mglur5 agc 007  (Alomone Labs)


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    Alomone Labs mglur5 agc 007
    Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The <t>mGluR5</t> antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .
    Mglur5 Agc 007, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala"

    Article Title: Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025639

    Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .
    Figure Legend Snippet: Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .

    Techniques Used:

    The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .
    Figure Legend Snippet: The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .

    Techniques Used: Activity Assay

    rabbit anti mglur5  (Alomone Labs)


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    Alomone Labs rabbit anti mglur5
    a Hippocampal neuron expressing <t>SEP-mGluR5.</t> Scale bar, 50 µm. b Representative confocal image of dendrite expressing mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. c Quantification of the ratio of spine over dendrite intensity of mCherry, surface SEP-mGluR5 co-expressing mCherry ( n = 10, p < 0.0001; two-sided paired t test), Homer1c-mCherry and surface SEP-mGluR5 co-expressing Homer1c-mCherry ( n = 13, p < 0.0001; two-sided paired t test). d Representative confocal image of dendrite expressing Homer1c-mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. e gSTED imaging of SEP-mGluR5 surface-labeled with an anti-GFP nanobody Atto647N (cyan) and the merged image showing the relative localization to confocal-resolved Homer1c-mChery (red), shown in d . Scale bar, 2 µm. This experiment was replicated in cultures from more than three independent preparations of hippocampal neurons. f Zooms of dendritic spines indicated in e with asterisks. Scale bar, 1 µm. g Line profiles of spine 1 and h spine 2, indicated with dotted line in f . i Hippocampal neuron with an ORANGE GFP knock-in (KI) endogenously tagging mGluR5 at the N-terminus, enhanced with anti-GFP Alexa488 labeling (left), co-stained for anti-PSD-95 Alexa594 (right). Scale bar, 20 µm. j Representative two-color gSTED image of dendrite with GFP-mGluR5 KI stained with anti-GFP Alexa488 to label surface-expressed receptors (cyan) and anti-PSD-95 Alexa594 (red). Scale bar, 2 µm. This experiment was replicated in cultures from three independent preparations of hippocampal neurons. k Zooms of dendritic spines indicated in j with asterisks. Scale bar, 1 µm. l Line profiles of spine 1 and m spine 2, indicated with dotted line in k . Data are represented as means ± SEM. *** p < 0.001. Source data are provided as a Source Data file.
    Rabbit Anti Mglur5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function"

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    Journal: Nature Communications

    doi: 10.1038/s41467-022-35680-w

    a Hippocampal neuron expressing SEP-mGluR5. Scale bar, 50 µm. b Representative confocal image of dendrite expressing mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. c Quantification of the ratio of spine over dendrite intensity of mCherry, surface SEP-mGluR5 co-expressing mCherry ( n = 10, p < 0.0001; two-sided paired t test), Homer1c-mCherry and surface SEP-mGluR5 co-expressing Homer1c-mCherry ( n = 13, p < 0.0001; two-sided paired t test). d Representative confocal image of dendrite expressing Homer1c-mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. e gSTED imaging of SEP-mGluR5 surface-labeled with an anti-GFP nanobody Atto647N (cyan) and the merged image showing the relative localization to confocal-resolved Homer1c-mChery (red), shown in d . Scale bar, 2 µm. This experiment was replicated in cultures from more than three independent preparations of hippocampal neurons. f Zooms of dendritic spines indicated in e with asterisks. Scale bar, 1 µm. g Line profiles of spine 1 and h spine 2, indicated with dotted line in f . i Hippocampal neuron with an ORANGE GFP knock-in (KI) endogenously tagging mGluR5 at the N-terminus, enhanced with anti-GFP Alexa488 labeling (left), co-stained for anti-PSD-95 Alexa594 (right). Scale bar, 20 µm. j Representative two-color gSTED image of dendrite with GFP-mGluR5 KI stained with anti-GFP Alexa488 to label surface-expressed receptors (cyan) and anti-PSD-95 Alexa594 (red). Scale bar, 2 µm. This experiment was replicated in cultures from three independent preparations of hippocampal neurons. k Zooms of dendritic spines indicated in j with asterisks. Scale bar, 1 µm. l Line profiles of spine 1 and m spine 2, indicated with dotted line in k . Data are represented as means ± SEM. *** p < 0.001. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Hippocampal neuron expressing SEP-mGluR5. Scale bar, 50 µm. b Representative confocal image of dendrite expressing mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. c Quantification of the ratio of spine over dendrite intensity of mCherry, surface SEP-mGluR5 co-expressing mCherry ( n = 10, p < 0.0001; two-sided paired t test), Homer1c-mCherry and surface SEP-mGluR5 co-expressing Homer1c-mCherry ( n = 13, p < 0.0001; two-sided paired t test). d Representative confocal image of dendrite expressing Homer1c-mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. e gSTED imaging of SEP-mGluR5 surface-labeled with an anti-GFP nanobody Atto647N (cyan) and the merged image showing the relative localization to confocal-resolved Homer1c-mChery (red), shown in d . Scale bar, 2 µm. This experiment was replicated in cultures from more than three independent preparations of hippocampal neurons. f Zooms of dendritic spines indicated in e with asterisks. Scale bar, 1 µm. g Line profiles of spine 1 and h spine 2, indicated with dotted line in f . i Hippocampal neuron with an ORANGE GFP knock-in (KI) endogenously tagging mGluR5 at the N-terminus, enhanced with anti-GFP Alexa488 labeling (left), co-stained for anti-PSD-95 Alexa594 (right). Scale bar, 20 µm. j Representative two-color gSTED image of dendrite with GFP-mGluR5 KI stained with anti-GFP Alexa488 to label surface-expressed receptors (cyan) and anti-PSD-95 Alexa594 (red). Scale bar, 2 µm. This experiment was replicated in cultures from three independent preparations of hippocampal neurons. k Zooms of dendritic spines indicated in j with asterisks. Scale bar, 1 µm. l Line profiles of spine 1 and m spine 2, indicated with dotted line in k . Data are represented as means ± SEM. *** p < 0.001. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Labeling, Imaging, Knock-In, Staining

