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: 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: The mGluR5 Fl/FL mouse was purchased from the Jackson Laboratory (B6.129- Grm5tm1.1Jixu /J, JAX stock #028626) and bred and maintained in house.

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: The mGluR5 Fl/FL mouse was purchased from the Jackson Laboratory (B6.129- Grm5tm1.1Jixu /J, JAX stock #028626) and bred and maintained in house.

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: The mGluR5 Fl/FL mouse was purchased from the Jackson Laboratory (B6.129- Grm5tm1.1Jixu /J, JAX stock #028626) and bred and maintained in house.

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: The mGluR5 Fl/FL mouse was purchased from the Jackson Laboratory (B6.129- Grm5tm1.1Jixu /J, JAX stock #028626) and bred and maintained in house.

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: The mGluR5 Fl/FL mouse was purchased from the Jackson Laboratory (B6.129- Grm5tm1.1Jixu /J, JAX stock #028626) and bred and maintained in house.

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: The mGluR5 Fl/FL mouse was purchased from the Jackson Laboratory (B6.129- Grm5tm1.1Jixu /J, JAX stock #028626) and bred and maintained in house.

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

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: Oral preclinical treatment with mGluR5 inhibitors significantly prolong survival in a prion disease mouse model. a Schematic overview of the experimental outline including intracerebral inoculation with RML5.0 prions (at experimental day 0), oral CTEP and SAM treatments starting at 60- or 90-days post inoculation (dpi), and time points of analyses. The majority of animals were allowed to progress to terminal disease to assess survival kinetics. A subset of animals was analyzed at early clinical time points to determine the impact of oral treatment on prion-induced brain pathology. b Kaplan-Meier survival curve of prion-infected mice without (RML; n = 10) or with chronic oral CTEP treatment starting at 60 dpi (CTEP60; n = 9) or 90 dpi (CTEP90; n = 12). Female and male mice were both used in this experiment and distributed equally in the curves. No gender effect could be seen; *** p = 0.0002. c Kaplan-Meier survival curve of prion-infected mice without treatment (RML; n = 8) or with chronic oral SAM treatment from day 60 (SAM60; n = 9) or day 90 (SAM90; n = 9) post infection. Only female mice were used in this study; ** p = 0.0051

Article Snippet: The highly selective mGluR5 antagonist CTEP was purchased at MCE (MedChem Express, USA) and applied via a standard rodent diet.

Techniques: Infection

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: Preclinical treatment of prion-infected mice with CTEP does not change levels of PrP Res or deposition of PrP Sc . a Representative IHC staining for deposition of PrP Sc in different brain regions in clinically diseased mice and age-matched controls does not reveal major differences in PrP Sc amounts and distribution pattern. Scale bar: 30 μm. b Quantification of PrP Sc staining signal confirmed that treatment with CTEP did not change the formation of PrP Sc . c Representative western blot of PrP Res after PK digestion at terminal and age-matched time points respectively, confirms that PrP Res levels are unchanged by treatment. d Quantification of total signal of western blot was performed on a limited number of mice. e Quantification of total PrP Res signal of western blot was performed at the early clinical day 90 after 30 days of treatment with CTEP in the mGluR5 abundant cortex and in the mGluR5 low-abundant thalamus. f Representative western blot of PrP Res after PK digestion of cortex tissue show that CTEP treatment has no influence on PrP Res levels

Article Snippet: The highly selective mGluR5 antagonist CTEP was purchased at MCE (MedChem Express, USA) and applied via a standard rodent diet.

Techniques: Infection, Immunohistochemistry, Staining, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: mGluR5 is dysregulated during the course of prion disease in mice. a Representative western blot of mGluR5, total PrP, and β-actin (Actin) from cortical tissues of SAM60, SAM90, RML, and control mice with (ConSAM) and without SAM treatment (Control) at 115 dpi ( n = 3–6 per group). mGluR5 monomers are labeled with m, whereas mGluR5 dimers are labeled with d. Total protein staining served as a loading control. b Quantification of western blot signal of mGluR5 normalized to β-actin (actin) abundance shows a significant upregulation of dimeric mGluR5 in the untreated prion-infected RML mice that was absent upon SAM treatment. One-way ANOVA with Tukey’s multiple comparison test: Monomers ** p = 0.0042; * p = 0.0293; ** p = 0.0015, Dimers * p = 0.0285; ** p = 0.0045. c Representative immunofluorescence staining of brain tissue from control and prion-infected RML mice at 90 dpi shows abundant expression of mGluR5 in the hippocampus region (mGluR5 (green), IBA1 (red), nuclei/DAPI (blue)). Scale bar: 90 μm. d Quantification of fluorescence signal showing a trend towards upregulated mGluR5 in the untreated prion-infected RML mice at 90 dpi. ( n = 3 per group). e Expansion microscopy of hippocampus tissue (stratum radiatum of the CA1 region) from selected control and prion-infected RML mice at 90 dpi shows mGluR5 puncta (green) with increased size and signal intensity surrounding neuronal processes (MAP2, magenta) in prion-infected RML mice at 90 dpi. Scale bar: 0.8 μm

Article Snippet: The highly selective mGluR5 antagonist CTEP was purchased at MCE (MedChem Express, USA) and applied via a standard rodent diet.

Techniques: Western Blot, Control, Labeling, Staining, Infection, Comparison, Immunofluorescence, Expressing, Fluorescence, Microscopy

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: mGluR5 is dysregulated during the subclinical phase in a preclinical non-human primate model of prion disease. a Representative immunohistochemical staining of PrP Sc in the brains of rhesus macaques is shown at given time points after intraperitoneal infection with human vCJD prions. Note the intra-neuronal thread-like PrP Sc pattern in the hippocampus and the plaque-like PrP Sc depositions in the temporal cortex in one of the preclinical and the clinical animal. Scale bar: 50 μm. b Representative IHC staining of mGluR5 in the hippocampus and temporal cortex of rhesus macaques infected with human vCJD prions. Of note, mGluR5 is reduced in preclinical prion disease at 30 months post infection when compared to a mock-infected control animal. At a late disease stage, mGluR5 abundance is still reduced in the temporal cortex, but is displaying a peri-neuronal and plaque-like staining pattern in both hippocampus and temporal cortex (green arrows). The ages of the individual rhesus macaques at the time of termination of the experiments in years are noted. Scale bar: 50 μm

Article Snippet: The highly selective mGluR5 antagonist CTEP was purchased at MCE (MedChem Express, USA) and applied via a standard rodent diet.

Techniques: Immunohistochemical staining, Staining, Infection, Immunohistochemistry, Control

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: Oral preclinical treatment with mGluR5 inhibitors significantly prolong survival in a prion disease mouse model. a Schematic overview of the experimental outline including intracerebral inoculation with RML5.0 prions (at experimental day 0), oral CTEP and SAM treatments starting at 60- or 90-days post inoculation (dpi), and time points of analyses. The majority of animals were allowed to progress to terminal disease to assess survival kinetics. A subset of animals was analyzed at early clinical time points to determine the impact of oral treatment on prion-induced brain pathology. b Kaplan-Meier survival curve of prion-infected mice without (RML; n = 10) or with chronic oral CTEP treatment starting at 60 dpi (CTEP60; n = 9) or 90 dpi (CTEP90; n = 12). Female and male mice were both used in this experiment and distributed equally in the curves. No gender effect could be seen; *** p = 0.0002. c Kaplan-Meier survival curve of prion-infected mice without treatment (RML; n = 8) or with chronic oral SAM treatment from day 60 (SAM60; n = 9) or day 90 (SAM90; n = 9) post infection. Only female mice were used in this study; ** p = 0.0051

Article Snippet: For detection of glial fibrillary acidic protein (GFAP, Dako M0761), ionized calcium binding adaptor molecule 1 (IBA1, Wako Pure Chemical Industries 019-19741) in mouse brain tissue, or mGluR5 (1:200; #55920 Cell Signaling) in mouse and non-human primate brain tissue by immunohistochemical staining, a Ventana Benchmark XT autostainer was used (Ventana, Tuscon, Arizona, USA).

