Journal: bioRxiv

Article Title: GluN2D-containing NMDA receptors regulate dentate gyrus function by facilitating granule cell activity and mediating synaptic plasticity

doi: 10.64898/2026.03.06.710109

Figure Lengend Snippet: (A) Grin2d f l/fl mice were injected with AAV5-CamKII-mCherry (Control) or AAV-CamKII-mCherry-Cre ( Grin2d cKO). NMDAR-LTP was abolished in Grin2d cKO compared with control mice (Control: 149.5 ± 6.0 %, p < 0.01, n = 5, paired t-test; cKO: 92.5 ± 5.3 %, p = 0.12201, n = 6, paired t-test; Control vs cKO: p < 0.001, unpaired t-test). (B) WT mice were bilaterally injected with an anti-GluN2D antibody or control Ab into the dentate gyrus. After one hour, animals were euthanized, and slices were prepared. Injection was confirmed by the presence of methylene blue. NMDAR-LTP was abolished in mice injected with the anti-GluN2D antibody (cKO: 110.4 ± 8.5 %, p = 0.2952, n = 6, paired t-test) compared with control mice (Control: 149.8 ± 8.1 %, p < 0.001, n = 7, paired t-test; Control vs cKO: p < 0.01, unpaired t-test). (C) NMDAR-LTP was impaired in Grid1 KO mice (KO: 117.7 ± 5.3, p < 0.05%, n = 8, Wilcoxon signed-rank test) compared with controls (Control: 147.5 ± 6.7 %, p < 0.001, n = 7, paired t-test; Control vs cKO: p < 0.05, Mann-Whitney U test). Data are presented as mean ± s.e.m.

Article Snippet: For GluN2D cross-linking experiments in C57BL/6J, the control group received 1 μL of anti-rabbit Alexa 568 (control IgG, 1/5), while the GluN2D-cross-link group received 1 μg of polyclonal rabbit anti-GluN2D subunit (Alomone Labs, cat #AGC-020), both diluted in PBS with 1% methylene blue (1 μL final volume).

Techniques: Injection, Control, MANN-WHITNEY

Journal: Science Advances

Article Title: TrkB/mGluR5 cross-talk underlies a synaptic metaplasticity mechanism of ketamine

doi: 10.1126/sciadv.aec1444

Figure Lengend Snippet: ( A ) Hippocampal CA1 fEPSP slope time course showing that blockade of TrkB with ANA-12 or mGluR5 with MPEP prevents ketamine-induced synaptic potentiation of CA3-CA1 synapses. Gray bars show regions averaged for baseline and post-ketamine values in (B). ( B ) Summary graph showing a lack of ketamine-induced potentiation in the presence of ANA-12 or MPEP. ( C and D ) Representative images (C) and summary graph (D) showing that ketamine-induced (20 μM) spine density increases in 21-day in vitro (DIV-21) hippocampal neurons are blocked by ANA-12 or MPEP. Neurons visualized with phalloidin (grayscale), with enrichment of F-actin at spine heads (arrowheads). Scale bars, 1 μm. ( E ) Summary graph showing that the decrease in immobility time observed in the TST 60 min post–ketamine (30 mg/kg) injection is abolished following cKO of mGluR5 in CA1 excitatory neurons. Individual points represent independent slices taken from distinct mice (B), separate neurons taken from at least three separate culture preparations (D), or individual mice (E; females = open circles; males = closed circles). One-way analysis of variance (ANOVA) [(B) and (D)] or two-way ANOVA (E) with Šidák’s multiple-comparison test is used. (D) and (E) display median, interquartile range, and minimum and maximum values. All data shown as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Primary antibodies for endogenous TrkB (R&D Systems, AF1494) and mGluR5 (Alomone, #AGC-007) were added at 1:1000 overnight at 4°C.

Techniques: In Vitro, Injection, Comparison

Journal: Science Advances

Article Title: TrkB/mGluR5 cross-talk underlies a synaptic metaplasticity mechanism of ketamine

doi: 10.1126/sciadv.aec1444

Figure Lengend Snippet: ( A and B ) Representative calcium imaging traces for individual HEK 293 cells showing lack of response to ketamine and clear response to BDNF in cells expressing TrkB (A) or TrkB and mGluR5 (B). ( C ) Summary graph showing a lack of ketamine-driven calcium responses in TrkB-expressing cells but a clear potentiation of BDNF responses by mGluR5. n.s., not significant. ( D and E ) Representative images (D) and average time course (E) showing that BDNF, but not ketamine, drives KTR relocalization from the nucleus to the cytosol. Line scans before and 20 min after ligand application are shown in (D). Scale bars, 30 μm. a.u., arbitrary units; Ket, ketamine. ( F ) Summary graph showing a lack of ketamine-driven ERK response in TrkB-expressing cells but a clear potentiation of BDNF responses by mGluR5. ( G ) Representative calcium imaging traces for individual cells showing test of ketamine’s ability to potentiate. ( H ) Summary graph showing a lack of ketamine-driven increases in BDNF responsiveness in the absence or presence of mGluR5 coexpression using calcium imaging. ( I ) Average time course of ERK response to BDNF in the absence or presence of ketamine. ( J ) Summary graph showing a lack of ketamine-driven increases in BDNF responsiveness in the absence or presence of mGluR5 coexpression using ERK imaging. Individual points represent independent coverslips each containing many cells from separate biological replicates [(C), (F), (H), and (J)]. Two-way ANOVA with Šidák’s multiple comparisons is used. All data shown as mean ± SEM; * P < 0.05 and ** P < 0.01.