    a Reconstruction of single-molecule localizations obtained for SEP-mGluR5 anti-GFP nanobody Alexa647 using dSTORM (orange hot) and PSD FingR -mEos3.2 using PALM (cyan hot). Same dendritic region is shown in Fig. . Scale bar, 2 µm. b Zooms of spines shown in a . Scale bar, 500 nm. c Representative spine with single-molecule localizations of PSD FingR (cyan) and mGluR5 (orange) with indicated PSD border (black line) determined using DBScan. Scale bar, 500 nm. d Relative frequency distribution (fractions) of the distance of individual mGluR5 localizations to the PSD border ( n = 13 neurons, 253 PSDs). e For each PSD, eight rings, proportionally scaled based on its PSD border, defined the synapse (ring 1 and 2; black), perisynaptic zone (ring 3–5; orange), and extrasynaptic region (ring 6–8; blue). f Fraction of PSD FingR (black; plotted on left y axis) and mGluR5 (orange; plotted on right y-axis) localizations in rings 1–8. For each PSD, the number of localizations was normalized to the maximum number per ring, and the area per ring was calculated and corrected for. g Example spines with mGluR5 localizations (orange) belonging to clusters (black outline) as determined using DBScan, relative to PSD FingR localizations (magenta). Scale bars, 500 nm. h Relative frequency distribution (fractions) of the distance from the center of mGluR5 clusters to the border of the PSD (as indicated in g by the black lines) ( n = 273 clusters). i Relative frequency distribution (fractions) of the mGluR5 cluster area. j FWTM analysis comparing the width and length (in nm) of individual mGluR5 clusters. Data are represented as means ± SEM. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Reconstruction of single-molecule localizations obtained for SEP-mGluR5 anti-GFP nanobody Alexa647 using dSTORM (orange hot) and PSD FingR -mEos3.2 using PALM (cyan hot). Same dendritic region is shown in Fig. . Scale bar, 2 µm. b Zooms of spines shown in a . Scale bar, 500 nm. c Representative spine with single-molecule localizations of PSD FingR (cyan) and mGluR5 (orange) with indicated PSD border (black line) determined using DBScan. Scale bar, 500 nm. d Relative frequency distribution (fractions) of the distance of individual mGluR5 localizations to the PSD border ( n = 13 neurons, 253 PSDs). e For each PSD, eight rings, proportionally scaled based on its PSD border, defined the synapse (ring 1 and 2; black), perisynaptic zone (ring 3–5; orange), and extrasynaptic region (ring 6–8; blue). f Fraction of PSD FingR (black; plotted on left y axis) and mGluR5 (orange; plotted on right y-axis) localizations in rings 1–8. For each PSD, the number of localizations was normalized to the maximum number per ring, and the area per ring was calculated and corrected for. g Example spines with mGluR5 localizations (orange) belonging to clusters (black outline) as determined using DBScan, relative to PSD FingR localizations (magenta). Scale bars, 500 nm. h Relative frequency distribution (fractions) of the distance from the center of mGluR5 clusters to the border of the PSD (as indicated in g by the black lines) ( n = 273 clusters). i Relative frequency distribution (fractions) of the mGluR5 cluster area. j FWTM analysis comparing the width and length (in nm) of individual mGluR5 clusters. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Techniques Used:

    a Widefield of dendrite expressing Homer1c-mCherry and SEP-mGluR5. Scale bar, 5 µm. b Single-molecule trajectories (SMTs) of mGluR5 (each trajectory is assigned a random color) relative to the Homer1c PSD mask (gray) in the same dendrite as shown in a. Scale bar, 2 µm. c Example PSDs (gray) with their perisynaptic zone (orange ring) and mGluR5 SMTs color-coded for their subsynaptic localization. Scale bar, 1 µm. d Fraction of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). e Mean log D eff per neuron of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). f Relative frequency distributions of D effs of individual synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. g Fraction of perisynaptic SMTs that stay within perisynaptic/synaptic region once entered (captured), exited at least once, but end up staying inside the perisynaptic/synaptic region (returned) and perisynaptic tracks that escaped the perisynaptic/synaptic region (escaped) ( n = 25 neurons). h Mean MSD curve over time of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. Data are represented as means ± SEM. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Widefield of dendrite expressing Homer1c-mCherry and SEP-mGluR5. Scale bar, 5 µm. b Single-molecule trajectories (SMTs) of mGluR5 (each trajectory is assigned a random color) relative to the Homer1c PSD mask (gray) in the same dendrite as shown in a. Scale bar, 2 µm. c Example PSDs (gray) with their perisynaptic zone (orange ring) and mGluR5 SMTs color-coded for their subsynaptic localization. Scale bar, 1 µm. d Fraction of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). e Mean log D eff per neuron of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). f Relative frequency distributions of D effs of individual synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. g Fraction of perisynaptic SMTs that stay within perisynaptic/synaptic region once entered (captured), exited at least once, but end up staying inside the perisynaptic/synaptic region (returned) and perisynaptic tracks that escaped the perisynaptic/synaptic region (escaped) ( n = 25 neurons). h Mean MSD curve over time of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Techniques Used: Expressing

