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: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 1. mGluR5 had higher expression in multiple myeloma (MM) cell lines. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate the mGluR5 expression in MM cell lines and MM1S, OPM-2, U266, NCI-H929, and RPMI-8226, and normal cell line CD143; B,D. MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group. The qRT-PCR was used to measure mGluR5 RNA expression in each group in MM1S cell line. Western blot was used to measure mGluR5 protein expression in each group in MM1S cell line; C,E. OPM2 cell line was treated with DMSO as a control group, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. The qRT-PCR was used to measure mGluR5 RNA expression in each group in the MM1S cell line. Western blot was used for mGluR5 protein expression in each group in MM1S cell line. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 1A. human umbilical vein endothelial cells (HUVECs) compared to RPMI82226, p = 0.0197; HUVECs compared to U266, p = 0.0324; HUVECs compared to OPM2, p = 0.0029; HUVECs compared to MM1S, p = 0.0452; HUVECs compared to NCI-H929, p = 0.0298; K–W, Dunn’s post hoc. Fig. 1B. p = 0.0237, K–S; Fig. 1C. DMSO compared to MPEP, p = 0.0376; DMSO compared to MTEP, p = 0.0425; K–W, Dunn’s post hoc; Fig. 1D. p = 0.0198, K–S; Fig. 1E. DMSO compared to MPEP1. p = 0.0487; DMSO compared to MTEP2, p = 0.0341; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, RNA Expression, Western Blot

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 2. Agonist-induced mGluR5 upregulation promoted cell viability in multiple myeloma (MM) cells. A,B. The MTT assay measured the cell viability situation when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl) ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 2A. p = 0.0032, K–S; Fig. 2B. DMSO compared to MPEP, p = 0.0356; DMSO compared to MTEP, p = 0.0225; K–W, Dunn’s post hoc)Fig. 1. mGluR5 had higher expression in multiple myeloma (MM) cell lines. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate the mGluR5 expression in MM cell lines and MM1S, OPM-2, U266, NCI-H929, and RPMI-8226, and normal cell line CD143; B,D. MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group. The qRT-PCR was used to measure mGluR5 RNA expression in each group in MM1S cell line. Western blot was used to measure mGluR5 protein expression in each group in MM1S cell line; C,E. OPM2 cell line was treated with DMSO as a control group, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. The qRT-PCR was used to measure mGluR5 RNA expression in each group in the MM1S cell line. Western blot was used for mGluR5 protein expression in each group in MM1S cell line. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 1A. human umbilical vein endothelial cells (HUVECs) compared to RPMI82226, p = 0.0197; HUVECs compared to U266, p = 0.0324; HUVECs compared to OPM2, p = 0.0029; HUVECs compared to MM1S, p = 0.0452; HUVECs compared to NCI-H929, p = 0.0298; K–W, Dunn’s post hoc. Fig. 1B. p = 0.0237, K–S; Fig. 1C. DMSO compared to MPEP, p = 0.0376; DMSO compared to MTEP, p = 0.0425; K–W, Dunn’s post hoc; Fig. 1D. p = 0.0198, K–S; Fig. 1E. DMSO compared to MPEP1. p = 0.0487; DMSO compared to MTEP2, p = 0.0341; K–W, Dunn’s post hoc)Fig. 2. Agonist-induced mGluR5 upregulation promoted cell viability in multiple myeloma (MM) cells. A,B. The MTT assay measured the cell viability situation when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 2A. p = 0.0032, K–S; Fig. 2B. DMSO compared to MPEP, p = 0.0356; DMSO compared to MTEP, p = 0.0225; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: MTT Assay, Control, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Expression, Western Blot