Article Snippet: Primary antibodies for endogenous TrkB (R&D Systems, AF1494) and mGluR5 (Alomone, #AGC-007) were added at 1:1000 overnight at 4°C.

Techniques: Imaging, Expressing

Journal: Science Advances

Article Title: TrkB/mGluR5 cross-talk underlies a synaptic metaplasticity mechanism of ketamine

doi: 10.1126/sciadv.aec1444

Figure Lengend Snippet: ( A ) SNAP-tag–based surface labeling assay to quantify the change in TrkB surface levels following ligand treatment in HEK 293 cells. ( B ) Summary graph showing ketamine (20 μM)–induced increase and BDNF (100 ng/ml)–induced decrease in TrkB surface level with or without the TrkB inhibitor ANA-12 (10 μM). ( C ) Dose response to ketamine in the surface labeling assay for wild-type (WT), K571N (kinase-dead), and Y433F (impaired ketamine/cholesterol binding) variants. ( D ) SNAP-tag–based forward trafficking assay used to quantify the emergence of new TrkB receptors on the cell surface after vehicle or drug incubation. ( E ) Summary graph showing a ketamine-induced increase in TrkB forward trafficking. ( F and G ) Representative images (F) and quantification (G) of surface levels of TrkB following 30 min treatment with vehicle (control) or ketamine (20 μM). Tetrodotoxin (TTX) was applied to prevent action potential firing and subsequent BDNF release. F-actin in spines and dendrites are visualized with phallodin (grayscale), with endogenous TrkB (green) costained with an extracellular-targeting antibody. ( H ) Functional experiments assessing the effect of ketamine incubation on BDNF responses of TrkB. ( I ) Summary graph showing that ketamine and mGluR5 coexpression both increase the calcium response of TrkB to BDNF (25 ng/ml) in an additive manner. ( J and K ) Dose-response curve (J) and summary graph (K) showing that ketamine preincubation increases the sensitivity of TrkB to BDNF. Individual points represent independent coverslips from separate biological replicates [(B), (E), (I), and (K)] or separate neurons taken from at least three separate culture preparations (G). EC 50 , median effective concentration. One-way ANOVA (B), two-way ANOVA [(C) and (I)] with Šidák’s multiple-comparison test, or unpaired t test [(E), (G), and (K)] is used. All data shown as mean ± SEM; norm., normalized to vehicle condition; * P < 0.05, ** P < 0.01, and *** P < 0.001. Scale bar, 1 μm (F).

Article Snippet: Primary antibodies for endogenous TrkB (R&D Systems, AF1494) and mGluR5 (Alomone, #AGC-007) were added at 1:1000 overnight at 4°C.

Techniques: Labeling, Binding Assay, Incubation, Control, Functional Assay, Concentration Assay, Comparison

Journal: Science Advances

Article Title: TrkB/mGluR5 cross-talk underlies a synaptic metaplasticity mechanism of ketamine

doi: 10.1126/sciadv.aec1444

Figure Lengend Snippet: ( A ) Confocal images showing endogenous mGluR5 (red) and TrkB (green) in hippocampal neurons transduced with mGreenLantern (blue) with or without BDNF (100 ng/ml; 30 min; 37°C). Insets show dendritic spines. ( B ) Summary graph showing BDNF-induced increase in TrkB/mGluR5 colocalization, as measured via Pearson’s correlation coefficient (PCC). ( C ) Summary graph showing an increase in proportion of spines containing both receptors following BDNF treatment. ( D and E ) Representative images of HEK 293 cells showing mGluR5 internalization following treatment with BDNF only in the presence of TrkB and the absence of the TrkB inhibitor K252a. ( F ) Summary graph of mGluR5 surface levels following 30 min treatment with ligands [1 mM Glu, BDNF (50 ng/ml), 200 nM K252a, and 1 μM MPEP]. ( G ) TrkB activation drives mGluR5 endocytosis in a kinase-dependent way, which also depends on mGluR5 constitutive activity. ( H ) Confocal images (left), zoomed insets (middle), and line scans (right) showing colocalization of mGluR5, TrkB, and endosomal recycling complex marker Tf-Cy3 following BDNF treatment in HEK 293 cells. ( I and J ) PCC analysis showing an increase in TrkB/mGluR5 colocalization (G) and mGluR5 colocalization with endosomal markers (H). ( K ) Secondary dendritic segment (gray outline) images with surface hemagglutinin (HA)–labeled mGluR5 (left) and internalized HA-labeled mGluR5 (right). ( L to N ) Summary of normalized mean intensity of surface (L), internalized (M), and the ratio of internalized to total (N) mGluR5 within dendritic spines. Individual points represent individual cells pooled from multiple biological replicates [(B), (C), (H), and (I)], coverslips from separate biological replicates (F), or secondary dendrites from distinct neurons [(K) to (N)]. Unpaired t test [(B), (I), (L), (M), and (N)], one-way ANOVA (F), or two-way ANOVA [(C), (J)] with Šidák’s multiple comparisons. All data shown as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001. Scale bars, 1 μm [(A) and (K)] or 10 μm [(D), (E), and (H)].