    a Widefield image of a dendritic spine expressing SEP-mGluR5 (cyan) and Homer1c-mCherry (red). (larger ROI shown in Fig. ). Scale bar, 1 µm. b The same spine as in a showing immobile (black) and mobile (red) SMTs of mGluR5 relative to the Homer1c PSD mask (larger ROI shown in Fig. ). Scale bar, 500 nm. c Transient confinement zones (red circles) of the mobile trajectories (random colors) shown in B (larger ROI shown in Fig. ). Scale bar, 500 nm. d Relative frequency plots of the dwell time (s) and e average radius of confinement zones for mGluR5 SMTs. f Example trajectory (assigned to perisynaptic fraction) that undergoes transient confinement in the perisynaptic zone, color-coded for entering the synapse (black) and perisynaptic zone (orange) and the confinement zones (red). The trajectory starts (green dot) and ends (red dot) in perisynaptic transient confinement zones. Scale bar, 100 nm. g The diffusion coefficient and h confinement index L over time for the trajectory shown in f , using the same color-coding. i The same example synapse as in a – c with hotspots of transient confinement zones, immobile tracks, and both images combined, color-coded for the frequency of confinement zones and/or immobile tracks (larger ROI shown in Fig. ). Scale bar, 500 nm. j Relative frequency distribution of the distance of confinement zones of mGluR5 and k center of immobile mGluR5 trajectories to the border of the PSD ( = 0 and indicated by dashed line). Data in this figure is the same dataset as used in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Widefield image of a dendritic spine expressing SEP-mGluR5 (cyan) and Homer1c-mCherry (red). (larger ROI shown in Fig. ). Scale bar, 1 µm. b The same spine as in a showing immobile (black) and mobile (red) SMTs of mGluR5 relative to the Homer1c PSD mask (larger ROI shown in Fig. ). Scale bar, 500 nm. c Transient confinement zones (red circles) of the mobile trajectories (random colors) shown in B (larger ROI shown in Fig. ). Scale bar, 500 nm. d Relative frequency plots of the dwell time (s) and e average radius of confinement zones for mGluR5 SMTs. f Example trajectory (assigned to perisynaptic fraction) that undergoes transient confinement in the perisynaptic zone, color-coded for entering the synapse (black) and perisynaptic zone (orange) and the confinement zones (red). The trajectory starts (green dot) and ends (red dot) in perisynaptic transient confinement zones. Scale bar, 100 nm. g The diffusion coefficient and h confinement index L over time for the trajectory shown in f , using the same color-coding. i The same example synapse as in a – c with hotspots of transient confinement zones, immobile tracks, and both images combined, color-coded for the frequency of confinement zones and/or immobile tracks (larger ROI shown in Fig. ). Scale bar, 500 nm. j Relative frequency distribution of the distance of confinement zones of mGluR5 and k center of immobile mGluR5 trajectories to the border of the PSD ( = 0 and indicated by dashed line). Data in this figure is the same dataset as used in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Diffusion-based Assay

    a Schematic of monomeric full-length mGluR5WT (top) and mGluR5∆C (bottom) lacking its C-terminal tail. b Representative gSTED images of dendrite expressing SEP-mGluR5WT and SEP-mGluR5∆C, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16) and mGluR5∆C (orange; n = 14) localization in spines: (1) synaptic enrichment ( p = 0.1565), (2) synaptic + perisynaptic enrichment ( p = 0.1488), (3) perisynaptic enrichment (0.3696) and (4) homogeneous distribution ( p = 0.0204; two-sided unpaired t test for each category). On top are representative images of the different categories of mGluR5 localization (cyan), relative to Homer1c (red), at spines. Scale bar, 1 µm. e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11) and mGluR5∆C ( n = 13, p < 0.0001; unpaired t test). f Fraction of synaptic ( p = 0.3529), perisynaptic ( p = 0.0218) and transient perisynaptic trajectories ( p = 0.0254) of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). g Mean log D eff per neuron of synaptic ( p = 0.5923), perisynaptic ( p = 0.0008), and transient perisynaptic ( p = 0.0025) trajectories of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). h Mean log D eff per neuron of trajectories inside confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11, p = 0.0053; two-sided unpaired t test). i Relative frequency distribution of the distance of confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11) to the border of the PSD (=0 and indicated by dashed line) (two-way repeated measures ANOVA with Bonferroni’s multiple comparisons test, at 25 nm distance from PSD border: p = 0.0003). j Example synapses of mGluR5WT and mGluR5∆C with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), k for being mobile (red) and immobile (black), l for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and m hotspots of immobile tracks (shown in k ) and confinement zones (shown in l ), color-coded for their frequency. Scale bars, 500 nm. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Schematic of monomeric full-length mGluR5WT (top) and mGluR5∆C (bottom) lacking its C-terminal tail. b Representative gSTED images of dendrite expressing SEP-mGluR5WT and SEP-mGluR5∆C, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16) and mGluR5∆C (orange; n = 14) localization in spines: (1) synaptic enrichment ( p = 0.1565), (2) synaptic + perisynaptic enrichment ( p = 0.1488), (3) perisynaptic enrichment (0.3696) and (4) homogeneous distribution ( p = 0.0204; two-sided unpaired t test for each category). On top are representative images of the different categories of mGluR5 localization (cyan), relative to Homer1c (red), at spines. Scale bar, 1 µm. e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11) and mGluR5∆C ( n = 13, p < 0.0001; unpaired t test). f Fraction of synaptic ( p = 0.3529), perisynaptic ( p = 0.0218) and transient perisynaptic trajectories ( p = 0.0254) of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). g Mean log D eff per neuron of synaptic ( p = 0.5923), perisynaptic ( p = 0.0008), and transient perisynaptic ( p = 0.0025) trajectories of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). h Mean log D eff per neuron of trajectories inside confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11, p = 0.0053; two-sided unpaired t test). i Relative frequency distribution of the distance of confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11) to the border of the PSD (=0 and indicated by dashed line) (two-way repeated measures ANOVA with Bonferroni’s multiple comparisons test, at 25 nm distance from PSD border: p = 0.0003). j Example synapses of mGluR5WT and mGluR5∆C with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), k for being mobile (red) and immobile (black), l for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and m hotspots of immobile tracks (shown in k ) and confinement zones (shown in l ), color-coded for their frequency. Scale bars, 500 nm. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Labeling

    a Schematic of mGluR5-STGtail (top) and mGluR5∆C-STGtail (bottom). b Representative gSTED images of dendrite expressing SEP-mGluR5-STGtail and SEP-mGluR5∆C-STGtail, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16), mGluR5-STGtail (blue; n = 11), and mGluR5∆C-STGtail (green; n = 25) localization in spines: (1) synaptic enrichment, (2) synaptic + perisynaptic enrichment, (3) perisynaptic enrichment and (4) homogeneous distribution ( p < 0.0001, Kruskal–Wallis test for each category with Dunn’s multiple comparisons test: p = 0.5398 for mGluR5WT vs. mGluR5-STGtail, p < 0.0001 for mGluR5WT vs. mGluR5∆C-STGtail and p = 0.0011 for mGluR5-STGtail vs. mGluR5∆C-STGtail). e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11), mGluR5-STGtail ( n = 12) and mGluR5∆C-STGtail ( n = 27; p < 0.0001, one-way ANOVA with Dunnet’s multiple comparisons test: compared to mGluR5WT p = 0.4700 for mGluR5-STGtail and p < 0.0001 for mGluR5∆C-STGtail). f Example synapses of mGluR5-STGtail and mGluR5∆C-STGtail with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), g for being mobile (red) and immobile (black), h for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and i hotspots of immobile tracks (shown in g ) and confinement zones (shown in h ), color-coded for their frequency. Scale bar, 500 nm. The mGluR5WT dataset shown in d and e is also shown in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Schematic of mGluR5-STGtail (top) and mGluR5∆C-STGtail (bottom). b Representative gSTED images of dendrite expressing SEP-mGluR5-STGtail and SEP-mGluR5∆C-STGtail, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16), mGluR5-STGtail (blue; n = 11), and mGluR5∆C-STGtail (green; n = 25) localization in spines: (1) synaptic enrichment, (2) synaptic + perisynaptic enrichment, (3) perisynaptic enrichment and (4) homogeneous distribution ( p < 0.0001, Kruskal–Wallis test for each category with Dunn’s multiple comparisons test: p = 0.5398 for mGluR5WT vs. mGluR5-STGtail, p < 0.0001 for mGluR5WT vs. mGluR5∆C-STGtail and p = 0.0011 for mGluR5-STGtail vs. mGluR5∆C-STGtail). e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11), mGluR5-STGtail ( n = 12) and mGluR5∆C-STGtail ( n = 27; p < 0.0001, one-way ANOVA with Dunnet’s multiple comparisons test: compared to mGluR5WT p = 0.4700 for mGluR5-STGtail and p < 0.0001 for mGluR5∆C-STGtail). f Example synapses of mGluR5-STGtail and mGluR5∆C-STGtail with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), g for being mobile (red) and immobile (black), h for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and i hotspots of immobile tracks (shown in g ) and confinement zones (shown in h ), color-coded for their frequency. Scale bar, 500 nm. The mGluR5WT dataset shown in d and e is also shown in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Labeling

    a Schematic of the FKBP-rapalog-FRB heterodimerization system (left) and how this system is used to recruit mGluR5 to the PSD (right). b Live-cell time-lapse images of SEP-mGluR5-FRB before (0’) and 20 and 30 min after rapalog application. The dendrites are color-coded for the fluorescence intensity of SEP-mGluR5-FRB. Scale bar, 2 µm. c Live-cell time-lapse images of the same dendrite as shown in b showing the relative localization SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) before (0’) and 20 and 30 min after rapalog application. Scale bar, 2 µm. d Zoom of spine indicated in c with asterisk before (0’) and 20 min after rapalog application. Scale bar, 500 nm. e Line profile of the spine in d , indicated with dotted line, showing the localization of mGluR5 (cyan) relative to Homer1c (red) before (0’) and 20 min after rapalog application. f Quantification of SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) intensity in spines over time upon rapalog application at t = 0 ( n = 17). Data are represented as means ± SEM. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Schematic of the FKBP-rapalog-FRB heterodimerization system (left) and how this system is used to recruit mGluR5 to the PSD (right). b Live-cell time-lapse images of SEP-mGluR5-FRB before (0’) and 20 and 30 min after rapalog application. The dendrites are color-coded for the fluorescence intensity of SEP-mGluR5-FRB. Scale bar, 2 µm. c Live-cell time-lapse images of the same dendrite as shown in b showing the relative localization SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) before (0’) and 20 and 30 min after rapalog application. Scale bar, 2 µm. d Zoom of spine indicated in c with asterisk before (0’) and 20 min after rapalog application. Scale bar, 500 nm. e Line profile of the spine in d , indicated with dotted line, showing the localization of mGluR5 (cyan) relative to Homer1c (red) before (0’) and 20 min after rapalog application. f Quantification of SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) intensity in spines over time upon rapalog application at t = 0 ( n = 17). Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Techniques Used: Fluorescence

    a Maximum projections of the GCaMP6f stream (50 s) in a representative dendrite before (baseline) and after 30 min rapalog application. Scale bar, 5 µm. b Zoom of spine 2 indicated in a with asterisk, expressing SNAP-mGluR5-FRB labeled with the cell-impermeable SNAPdye JF646 (cyan) and 2xFKBP-Homer1c-mCherry (red) before and 30 min after rapalog-induced recruitment. Scale bar, 1 µm. c ∆F/F 0 traces of GCaMP6f signal from two spines indicated in a with asterisks before and after 30 min of rapalog-induced recruitment of mGluR5 to Homer1c. d Quantification of mSCT frequencies upon application of rapalog in neurons expressing SNAP-mGluR5-FRB and 2xFKBP-Homer1c-mCherry ( n = 43 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test) and e in neurons expressing SNAP-mGluR5 and 2xFKBP-Homer1c-mCherry (control; n = 37 neurons, p = 0.5819, two-sided Wilcoxon matched-pairs signed rank test). f Average traces of all mSCTs per neuron (gray) and average mSCT trace of all neurons (red) before and after 30 min of rapalog. g Quantification of mSCT decay tau times (s) upon application of rapalog ( n = 37 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test). h Model of deregulated calcium signaling upon mGluR5 recruitment to the synapse during spontaneous synaptic activity. Medians are indicated by the red lines. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Maximum projections of the GCaMP6f stream (50 s) in a representative dendrite before (baseline) and after 30 min rapalog application. Scale bar, 5 µm. b Zoom of spine 2 indicated in a with asterisk, expressing SNAP-mGluR5-FRB labeled with the cell-impermeable SNAPdye JF646 (cyan) and 2xFKBP-Homer1c-mCherry (red) before and 30 min after rapalog-induced recruitment. Scale bar, 1 µm. c ∆F/F 0 traces of GCaMP6f signal from two spines indicated in a with asterisks before and after 30 min of rapalog-induced recruitment of mGluR5 to Homer1c. d Quantification of mSCT frequencies upon application of rapalog in neurons expressing SNAP-mGluR5-FRB and 2xFKBP-Homer1c-mCherry ( n = 43 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test) and e in neurons expressing SNAP-mGluR5 and 2xFKBP-Homer1c-mCherry (control; n = 37 neurons, p = 0.5819, two-sided Wilcoxon matched-pairs signed rank test). f Average traces of all mSCTs per neuron (gray) and average mSCT trace of all neurons (red) before and after 30 min of rapalog. g Quantification of mSCT decay tau times (s) upon application of rapalog ( n = 37 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test). h Model of deregulated calcium signaling upon mGluR5 recruitment to the synapse during spontaneous synaptic activity. Medians are indicated by the red lines. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Labeling, Activity Assay

    mglur5 agc 007  (Alomone Labs)


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    Alomone Labs mglur5 agc 007
    Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The <t>mGluR5</t> antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .
    Mglur5 Agc 007, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala"

    Article Title: Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025639

    Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .
    Figure Legend Snippet: Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .

    Techniques Used:

    The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .
    Figure Legend Snippet: The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .

    Techniques Used: Activity Assay

    anti mglur5 antibody  (Alomone Labs)


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    Alomone Labs anti mglur5 antibody
    Transcripts encoding metabotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Metabotropic glutamate receptor types are listed in the tables on the left. In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+Grm5 , Glutamate receptor, metabotropic 5; Grm6 , Glutamate receptor, metabotropic 6; Grm7 , Glutamate receptor, metabotropic 7; Grm8 , Glutamate receptor, metabotropic 8; MW, molecular weight markers; A, positive control tissue; B, ovulated oocytes; C, blastocysts. The MWs in base pairs (bp) are indicated to the right of the panels. * Primers for Grm5 receptor type were designed in this study. " width="250" height="auto" />
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    1) Product Images from "Glutamate can act as a signaling molecule in mouse preimplantation embryos"

    Article Title: Glutamate can act as a signaling molecule in mouse preimplantation embryos

    Journal: Biology of Reproduction

    doi: 10.1093/biolre/ioac126

    Transcripts encoding metabotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Metabotropic glutamate receptor types are listed in the tables on the left. In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+
    Figure Legend Snippet: Transcripts encoding metabotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Metabotropic glutamate receptor types are listed in the tables on the left. In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+" means upregulation and “−" means downregulation in blastocysts compared to oocytes) and corresponding P -values are shown. Transcripts were detected by RT-PCR and representative agarose gels with separated PCR products are shown in the panels on the right. Lanes: Grm1 , Glutamate receptor, metabotropic 1; Grm2 , Glutamate receptor, metabotropic 2; Grm3 , Glutamate receptor, metabotropic 3; Grm4 , Glutamate receptor, metabotropic 4; Grm5 , Glutamate receptor, metabotropic 5; Grm6 , Glutamate receptor, metabotropic 6; Grm7 , Glutamate receptor, metabotropic 7; Grm8 , Glutamate receptor, metabotropic 8; MW, molecular weight markers; A, positive control tissue; B, ovulated oocytes; C, blastocysts. The MWs in base pairs (bp) are indicated to the right of the panels. * Primers for Grm5 receptor type were designed in this study.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Positive Control

    Glutamate effects are blocked with AMPA/kainate and GRM5 receptor antagonists. Cell numbers and proportions of dead cells in blastocysts pretreated with the mixture of AMPA, kainate, and GRM5 receptor antagonists (CNQX and MPEP) prior to L-glutamic acid exposure. GlAc 5 mM, blastocysts incubated with L-glutamic acid (at 5 mM final concentration) for 24 h; CNQX + MPEP, blastocysts incubated with CNQX and MPEP (at 300 μM and 10 μM final concentrations, respectively) for 24 h; GlAc 5 mM + (CNQX + MPEP), blastocysts incubated with CNQX + MPEP antagonists for 20 min prior to addition of L-glutamic acid (for the following 24 h incubation). TE, trophectoderm, ICM, inner cell mass. The number of blastocysts in the groups ( n ): Control, n = 32; GlAc 5 mM, n = 33; CNQX + MPEP, n = 28; GlAc 5 mM + (CNQX + MPEP), n = 34. The values are arithmetical mean + SEM. Statistical significance of differences: * P < 0.05, * * P < 0.01, * * * P < 0.001.
    Figure Legend Snippet: Glutamate effects are blocked with AMPA/kainate and GRM5 receptor antagonists. Cell numbers and proportions of dead cells in blastocysts pretreated with the mixture of AMPA, kainate, and GRM5 receptor antagonists (CNQX and MPEP) prior to L-glutamic acid exposure. GlAc 5 mM, blastocysts incubated with L-glutamic acid (at 5 mM final concentration) for 24 h; CNQX + MPEP, blastocysts incubated with CNQX and MPEP (at 300 μM and 10 μM final concentrations, respectively) for 24 h; GlAc 5 mM + (CNQX + MPEP), blastocysts incubated with CNQX + MPEP antagonists for 20 min prior to addition of L-glutamic acid (for the following 24 h incubation). TE, trophectoderm, ICM, inner cell mass. The number of blastocysts in the groups ( n ): Control, n = 32; GlAc 5 mM, n = 33; CNQX + MPEP, n = 28; GlAc 5 mM + (CNQX + MPEP), n = 34. The values are arithmetical mean + SEM. Statistical significance of differences: * P < 0.05, * * P < 0.01, * * * P < 0.001.