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 3. Agonist-induced mGluR5 upregulation inhibited apoptosis in multiple myeloma (MM) cells. A,B. Flow cytometry (FCM) apoptosis assays measured the cell apoptosis changes when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as a control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 3A. p = 0.0269, K–S; Fig. 3B. DMSO compared to MPEP, p = 0.0274; DMSO compared to MPTP, p = 0.0225; K–W, Dunn’s post hoc)Fig. 3. Agonist-induced mGluR5 upregulation inhibited apoptosis in multiple myeloma (MM) cells. A,B. Flow cytometry (FCM) apoptosis assays measured the cell apoptosis changes when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as a control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 3A. p = 0.0269, K–S; Fig. 3B. DMSO compared to MPEP, p = 0.0274; DMSO compared to MPTP, p = 0.0225; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Flow Cytometry, Control

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 4. Transfection-induced upregulation of mGluR5 promoted cell viability and inhibited cell death in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids while OPM2 cell line was selected to knock down by transfection with oe-NC and oe-mGluR5 plasmids. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the relative mGluR5 mRNA expression among all the groups in both cell lines; B. Cell viability was measured with MTT method; C,D. Flow cytometry (FCM) apoptosis assay was used to measure cell apoptosis. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 4A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0163; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0319; K–W, Dunn’s post hoc; Fig. 4B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0308; K–W, Dunn’s post hoc. Fig. 4C; oe-NC(MMIS) compared to oe-mGluR5 (MMIS), p = 0.0268; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0325; K–W, Dunn’s post hoc)Fig. 4. Transfection-induced upregulation of mGluR5 promoted cell viability and inhibited cell death in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids while OPM2 cell line was selected to knock down by transfection with oe-NC and oe-mGluR5 plasmids. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the relative mGluR5 mRNA expression among all the groups in both cell lines; B. Cell viability was measured with MTT method; C,D. Flow cytometry (FCM) apoptosis assay was used to measure cell apoptosis. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 4A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0163; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0319; K–W, Dunn’s post hoc; Fig. 4B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0308; K–W, Dunn’s post hoc. Fig. 4C; oe-NC(MMIS) compared to oe-mGluR5 (MMIS), p = 0.0268; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0325; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Transfection, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Flow Cytometry, Apoptosis Assay

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 5. Transfection-induced upregulation activated the Ras–mitogen activated protein kinase (MAPK) signaling pathway in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids, while the OPM2 cell line was selected to be knocked down by transfection with oe-NC and oe-mGluR5 plasmids. Western blot was used to examine the protein levels of mGluR5 (A) and RAS (B) as well as phosphorylation of Raf1 (C) among all the groups in both cell lines after transfection. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 5A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0039, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0254; K–W, Dunn’s post hoc; Fig. 5B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0174, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0232; K–W, Dunn’s post hoc; Fig. 5D. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0163; K–W, Dunn’s post hoc)Fig. 5. Transfection-induced upregulation activated the Ras–mitogen activated protein kinase (MAPK) signaling pathway in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids, while the OPM2 cell line was selected to be knocked down by transfection with oe-NC and oe-mGluR5 plasmids. Western blot was used to examine the protein levels of mGluR5 (A) and RAS (B) as well as phosphorylation of Raf1 (C) among all the groups in both cell lines after transfection. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 5A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0039, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0254; K–W, Dunn’s post hoc; Fig. 5B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0174, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0232; K–W, Dunn’s post hoc; Fig. 5D. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0163; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Transfection, Western Blot, Phospho-proteomics

Journal: Learning & Memory

Article Title: The eIF2α kinase PERK limits the expression of hippocampal metabotropic glutamate receptor-dependent long-term depression

doi: 10.1101/lm.032219.113

Figure Lengend Snippet: PERK is required for the distinct regulation of eIF2α phosphorylation following mGluR-LTD induction. ( A ) E-LTP and L-LTP stimulation decreases eIF2α phosphorylation in hippocampal area CA1. Untetanized (control) slices vs. 1 × 100 Hz (E-LTP) and 4 × 100 Hz (L-LTP) slices are compared ( n = 5 slices per condition, [*] P < 0.05, [**] P < 0.01, one-way ANOVA). ( B ) DHPG application (50 µM, 10 min) increases eIF2α phosphorylation in hippocampal area CA1 ( n = 6 slices per condition, [*] P < 0.05, Student's t -test). ( C ) The DHPG-induced increase in eIF2α phosphorylation in WT slices is blocked in PERK cKO slices. Hippocampal slices from PERK cKO and WT mice were treated with or without DHPG (50 µM, 10 min, n = 6 slices per condition, [*] P < 0.05, one-way ANOVA). ( D ) mGluR1 and mGluR5 expression were unaltered in hippocampal lysates from PERK cKO mice compared to wild-type littermates (mGluR1, n = 4, P = 0.6; mGluR5, n = 6, P = 0.8, Student's t -test). Representative Western blots are shown in each panel.

