Journal: PLoS ONE

Article Title: Olfactory Bulb Glomerular NMDA Receptors Mediate Olfactory Nerve Potentiation and Odor Preference Learning in the Neonate Rat

doi: 10.1371/journal.pone.0035024

Figure Lengend Snippet: Lateral drug infusion site that selectively affects glomerular ON-MC synapses. A1 . Mid-lateral infusion sites at either coronal or horizontal views of the olfactory bulbs, infused with methylene blue dye (4%). Black arrows indicate the sites of infusions. A2 . pCREB immunohistochemistry staining shows that MC staining on the D-APV infusion site were suppressed compared to the control ACSF site, suggesting D-APV mainly acts on the ON-MC synapses in this preparation. Red arrows in the upper panel indicate the difference in pCREB staining in the lateral regions of the MC layers. Lower panel shows enlarged lateral regions of MC layers. Scale bars, 500 µm (upper panel) and 200 µm (lower panel). B1 . Central infusion sites at either coronal or horizontal views of the olfactory bulbs, B2 . pCREB immunohistochemistry staining shows that MC staining on the D-APV infusion sites was enhanced compared to the control ACSF sites, suggesting D-APV mainly acts on the GC-MC synapses in this preparation. Same labeling and enlargement were used as in A2 . C . Early odor preference learning is blocked when an NMDAR antagonist D-APV is infused during training. Bars show the percentage of time spent in the peppermint side of a two-choice test box across different experimental groups. * p <0.05. Error bars, mean±SEM. D. D-APV infusion 10 min before testing does not prevent the odor preference formed with ISO+Odor pairing. * p <0.05. Error bars, mean±SEM.

Article Snippet: During training on PND 6, animals received bilateral intrabulbar infusion of either isoproterenol, isoproterenol together with muscimol (20 mM, Tocris), gabazine (0.1 mM, 1 mM, Tocris), or gabazine together with D-APV (0.5 mM).

Techniques: Immunohistochemistry, Staining, Labeling

Journal: PLoS ONE

Article Title: Olfactory Bulb Glomerular NMDA Receptors Mediate Olfactory Nerve Potentiation and Odor Preference Learning in the Neonate Rat

doi: 10.1371/journal.pone.0035024

Figure Lengend Snippet: D-APV effect on MC spiking. A1 . Single MC spiking patterns before, in the presence of, and 30 min following D-APV (50 µM) bath application. Arrow indicates the time of the single olfactory nerve test stimulus. A2&A3 . Peristimulus spike frequency histograms (binning 50 ms) under the control condition (red), in the presence of D-APV (green) and 20–30 min following D-APV washout of the example cell in A1 ( A2 ); and of the average of n = 4 cells in the same condition ( A3 ). A4 . Histogram comparing mean spike frequencies during the 250 ms intervals before (spontaneous) and at two time intervals after the olfactory nerve stimulation (evoked) under control conditions (red), during D-APV application (green) and 20–30 min following D-APV washout (black). B1-B3 . D-APV bath application blocked MC spike potentiation induced by paring ISO with TBS. B1 . Single MC spiking patterns before and 30 min following TBS induction in the presence of ISO and D-APV. B2&B3 . Peristimulus spike frequency histograms under the control condition and 20–30 min after TBS induction in the presence of ISO and D-APV of the example cell in B1 ( B2 ); and of the average of n = 6 cells ( B3 ). C. Histogram comparing mean spike frequencies during the 250 ms intervals before (spontaneous) and after the olfactory nerve stimulation (evoked) under control conditions (red) and 20–30 min post-induction (black). ** p <0.01. Error bars, mean±SEM.

Article Snippet: During training on PND 6, animals received bilateral intrabulbar infusion of either isoproterenol, isoproterenol together with muscimol (20 mM, Tocris), gabazine (0.1 mM, 1 mM, Tocris), or gabazine together with D-APV (0.5 mM).

Techniques:

Journal: PLoS ONE

Article Title: Olfactory Bulb Glomerular NMDA Receptors Mediate Olfactory Nerve Potentiation and Odor Preference Learning in the Neonate Rat

doi: 10.1371/journal.pone.0035024

Figure Lengend Snippet: A1-A3 . 2 µM gabazine (GABAA antagonist) induced MC spike potentiation following TBS. A1 . Single MC spiking patterns in the control condition and in the presence of 2 µM gabazine locally puffed to the glomerular layer. Arrow indicates the time of the single olfactory nerve test stimulus. A2&A3 . Peristimulus spike frequency histograms (binning 50 ms) under the control condition (red) and in the presence of 2 µM gabazine (black) of the example cell in A1 ( A2 ), and of the average of n = 6 cells in the same condition ( A3 ). B . Peristimulus spike frequency histograms showing that a higher dose (10 µM) of gabazine local application failed to induce MC spike potentiation. C1-C3 . D-APV application blocked MC spike potentiation induced by 2 µM gabazine. C1 . Single MC spiking patterns before and 30 min following TBS induction in the presence of a 2 µM gabazine and 500 µM D-APV local puff. C2&C3. Peristimulus spike frequency histograms under the control condition and 20–30 min after TBS induction in the presence of gabazine and D-APV of the example cell in C1 ( C2 ); and of the average of n = 7 cells ( C3 ). D. Histogram comparing mean spike frequencies during the 250 ms intervals before (spontaneous), and after, olfactory nerve stimulation (evoked) under control conditions (red) and 20–30 min post-induction (black). * p <0.05. Error bars, mean±SEM.

Article Snippet: During training on PND 6, animals received bilateral intrabulbar infusion of either isoproterenol, isoproterenol together with muscimol (20 mM, Tocris), gabazine (0.1 mM, 1 mM, Tocris), or gabazine together with D-APV (0.5 mM).

Techniques:

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