    Techniques Used: Incubation, Concentration Assay

    rabbit polyclonal anti mglur5  (Alomone Labs)


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    1) Product Images from "Alzheimer’s vulnerable brain region relies on a distinct retromer core dedicated to endosomal recycling"

    Article Title: Alzheimer’s vulnerable brain region relies on a distinct retromer core dedicated to endosomal recycling

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.110182

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Construct, Plasmid Preparation, Software

    guinea pig polyclonal  (Alomone Labs)


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    Alomone Labs guinea pig polyclonal
    List of TRPV1 antibodies tested on sheep DRGs
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    1) Product Images from "Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect"

    Article Title: Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect

    Journal: eNeuro

    doi: 10.1523/ENEURO.0237-21.2021

    List of TRPV1 antibodies tested on sheep DRGs
    Figure Legend Snippet: List of TRPV1 antibodies tested on sheep DRGs

    Techniques Used:

    anti mglur5  (Alomone Labs)


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    Anti Mglur5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mglur5  (Alomone Labs)


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    mglur5  (Alomone Labs)


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    Alomone Labs mglur5
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    anti mglur5  (Alomone Labs)


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    Alomone Labs anti mglur5
    Anti Mglur5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti mglur5  (Alomone Labs)


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    A. Example responses of the CINs to a positive current pulse in the control and the Er81 cKO conditions. B. Average action potential rate during across all positive current pulses. The inset shows a time-expanded view of the early response (0-0.1 s; p = 0.280, n = 16 cells from 5 control mice and 18 cells from 2 Er81 cKO mice). C. Average action potential rate during positive current steps (sample size as in B). D. Adaptation index as a function of the current step amplitude (sample size as in B). E. Example trace of the putative I Kr following a depolarising voltage pulse. F. Mean traces of I Kr as a function of time at different holding potentials (-50 to −20 mV, 10 mV steps). G. Average I Kr as a function of the holding potential (control; n = 14, 3 mice and cKO; n = 16, 2 mice, -30 mV; p = 0.138, −20 mV; p = 0.272). H-J . Expression of KCNQ2 mRNA ( H , n = 6 control and n = 5 Er81 cKO), examples of CINs ( I , green) stained for KCNQ2 protein ( I , red) and the summary of the protein expression ( J , n = 5 control mice and n = 4 Er81 cKO mice, p = 0.302). K-M. Expression of <t>mGluR5</t> mRNA ( K , n = 7 control and n = 6 Er81 cKO), examples of CINs ( L , green) stained for <t>mGluR5</t> <t>protein</t> ( L , red) and the summary of the protein expression ( M , n = 5 control mice and n = 4 Er81 cKO mice). Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.
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    1) Product Images from "Loss of the Er81 Transcription Factor in Cholinergic Cells Alters Striatal Activity and Habit Formation"

    Article Title: Loss of the Er81 Transcription Factor in Cholinergic Cells Alters Striatal Activity and Habit Formation

    Journal: bioRxiv

    doi: 10.1101/2020.01.14.905497

    A. Example responses of the CINs to a positive current pulse in the control and the Er81 cKO conditions. B. Average action potential rate during across all positive current pulses. The inset shows a time-expanded view of the early response (0-0.1 s; p = 0.280, n = 16 cells from 5 control mice and 18 cells from 2 Er81 cKO mice). C. Average action potential rate during positive current steps (sample size as in B). D. Adaptation index as a function of the current step amplitude (sample size as in B). E. Example trace of the putative I Kr following a depolarising voltage pulse. F. Mean traces of I Kr as a function of time at different holding potentials (-50 to −20 mV, 10 mV steps). G. Average I Kr as a function of the holding potential (control; n = 14, 3 mice and cKO; n = 16, 2 mice, -30 mV; p = 0.138, −20 mV; p = 0.272). H-J . Expression of KCNQ2 mRNA ( H , n = 6 control and n = 5 Er81 cKO), examples of CINs ( I , green) stained for KCNQ2 protein ( I , red) and the summary of the protein expression ( J , n = 5 control mice and n = 4 Er81 cKO mice, p = 0.302). K-M. Expression of mGluR5 mRNA ( K , n = 7 control and n = 6 Er81 cKO), examples of CINs ( L , green) stained for mGluR5 protein ( L , red) and the summary of the protein expression ( M , n = 5 control mice and n = 4 Er81 cKO mice). Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: A. Example responses of the CINs to a positive current pulse in the control and the Er81 cKO conditions. B. Average action potential rate during across all positive current pulses. The inset shows a time-expanded view of the early response (0-0.1 s; p = 0.280, n = 16 cells from 5 control mice and 18 cells from 2 Er81 cKO mice). C. Average action potential rate during positive current steps (sample size as in B). D. Adaptation index as a function of the current step amplitude (sample size as in B). E. Example trace of the putative I Kr following a depolarising voltage pulse. F. Mean traces of I Kr as a function of time at different holding potentials (-50 to −20 mV, 10 mV steps). G. Average I Kr as a function of the holding potential (control; n = 14, 3 mice and cKO; n = 16, 2 mice, -30 mV; p = 0.138, −20 mV; p = 0.272). H-J . Expression of KCNQ2 mRNA ( H , n = 6 control and n = 5 Er81 cKO), examples of CINs ( I , green) stained for KCNQ2 protein ( I , red) and the summary of the protein expression ( J , n = 5 control mice and n = 4 Er81 cKO mice, p = 0.302). K-M. Expression of mGluR5 mRNA ( K , n = 7 control and n = 6 Er81 cKO), examples of CINs ( L , green) stained for mGluR5 protein ( L , red) and the summary of the protein expression ( M , n = 5 control mice and n = 4 Er81 cKO mice). Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Expressing, Staining