Article Snippet: Anti-mGluR5 antibody was from R&D Systems.

Techniques: Phospho-proteomics, Control, Expressing, Western Blot

Journal: Nature medicine

Article Title: The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption.

doi: 10.1038/nm1163

Figure Lengend Snippet: Figure 2 Protein levels of mGluRs1, 2, 3, 5 and Eaat2. Western blot analysis of wild type (wt, solid line) and Per2Brdm1 mutant (hatched line) mouse brains over 24 h. (a) mGluR1, no significant difference observed. (b) mGluR2/3, no difference detected between the two genotypes. (c) mGluR5 is similar in Per2Brdm1 mutant and wild-type mice. (d) Eaat2 expression is shifted in Per2Brdm1 mutant mice. Values are shown as mean ± s.e.m. of three animals per time point. Data at ZT0 are plotted twice (ZT0 and ZT24). Photographs below the plots depict representative western blots with the corresponding actin controls (bottom, labeled A). *P < 0.05 compared to wild-type mice.

Article Snippet: Antibodies used for detection were: rabbit Eaat1-specific antibody diluted 1:1,000 (Abcam, Cambridge, ab 416); Eaat2 (Alpha Diagnostics, # GLT-1 1-A); mGluR1; mGluR5 (Upstate, #06-310 and #06- 451) and mGluR 2/3 (Novus Biologicals, NB 300-126).

Techniques: Western Blot, Mutagenesis, Expressing, Labeling

Journal: PLOS ONE

Article Title: Genetic ablation of metabotropic glutamate receptor 5 in rats results in an autism-like behavioral phenotype

doi: 10.1371/journal.pone.0275937

Figure Lengend Snippet: The mGluR5 Ko rats do not show differences in time spent in center ( A ), and in perimeter ( B ) compared to WT animals. p > 0.05 Ko versus WT animals. ns: non-significant.

Article Snippet: Heterozygous breeders of mGluR5 Ko rats on Sprague Dawley background were obtained from the Envigo company (Indianapolis, IN, USA).

Techniques:

Journal: PLOS ONE

Article Title: Genetic ablation of metabotropic glutamate receptor 5 in rats results in an autism-like behavioral phenotype

doi: 10.1371/journal.pone.0275937

Figure Lengend Snippet: A) Schematic representation of three-chamber sociability test. B) The mGluR5 Ko rats do not exhibit sociability as there was no statistically significant difference between time spent with the stranger rat over the empty cage ( p > 0.05). On the other hand, WT rats preferred spending time with the stranger rat compared to the empty cage (*** p < 0.001). ns: non-significant.

Article Snippet: Heterozygous breeders of mGluR5 Ko rats on Sprague Dawley background were obtained from the Envigo company (Indianapolis, IN, USA).

Techniques:

Journal: PLOS ONE

Article Title: Genetic ablation of metabotropic glutamate receptor 5 in rats results in an autism-like behavioral phenotype

doi: 10.1371/journal.pone.0275937

Figure Lengend Snippet: A) Schematic representation of social-novelty test. B) WT rats showed the preference for social novelty as they spent significantly more time with novel rat compared to the familiar rat (*** p < 0.001). On the other hand, the mGluR5 Ko rats showed no preference and there was no statistically significant difference in total time spent with the novel rat over the familiar rat suggesting reluctance to social novelty ( p > 0.05). ns: non-significant.

Article Snippet: Heterozygous breeders of mGluR5 Ko rats on Sprague Dawley background were obtained from the Envigo company (Indianapolis, IN, USA).

Techniques: