitive and behavioral abnormalities in Mecp2 and Disc1 transgenic mice

doi: 10.1126/sciadv.ady3891

Figure Lengend Snippet: ( A ) The predicted binding site of PAM 175 (blue) overlaps with the binding site of the NAM Ro25-6981 (red). ( B ) Dose-dependent potentiation of 175 on currents gated by WT GluN1/GluN2B-NMDARs ( n = 5 cells) or various putative binding pocket mutational NMDARs including 2B Q110A ( n = 6 cells; top value: 1.79 ± 0.12, * P < 0.05, logEC 50 = –7.58 ± 0.38, P = 0.73 versus WT), 2B F114 ( n = 6 cells; top value: 1.08 ± 0.06, P = 0.76, logEC 50 = –7.48 ± 1.66, combined top value and logEC 50 : *** P < 0.001 versus WT), and N1 L135Q ( n = 5 cells; top value: 4.10 ± 0.30, P = 0.83, logEC 50 = –7.42 ± 0.24, P = 0.86 versus WT). The same dataset from the GluN2B condition in is used as the GluN2B WT condition for comparison. ( C ) Dose-response curves of glutamate on GluN1/GluN2B-NMDAR currents with (blue; n = 5 cells) and without (black; n = 7 cells) 175 (1 μM). Top value: 4.30 ± 0.38 versus 2.00 ± 0.02, P = 0.07, logEC 50 = −5.51 ± 0.23 versus −5.87 ± 0.05, P = 0.60, combined top value and logEC 50 : *** P < 0.001 for Glu + 175 versus Glu. ( D ) Representative current traces related to (C). ( E ) Dose-response curves of 175 on currents induced by high (100 μM; light blue; n = 5 cells) and low (1 μM; blue; n = 7 cells) concentrations of glutamate. Top value: 4.01 ± 0.30 versus 1.15 ± 0.04, P = 0.52, logEC 50 = −7.36 ± 0.24 versus −7.29 ± 0.50, P = 0.97, combined top value and logEC 50 : *** P < 0.001 for 100 μM Glu versus 1 μM Glu. The same dataset from the GluN2B condition in is used as the 100 μM glutamate condition for comparison. ( F ) Representative current traces related to (E). (B to F) NMDAR-gated currents are induced by a short glutamate exposure (100 or 1 μM for 2 s) with glycine (30 μM) present in the ECS. All data are reported as mean ± SEM. Dose-response curves are fitted using a three-parameter Hill equation. LogEC 50 and top values are compared using an extra sum-of-squares F test.

Article Snippet: An NMDAR antagonist d -APV (100 μM; Hello Bio) was added to the cell culture medium to improve cell viability, and the transfected cells were cultured for an additional 18 to 30 hours before electrophysiology experiments.

Techniques: Binding Assay, Comparison

Journal: Science Advances

Article Title: GluN2B-specific NMDAR positive allosteric modulation reverses cognitive and behavioral abnormalities in Mecp2 and Disc1 transgenic mice