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    Alomone Labs mglur5 agc 007
    Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The <t>mGluR5</t> antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .
    Mglur5 Agc 007, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Hippocampal neuron expressing <t>SEP-mGluR5.</t> Scale bar, 50 µm. b Representative confocal image of dendrite expressing mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. c Quantification of the ratio of spine over dendrite intensity of mCherry, surface SEP-mGluR5 co-expressing mCherry ( n = 10, p < 0.0001; two-sided paired t test), Homer1c-mCherry and surface SEP-mGluR5 co-expressing Homer1c-mCherry ( n = 13, p < 0.0001; two-sided paired t test). d Representative confocal image of dendrite expressing Homer1c-mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. e gSTED imaging of SEP-mGluR5 surface-labeled with an anti-GFP nanobody Atto647N (cyan) and the merged image showing the relative localization to confocal-resolved Homer1c-mChery (red), shown in d . Scale bar, 2 µm. This experiment was replicated in cultures from more than three independent preparations of hippocampal neurons. f Zooms of dendritic spines indicated in e with asterisks. Scale bar, 1 µm. g Line profiles of spine 1 and h spine 2, indicated with dotted line in f . i Hippocampal neuron with an ORANGE GFP knock-in (KI) endogenously tagging mGluR5 at the N-terminus, enhanced with anti-GFP Alexa488 labeling (left), co-stained for anti-PSD-95 Alexa594 (right). Scale bar, 20 µm. j Representative two-color gSTED image of dendrite with GFP-mGluR5 KI stained with anti-GFP Alexa488 to label surface-expressed receptors (cyan) and anti-PSD-95 Alexa594 (red). Scale bar, 2 µm. This experiment was replicated in cultures from three independent preparations of hippocampal neurons. k Zooms of dendritic spines indicated in j with asterisks. Scale bar, 1 µm. l Line profiles of spine 1 and m spine 2, indicated with dotted line in k . Data are represented as means ± SEM. *** p < 0.001. Source data are provided as a Source Data file.
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    Transcripts encoding metabotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Metabotropic glutamate receptor types are listed in the tables on the left. In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+Grm5 , Glutamate receptor, metabotropic 5; Grm6 , Glutamate receptor, metabotropic 6; Grm7 , Glutamate receptor, metabotropic 7; Grm8 , Glutamate receptor, metabotropic 8; MW, molecular weight markers; A, positive control tissue; B, ovulated oocytes; C, blastocysts. The MWs in base pairs (bp) are indicated to the right of the panels. * Primers for Grm5 receptor type were designed in this study. " width="250" height="auto" />
    Anti Mglur5 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    List of TRPV1 antibodies tested on sheep DRGs
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    Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .

    Journal: PLoS ONE

    Article Title: Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

    doi: 10.1371/journal.pone.0025639

    Figure Lengend Snippet: Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .

    Article Snippet: Primary antibodies (with the references provided where the antibodies were tested for specificity) included: metabotropic glutamate receptors [mGluR1 (AGC-006) and mGluR5 (AGC-007) ] from Alomone (Jerusalem, Israel); phospholipase D [PLD1 (sc-25512) and PLD2 (sc-25513), ] and actin (sc-1616) from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA); dopamine receptor D1R (AB20066) from Abcam (Cambridge, MA); D5R (MAB5292) from Millipore (Temecula, CA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clone 6C5) from Advanced Immunochemicals Inc. (Long Beach, CA).

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    The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .

    Journal: PLoS ONE

    Article Title: Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

    doi: 10.1371/journal.pone.0025639

    Figure Lengend Snippet: The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .

    Article Snippet: Primary antibodies (with the references provided where the antibodies were tested for specificity) included: metabotropic glutamate receptors [mGluR1 (AGC-006) and mGluR5 (AGC-007) ] from Alomone (Jerusalem, Israel); phospholipase D [PLD1 (sc-25512) and PLD2 (sc-25513), ] and actin (sc-1616) from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA); dopamine receptor D1R (AB20066) from Abcam (Cambridge, MA); D5R (MAB5292) from Millipore (Temecula, CA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clone 6C5) from Advanced Immunochemicals Inc. (Long Beach, CA).