Techniques: Infection

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: Preclinical treatment of prion-infected mice with CTEP does not change levels of PrP Res or deposition of PrP Sc . a Representative IHC staining for deposition of PrP Sc in different brain regions in clinically diseased mice and age-matched controls does not reveal major differences in PrP Sc amounts and distribution pattern. Scale bar: 30 μm. b Quantification of PrP Sc staining signal confirmed that treatment with CTEP did not change the formation of PrP Sc . c Representative western blot of PrP Res after PK digestion at terminal and age-matched time points respectively, confirms that PrP Res levels are unchanged by treatment. d Quantification of total signal of western blot was performed on a limited number of mice. e Quantification of total PrP Res signal of western blot was performed at the early clinical day 90 after 30 days of treatment with CTEP in the mGluR5 abundant cortex and in the mGluR5 low-abundant thalamus. f Representative western blot of PrP Res after PK digestion of cortex tissue show that CTEP treatment has no influence on PrP Res levels

Article Snippet: For detection of glial fibrillary acidic protein (GFAP, Dako M0761), ionized calcium binding adaptor molecule 1 (IBA1, Wako Pure Chemical Industries 019-19741) in mouse brain tissue, or mGluR5 (1:200; #55920 Cell Signaling) in mouse and non-human primate brain tissue by immunohistochemical staining, a Ventana Benchmark XT autostainer was used (Ventana, Tuscon, Arizona, USA).

Techniques: Infection, Immunohistochemistry, Staining, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: mGluR5 is dysregulated during the course of prion disease in mice. a Representative western blot of mGluR5, total PrP, and β-actin (Actin) from cortical tissues of SAM60, SAM90, RML, and control mice with (ConSAM) and without SAM treatment (Control) at 115 dpi ( n = 3–6 per group). mGluR5 monomers are labeled with m, whereas mGluR5 dimers are labeled with d. Total protein staining served as a loading control. b Quantification of western blot signal of mGluR5 normalized to β-actin (actin) abundance shows a significant upregulation of dimeric mGluR5 in the untreated prion-infected RML mice that was absent upon SAM treatment. One-way ANOVA with Tukey’s multiple comparison test: Monomers ** p = 0.0042; * p = 0.0293; ** p = 0.0015, Dimers * p = 0.0285; ** p = 0.0045. c Representative immunofluorescence staining of brain tissue from control and prion-infected RML mice at 90 dpi shows abundant expression of mGluR5 in the hippocampus region (mGluR5 (green), IBA1 (red), nuclei/DAPI (blue)). Scale bar: 90 μm. d Quantification of fluorescence signal showing a trend towards upregulated mGluR5 in the untreated prion-infected RML mice at 90 dpi. ( n = 3 per group). e Expansion microscopy of hippocampus tissue (stratum radiatum of the CA1 region) from selected control and prion-infected RML mice at 90 dpi shows mGluR5 puncta (green) with increased size and signal intensity surrounding neuronal processes (MAP2, magenta) in prion-infected RML mice at 90 dpi. Scale bar: 0.8 μm

Article Snippet: For detection of glial fibrillary acidic protein (GFAP, Dako M0761), ionized calcium binding adaptor molecule 1 (IBA1, Wako Pure Chemical Industries 019-19741) in mouse brain tissue, or mGluR5 (1:200; #55920 Cell Signaling) in mouse and non-human primate brain tissue by immunohistochemical staining, a Ventana Benchmark XT autostainer was used (Ventana, Tuscon, Arizona, USA).

Techniques: Western Blot, Control, Labeling, Staining, Infection, Comparison, Immunofluorescence, Expressing, Fluorescence, Microscopy

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: mGluR5 is dysregulated during the subclinical phase in a preclinical non-human primate model of prion disease. a Representative immunohistochemical staining of PrP Sc in the brains of rhesus macaques is shown at given time points after intraperitoneal infection with human vCJD prions. Note the intra-neuronal thread-like PrP Sc pattern in the hippocampus and the plaque-like PrP Sc depositions in the temporal cortex in one of the preclinical and the clinical animal. Scale bar: 50 μm. b Representative IHC staining of mGluR5 in the hippocampus and temporal cortex of rhesus macaques infected with human vCJD prions. Of note, mGluR5 is reduced in preclinical prion disease at 30 months post infection when compared to a mock-infected control animal. At a late disease stage, mGluR5 abundance is still reduced in the temporal cortex, but is displaying a peri-neuronal and plaque-like staining pattern in both hippocampus and temporal cortex (green arrows). The ages of the individual rhesus macaques at the time of termination of the experiments in years are noted. Scale bar: 50 μm

Article Snippet: For detection of glial fibrillary acidic protein (GFAP, Dako M0761), ionized calcium binding adaptor molecule 1 (IBA1, Wako Pure Chemical Industries 019-19741) in mouse brain tissue, or mGluR5 (1:200; #55920 Cell Signaling) in mouse and non-human primate brain tissue by immunohistochemical staining, a Ventana Benchmark XT autostainer was used (Ventana, Tuscon, Arizona, USA).

Techniques: Immunohistochemical staining, Staining, Infection, Immunohistochemistry, Control

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: Oral preclinical treatment with mGluR5 inhibitors significantly prolong survival in a prion disease mouse model. a Schematic overview of the experimental outline including intracerebral inoculation with RML5.0 prions (at experimental day 0), oral CTEP and SAM treatments starting at 60- or 90-days post inoculation (dpi), and time points of analyses. The majority of animals were allowed to progress to terminal disease to assess survival kinetics. A subset of animals was analyzed at early clinical time points to determine the impact of oral treatment on prion-induced brain pathology. b Kaplan-Meier survival curve of prion-infected mice without (RML; n = 10) or with chronic oral CTEP treatment starting at 60 dpi (CTEP60; n = 9) or 90 dpi (CTEP90; n = 12). Female and male mice were both used in this experiment and distributed equally in the curves. No gender effect could be seen; *** p = 0.0002. c Kaplan-Meier survival curve of prion-infected mice without treatment (RML; n = 8) or with chronic oral SAM treatment from day 60 (SAM60; n = 9) or day 90 (SAM90; n = 9) post infection. Only female mice were used in this study; ** p = 0.0051

Article Snippet: Antibodies against mGluR5 (1:200; #55920 Cell Signaling), IBA1 (1:200; #234308 Synaptic Systems or 1:500; #019-19741 Wako), or P2RY12 (1:5,000; #476011 Synaptic Systems) were incubated overnight at 4 °C.

Techniques: Infection

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: Preclinical treatment of prion-infected mice with CTEP does not change levels of PrP Res or deposition of PrP Sc . a Representative IHC staining for deposition of PrP Sc in different brain regions in clinically diseased mice and age-matched controls does not reveal major differences in PrP Sc amounts and distribution pattern. Scale bar: 30 μm. b Quantification of PrP Sc staining signal confirmed that treatment with CTEP did not change the formation of PrP Sc . c Representative western blot of PrP Res after PK digestion at terminal and age-matched time points respectively, confirms that PrP Res levels are unchanged by treatment. d Quantification of total signal of western blot was performed on a limited number of mice. e Quantification of total PrP Res signal of western blot was performed at the early clinical day 90 after 30 days of treatment with CTEP in the mGluR5 abundant cortex and in the mGluR5 low-abundant thalamus. f Representative western blot of PrP Res after PK digestion of cortex tissue show that CTEP treatment has no influence on PrP Res levels

Article Snippet: Antibodies against mGluR5 (1:200; #55920 Cell Signaling), IBA1 (1:200; #234308 Synaptic Systems or 1:500; #019-19741 Wako), or P2RY12 (1:5,000; #476011 Synaptic Systems) were incubated overnight at 4 °C.