Article Snippet: Primary antibodies for endogenous TrkB (R&D Systems, AF1494) and mGluR5 (Alomone, #AGC-007) were added at 1:1000 overnight at 4°C.

Techniques: Transduction, Activation Assay, Activity Assay, Marker, Labeling

Journal: Science Advances

Article Title: TrkB/mGluR5 cross-talk underlies a synaptic metaplasticity mechanism of ketamine

doi: 10.1126/sciadv.aec1444

Figure Lengend Snippet: ( A ) Schematic showing experimental timeline for experiments in (B) to (D). Following incubation with vehicle or ketamine, TrkB and mGluR5 coexpressing HEK 293 cells were treated with BDNF. ( B and C ) Dose-response curve (B) and summary graph (C) showing a leftward shift in the BDNF dose dependence of mGluR5 internalization following ketamine incubation as measured using the SNAP-tag surface labeling assay. ( D ) Summary graph showing that ketamine preincubation enhances the BDNF-driven decrease in responses to 1 μM glutamate. ( E ) Schematic (top), time course (bottom left), and summary graph (bottom right) showing that in vivo ketamine injection impairs DHPG-LTD in hippocampal slices prepared 1 hour later. Gray bars show regions averaged for baseline and post-ketamine values. ( F ) Summary cartoon defining working model of four simultaneous, synergistic effects of ketamine on TrkB/mGluR5 signaling. Ketamine acutely drives BDNF release (1) and forward trafficking of TrkB (2), which both facilitate signaling cross-talk with mGluR5 (3) to produce synaptic potentiation, which drives initial antidepressant effects. On overlapping but longer timescales, TrkB activation drives mGluR5 internalization (4), impairing mGluR-dependent synaptic depression, enabling antidepressant effects to be maintained. Individual points represent individual coverslips from separate biological replicates [(C) and (D)] or individual slices from separate mice (E). Unpaired t test [(C) and (E)] and two-way ANOVA with Šidák’s multiple-comparison test (D) are used. All data shown as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Primary antibodies for endogenous TrkB (R&D Systems, AF1494) and mGluR5 (Alomone, #AGC-007) were added at 1:1000 overnight at 4°C.

Techniques: Incubation, Labeling, In Vivo, Injection, Activation Assay, Comparison

Journal: Science Advances

Article Title: TrkB/mGluR5 cross-talk underlies a synaptic metaplasticity mechanism of ketamine

doi: 10.1126/sciadv.aec1444

Figure Lengend Snippet: ( A ) Left: Schematic showing experimental timeline. Right: Summary graph showing that following incubation with ketamine, the mGluR5 PAM VU-29 enhances BDNF-driven calcium responses. ( B ) Left: Schematic showing experimental timeline. Right: Summary graph showing that following incubation with ketamine, the mGluR5 PAM VU-29 enhances BDNF-driven mGluR5 internalization using the SNAP-tag surface labeling assay. ( C and D ) Time course and summary graph showing that VU-29 increases ketamine-induced synaptic potentiation. ( E and F ) Representative images of neurons visualized with phalloidin for F-actin (grayscale) (E) and summary graph (F) showing that ketamine-driven spine growth in cultured hippocampal neurons is enhanced by VU-29 coapplication. ( G ) Summary graph showing that VU-29 coinjection enables an antidepressant-like effect 24 hours after injection of a subthreshold ketamine dose. ip, intraperitoneally. ( H ) Left: Schematic showing experimental timeline. Right: Summary time course showing DHPG-induced depression. ( I ) Summary graph showing that coinjection with VU-29 leads to impaired DHPG-LTD 24 hours post–ketamine injection. Individual points represent individual coverslips from separate biological replicates [(A) and (B)], individual slices from separate mice [(D) and (I)], individual neurons from multiple biological replicates (F), or individual mice [(G); females = open circles; males = closed circles]. Unpaired t test [(A), (D), and (I)] or one-way ANOVA with Šidák’s multiple-comparison test [(B), (F), and (G)] is used. [(F) and (G)] Display median, interquartile range, and minimum and maximum values. Gray bars show time regions used for baseline fEPSP calculation and quantification of plasticity [(D) and (I)]. All data shown as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001. Scale bar, 1 μm (E).

Article Snippet: Primary antibodies for endogenous TrkB (R&D Systems, AF1494) and mGluR5 (Alomone, #AGC-007) were added at 1:1000 overnight at 4°C.

Techniques: Incubation, Labeling, Cell Culture, Injection, Comparison

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

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

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

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, MANN-WHITNEY, Immunostaining