doi: 10.1126/sciadv.ady3891

Figure Lengend Snippet: ( A to F ) NMDAR-gated currents are evoked by a short pulse of aspartate (100 μM for 0.5 s in the presence of 1 μM glycine) and recorded under whole-cell patch-clamp configuration at a holding membrane potential of –60 mV in DIV 9 and 10 primary cultured neurons (B) or at a holding potential of –40 mV in DIV 12 to 14 neurons [(C) to (F)]. (B) Dose-responsive curves (left) and representative current traces (right) show that 175 dose-dependently (blue; 0.0001 to 1 μM) potentiates the NMDAR-gated currents (black; n = 7 cells). [(C) and (D)] Bar graph (C) and representative traces (D) illustrate the potentiation of NMDAR-gated currents by 175 (0.1 μM; blue) following blockade of GluN2A component by NVP [0.2 μM; red; one-way analysis of variance (ANOVA), *** P < 0.001; Šidák’s, ** P < 0.01 for NVP + 175 versus NVP; n = 5 cells]. [(E) and (F)] Bar graph (E) and representative current traces (F) show that 175 (0.1 μM; blue) does not affect NMDAR currents following the specific blockade of GluN2B component by Ro25-6981 (10 μM; red; one-way ANOVA, *** P < 0.001; Šidák’s, P = 0.83 for Ro + 175 versus Ro; n = 6 cells). n.s., not significant. ( G and H ) Bar graph (G) and representative current traces (H) show that 175 (1 μM; blue) positively modulates, albeit with much lower efficacy on, γ-aminobutyric acid type A receptor (GABA A R)–mediated currents induced by GABA (10 μM; black; unpaired t test, ** P < 0.01; n = 6 cells). ( I and J ) Bar graph (I) and representative current traces (J) show that 175 (1 μM; blue) does not affect α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)–mediated currents induced by AMPA (30 μM; black; unpaired t test, P = 0.63; n = 6 cells). The black bars indicate the duration of the agonist (aspartate, GABA, or AMPA) application. All data are reported as mean ± SEM. The dose-response curve is fitted using a three-parameter Hill equation.

Article Snippet: An NMDAR antagonist d -APV (100 μM; Hello Bio) was added to the cell culture medium to improve cell viability, and the transfected cells were cultured for an additional 18 to 30 hours before electrophysiology experiments.

Techniques: Patch Clamp, Membrane, Cell Culture

Journal: Science Advances

Article Title: GluN2B-specific NMDAR positive allosteric modulation reverses cognitive and behavioral abnormalities in Mecp2 and Disc1 transgenic mice

doi: 10.1126/sciadv.ady3891

Figure Lengend Snippet: ( A to F ) Basal hippocampal CA1 EPSPs were evoked by stimulations of the Schaffer collateral inputs every 30 s in anesthetized rats. 175 (1 mg/kg, ip) or Veh was injected 30 min before the induction of synaptic plasticity. (A) One train of HFS (100 Hz for 1 s) induces a nonsaturated form of LTP in Veh-treated rats (black; n = 7 rats) and 175 injections (1 mg/kg, ip; blue; n = 7 rats) and facilitates the expression of the late phase of this LTP. Norm., Normalized. (B) Bar graph shows the mean normalized slope of fEPSPs during the last 10 min of the LTP recording (unpaired t test; * P < 0.05). (C) Four trains of HFS (4 × 100 Hz for 1 s with 1-min interval) induce a saturated form of LTP in Veh-treated rats (black; n = 5 rats) and 175 injections (blue; n = 5 rats) and do not change the magnitude of LTP. (D) Bar graph shows the mean normalized slope of fEPSPs during the last 10 min of the LTP recording (unpaired t test; P = 0.61). (E) LFS (1 Hz for 900 s) fails to induce LTD in the Veh-treated group (black; n = 7 rats) but produces reliable LTD in 175-treated rats (blue; n = 7 rats), and the facilitation is blocked by cotreatment of 175 with GluN2B-subunit–specific NMDAR NAM Ro25-6981 (10 mg/kg, ip; green; n = 6 rats). (F) Bar graph shows the mean normalized slope of fEPSPs during the last 10 min of the LTD recording (one-way ANOVA main effect, ** P < 0.01; Šidák’s, * P < 0.05 for 175 versus Veh; Šidák’s, ** P < 0.01 for 175 + Ro versus 175; Šidák’s, P = 0.96 for 175 + Ro versus Veh). All data are reported as mean ± SEM.

Article Snippet: An NMDAR antagonist d -APV (100 μM; Hello Bio) was added to the cell culture medium to improve cell viability, and the transfected cells were cultured for an additional 18 to 30 hours before electrophysiology experiments.