Techniques: Infection, Immunohistochemistry, Staining, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: mGluR5 is dysregulated during the course of prion disease in mice. a Representative western blot of mGluR5, total PrP, and β-actin (Actin) from cortical tissues of SAM60, SAM90, RML, and control mice with (ConSAM) and without SAM treatment (Control) at 115 dpi ( n = 3–6 per group). mGluR5 monomers are labeled with m, whereas mGluR5 dimers are labeled with d. Total protein staining served as a loading control. b Quantification of western blot signal of mGluR5 normalized to β-actin (actin) abundance shows a significant upregulation of dimeric mGluR5 in the untreated prion-infected RML mice that was absent upon SAM treatment. One-way ANOVA with Tukey’s multiple comparison test: Monomers ** p = 0.0042; * p = 0.0293; ** p = 0.0015, Dimers * p = 0.0285; ** p = 0.0045. c Representative immunofluorescence staining of brain tissue from control and prion-infected RML mice at 90 dpi shows abundant expression of mGluR5 in the hippocampus region (mGluR5 (green), IBA1 (red), nuclei/DAPI (blue)). Scale bar: 90 μm. d Quantification of fluorescence signal showing a trend towards upregulated mGluR5 in the untreated prion-infected RML mice at 90 dpi. ( n = 3 per group). e Expansion microscopy of hippocampus tissue (stratum radiatum of the CA1 region) from selected control and prion-infected RML mice at 90 dpi shows mGluR5 puncta (green) with increased size and signal intensity surrounding neuronal processes (MAP2, magenta) in prion-infected RML mice at 90 dpi. Scale bar: 0.8 μm

Article Snippet: Antibodies against mGluR5 (1:200; #55920 Cell Signaling), IBA1 (1:200; #234308 Synaptic Systems or 1:500; #019-19741 Wako), or P2RY12 (1:5,000; #476011 Synaptic Systems) were incubated overnight at 4 °C.

Techniques: Western Blot, Control, Labeling, Staining, Infection, Comparison, Immunofluorescence, Expressing, Fluorescence, Microscopy

Journal: Acta Neuropathologica Communications

Article Title: Presymptomatic pharmacological inhibition of mGluR5 improves survival in a mouse model of prion diseases

doi: 10.1186/s40478-026-02235-9

Figure Lengend Snippet: mGluR5 is dysregulated during the subclinical phase in a preclinical non-human primate model of prion disease. a Representative immunohistochemical staining of PrP Sc in the brains of rhesus macaques is shown at given time points after intraperitoneal infection with human vCJD prions. Note the intra-neuronal thread-like PrP Sc pattern in the hippocampus and the plaque-like PrP Sc depositions in the temporal cortex in one of the preclinical and the clinical animal. Scale bar: 50 μm. b Representative IHC staining of mGluR5 in the hippocampus and temporal cortex of rhesus macaques infected with human vCJD prions. Of note, mGluR5 is reduced in preclinical prion disease at 30 months post infection when compared to a mock-infected control animal. At a late disease stage, mGluR5 abundance is still reduced in the temporal cortex, but is displaying a peri-neuronal and plaque-like staining pattern in both hippocampus and temporal cortex (green arrows). The ages of the individual rhesus macaques at the time of termination of the experiments in years are noted. Scale bar: 50 μm

Article Snippet: Antibodies against mGluR5 (1:200; #55920 Cell Signaling), IBA1 (1:200; #234308 Synaptic Systems or 1:500; #019-19741 Wako), or P2RY12 (1:5,000; #476011 Synaptic Systems) were incubated overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Infection, Immunohistochemistry, Control