Techniques: Injection, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic plasticity at the dentate gyrus granule cell to somatostatin-expressing interneuron synapses supports object location memory

doi: 10.1073/pnas.2312752120

Figure Lengend Snippet: Induction of LTP at MF-SOMIs synapse requires activation of mGluR1α, triggering G-protein-associated molecular cascades. ( A ) Schematic illustration of the proposed molecular mechanism mediating LTP at MF-SOMI synapses. Glutamate is released from MF terminals and binds to mGluR1α and NMDAR at the post-synaptic membrane of a SOMI, inducing an increased intracellular concentration of Ca 2+ via several pathways. MGluR1α-mediated phosphorylation of G-proteins ultimately triggers activation of several kinases including PKC and ERK1/ERK2 MAP kinases. Intracellular Ca 2+ rise is supported not only by an influx of Ca 2+ via NMDAR, but also by interaction of ERK1/2 with TRP channels and Ca 2+ release from internal stores. ( B ) LTP at MF-SOMI synapses was completely abolished by the application of the selective mGluR1α antagonist LY 367385 (100 μM, 8 SOMIs, red circles), while a blockade of mGluR5 by the selective antagonist MPEP (10 μM, 7 SOMIs, white circles) had no effect on LTP induction or expression. Note that hippocampal slices were incubated with antagonist for 30 min prior to transfer into the recording chamber. The horizontal black bar indicates the time of application of the antagonist during recording. ( C and D ) Summary plots demonstrating the importance of G-protein-activated pathways for MF-LTP. Intracellular dialysis of the G-protein inhibitor GDP-β-S (0.5 mM, 6 SOMIs, white circles) and the PKC pseudosubstrate inhibitor PKC 19 to 36 (10 µM, 5 SOMIs, black circles), as well as inhibition of ERK1/2 kinases by bath-application of FR 180204 (20 μM, 6 SOMIs, gray circles), led to a significant reduction of aBFS-induced PTP and LTP of MF-SOMI synapses. Interestingly, inhibition of ERK1/2 kinases unmasked a depression of transmission. The MF-mediated nature of eEPSCs was confirmed by a further decrease in eEPSC amplitudes in response to the application of DCG IV (1 μM, 4 SOMIs). In ( B–D ), LTP was induced by applying the aBFS protocol (time-point indicated by arrows on all plots). Gray panels indicate intervals used to measure PTP and LTP. Insets show representative recorded eEPSC traces (individuals in gray, average as an overlay in green) before ( Left , baseline) and after PTP ( Middle ) and LTP ( Right ). (Scale bars, 5 ms, 100 pA.) ( E and F ) Bar-graphs summarizing changes in peak amplitudes compared to baseline of recorded eEPSCs immediately after aBFS (PTP, E ) and after 15 to 20 min of recording (LTP, F ) in controls (same as in Fig. 1 E and F ) or following pharmacological manipulations. * P < 0.05; ** P < 0.01; ns, not significant; one-way ANOVA test. Bars denote mean, error bars indicate SEM, and circles show individual cells.

Article Snippet: To dissect the cellular mechanism of potentiation at MF-SOMI synapse, we either incubated slices in or bath-applied the following agents: the group II mGluR agonist DCG IV ((2 S ,2′ R ,3′ R )-2-(2′,3′-Dicarboxycyclopropyl) glycine; 1 μM, Tocris); the GABA A R antagonist SR 95531 (6-Imino-3-(4-methoxyphenyl)-1(6 H )-pyridazinebutanoic acid hydrobromide; 5 μM, Tocris); the selective mGlu1α antagonist LY 367385 [( S )-(+)-α-Amino-4-carboxy-2-methyl benzeneacetic acid; 100 μM, Tocris]; the potent mGlu5 antagonist MPEP [2-Methyl-6-(phenylethynyl)pyridine hydrochloride; 10 μM, Tocris]; the ERK inhibitor FR 180204 (5-(2-Phenyl-pyrazolo[1,5- a ]pyridin-3-yl)-1 H -pyrazolo[3,4- c ]pyridazin-3-ylamine; 20 μM, Tocris); the TRPC inhibitor that also inhibits store-operated Ca 2+ entry SKF 96365 (1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1 H -imidazole hydrochloride; 30 μM, Tocris); the L - and R -type VGCC blockers Nimodipine [1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2-methyloxyethyl 1-methylethyl ester; 50 μM] with Ni 2+ (10 μM, Tocris); the NMDAR antagonist D-APV (also termed D-AP-5; 100 μM, Tocris); the inhibitor of the SERCA ATPase α-CPA (100 μM, Tocris).

Techniques: Activation Assay, Membrane, Concentration Assay, Phospho-proteomics, Expressing, Incubation, Inhibition, Transmission Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic plasticity at the dentate gyrus granule cell to somatostatin-expressing interneuron synapses supports object location memory

doi: 10.1073/pnas.2312752120

Figure Lengend Snippet: Contribution of different Ca 2+ sources to LTP of MF-SOMI synapse in DG. ( A ) Reducing the concentration of intracellular Ca 2+ by chelating cytoplasmic Ca 2+ by intracellularly dialyzing with BAPTA (30 μM, 5 SOMIs, black circles) or by depleting internal Ca 2+ stores with bath-applied CPA (7 SOMIs, white circles) led to striking reductions in both PTP and LTP. ( B ) Pharmacological block of NMDAR by application APV (100 μM, 5 SOMIs) for 15 min prior to aBFS protocol application failed to induce either PTP or LTP at MF-SOMI synapses. ( C ) Bath application of the TRP channel and T-type VGCC blocker SKF 96365 (30 μM, 6 SOMIs, black bordered circles) for 15 min strongly affected both PTP and LTP at MF-SOMIs. The inhibition of L - and R -type VGCCs by co-application of the corresponding blockers Nimodipine (50 μM) and Ni 2+ (10 μM) significantly reduced PTP and led to a non-significant decrease in the magnitude of LTP (6 SOMIs, gray bordered circles). ( A–C ) aBFS delivery is indicated by arrows, gray panels indicate the intervals used to measure PTP and LTP. Insets show representative recorded eEPSC traces (individuals in gray, average as an overlay in green) before ( Left , baseline) and immediately after aBFS ( Middle , PTP) and 20 mins later ( Right , LTP). (Scale bars, 5 ms, 100 pA.) ( D ) Comparison of changes in peak amplitudes of recorded eEPSCs immediately following aBFS ( Left , PTP) and after 20 to 25 min of recording ( Right , LTP) in control conditions (same as in Fig. 1 E and F ) or following pharmacological manipulations. * P < 0.05; ** P < 0.01; ns, not significant; one-way ANOVA test. Bars with lines denote mean ± SEM, circles represent individual data points.

Article Snippet: To dissect the cellular mechanism of potentiation at MF-SOMI synapse, we either incubated slices in or bath-applied the following agents: the group II mGluR agonist DCG IV ((2 S ,2′ R ,3′ R )-2-(2′,3′-Dicarboxycyclopropyl) glycine; 1 μM, Tocris); the GABA A R antagonist SR 95531 (6-Imino-3-(4-methoxyphenyl)-1(6 H )-pyridazinebutanoic acid hydrobromide; 5 μM, Tocris); the selective mGlu1α antagonist LY 367385 [( S )-(+)-α-Amino-4-carboxy-2-methyl benzeneacetic acid; 100 μM, Tocris]; the potent mGlu5 antagonist MPEP [2-Methyl-6-(phenylethynyl)pyridine hydrochloride; 10 μM, Tocris]; the ERK inhibitor FR 180204 (5-(2-Phenyl-pyrazolo[1,5- a ]pyridin-3-yl)-1 H -pyrazolo[3,4- c ]pyridazin-3-ylamine; 20 μM, Tocris); the TRPC inhibitor that also inhibits store-operated Ca 2+ entry SKF 96365 (1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1 H -imidazole hydrochloride; 30 μM, Tocris); the L - and R -type VGCC blockers Nimodipine [1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2-methyloxyethyl 1-methylethyl ester; 50 μM] with Ni 2+ (10 μM, Tocris); the NMDAR antagonist D-APV (also termed D-AP-5; 100 μM, Tocris); the inhibitor of the SERCA ATPase α-CPA (100 μM, Tocris).

Techniques: Concentration Assay, Blocking Assay, Inhibition, Comparison, Control