    Techniques: Activity Assay

    a Hippocampal neuron expressing SEP-mGluR5. Scale bar, 50 µm. b Representative confocal image of dendrite expressing mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. c Quantification of the ratio of spine over dendrite intensity of mCherry, surface SEP-mGluR5 co-expressing mCherry ( n = 10, p < 0.0001; two-sided paired t test), Homer1c-mCherry and surface SEP-mGluR5 co-expressing Homer1c-mCherry ( n = 13, p < 0.0001; two-sided paired t test). d Representative confocal image of dendrite expressing Homer1c-mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. e gSTED imaging of SEP-mGluR5 surface-labeled with an anti-GFP nanobody Atto647N (cyan) and the merged image showing the relative localization to confocal-resolved Homer1c-mChery (red), shown in d . Scale bar, 2 µm. This experiment was replicated in cultures from more than three independent preparations of hippocampal neurons. f Zooms of dendritic spines indicated in e with asterisks. Scale bar, 1 µm. g Line profiles of spine 1 and h spine 2, indicated with dotted line in f . i Hippocampal neuron with an ORANGE GFP knock-in (KI) endogenously tagging mGluR5 at the N-terminus, enhanced with anti-GFP Alexa488 labeling (left), co-stained for anti-PSD-95 Alexa594 (right). Scale bar, 20 µm. j Representative two-color gSTED image of dendrite with GFP-mGluR5 KI stained with anti-GFP Alexa488 to label surface-expressed receptors (cyan) and anti-PSD-95 Alexa594 (red). Scale bar, 2 µm. This experiment was replicated in cultures from three independent preparations of hippocampal neurons. k Zooms of dendritic spines indicated in j with asterisks. Scale bar, 1 µm. l Line profiles of spine 1 and m spine 2, indicated with dotted line in k . Data are represented as means ± SEM. *** p < 0.001. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Hippocampal neuron expressing SEP-mGluR5. Scale bar, 50 µm. b Representative confocal image of dendrite expressing mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. c Quantification of the ratio of spine over dendrite intensity of mCherry, surface SEP-mGluR5 co-expressing mCherry ( n = 10, p < 0.0001; two-sided paired t test), Homer1c-mCherry and surface SEP-mGluR5 co-expressing Homer1c-mCherry ( n = 13, p < 0.0001; two-sided paired t test). d Representative confocal image of dendrite expressing Homer1c-mCherry and SEP-mGluR5, surface-labeled with an anti-GFP nanobody Atto647N. Scale bar, 2 µm. e gSTED imaging of SEP-mGluR5 surface-labeled with an anti-GFP nanobody Atto647N (cyan) and the merged image showing the relative localization to confocal-resolved Homer1c-mChery (red), shown in d . Scale bar, 2 µm. This experiment was replicated in cultures from more than three independent preparations of hippocampal neurons. f Zooms of dendritic spines indicated in e with asterisks. Scale bar, 1 µm. g Line profiles of spine 1 and h spine 2, indicated with dotted line in f . i Hippocampal neuron with an ORANGE GFP knock-in (KI) endogenously tagging mGluR5 at the N-terminus, enhanced with anti-GFP Alexa488 labeling (left), co-stained for anti-PSD-95 Alexa594 (right). Scale bar, 20 µm. j Representative two-color gSTED image of dendrite with GFP-mGluR5 KI stained with anti-GFP Alexa488 to label surface-expressed receptors (cyan) and anti-PSD-95 Alexa594 (red). Scale bar, 2 µm. This experiment was replicated in cultures from three independent preparations of hippocampal neurons. k Zooms of dendritic spines indicated in j with asterisks. Scale bar, 1 µm. l Line profiles of spine 1 and m spine 2, indicated with dotted line in k . Data are represented as means ± SEM. *** p < 0.001. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Expressing, Labeling, Imaging, Knock-In, Staining

    a Reconstruction of single-molecule localizations obtained for SEP-mGluR5 anti-GFP nanobody Alexa647 using dSTORM (orange hot) and PSD FingR -mEos3.2 using PALM (cyan hot). Same dendritic region is shown in Fig. . Scale bar, 2 µm. b Zooms of spines shown in a . Scale bar, 500 nm. c Representative spine with single-molecule localizations of PSD FingR (cyan) and mGluR5 (orange) with indicated PSD border (black line) determined using DBScan. Scale bar, 500 nm. d Relative frequency distribution (fractions) of the distance of individual mGluR5 localizations to the PSD border ( n = 13 neurons, 253 PSDs). e For each PSD, eight rings, proportionally scaled based on its PSD border, defined the synapse (ring 1 and 2; black), perisynaptic zone (ring 3–5; orange), and extrasynaptic region (ring 6–8; blue). f Fraction of PSD FingR (black; plotted on left y axis) and mGluR5 (orange; plotted on right y-axis) localizations in rings 1–8. For each PSD, the number of localizations was normalized to the maximum number per ring, and the area per ring was calculated and corrected for. g Example spines with mGluR5 localizations (orange) belonging to clusters (black outline) as determined using DBScan, relative to PSD FingR localizations (magenta). Scale bars, 500 nm. h Relative frequency distribution (fractions) of the distance from the center of mGluR5 clusters to the border of the PSD (as indicated in g by the black lines) ( n = 273 clusters). i Relative frequency distribution (fractions) of the mGluR5 cluster area. j FWTM analysis comparing the width and length (in nm) of individual mGluR5 clusters. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Reconstruction of single-molecule localizations obtained for SEP-mGluR5 anti-GFP nanobody Alexa647 using dSTORM (orange hot) and PSD FingR -mEos3.2 using PALM (cyan hot). Same dendritic region is shown in Fig. . Scale bar, 2 µm. b Zooms of spines shown in a . Scale bar, 500 nm. c Representative spine with single-molecule localizations of PSD FingR (cyan) and mGluR5 (orange) with indicated PSD border (black line) determined using DBScan. Scale bar, 500 nm. d Relative frequency distribution (fractions) of the distance of individual mGluR5 localizations to the PSD border ( n = 13 neurons, 253 PSDs). e For each PSD, eight rings, proportionally scaled based on its PSD border, defined the synapse (ring 1 and 2; black), perisynaptic zone (ring 3–5; orange), and extrasynaptic region (ring 6–8; blue). f Fraction of PSD FingR (black; plotted on left y axis) and mGluR5 (orange; plotted on right y-axis) localizations in rings 1–8. For each PSD, the number of localizations was normalized to the maximum number per ring, and the area per ring was calculated and corrected for. g Example spines with mGluR5 localizations (orange) belonging to clusters (black outline) as determined using DBScan, relative to PSD FingR localizations (magenta). Scale bars, 500 nm. h Relative frequency distribution (fractions) of the distance from the center of mGluR5 clusters to the border of the PSD (as indicated in g by the black lines) ( n = 273 clusters). i Relative frequency distribution (fractions) of the mGluR5 cluster area. j FWTM analysis comparing the width and length (in nm) of individual mGluR5 clusters. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques:

    a Widefield of dendrite expressing Homer1c-mCherry and SEP-mGluR5. Scale bar, 5 µm. b Single-molecule trajectories (SMTs) of mGluR5 (each trajectory is assigned a random color) relative to the Homer1c PSD mask (gray) in the same dendrite as shown in a. Scale bar, 2 µm. c Example PSDs (gray) with their perisynaptic zone (orange ring) and mGluR5 SMTs color-coded for their subsynaptic localization. Scale bar, 1 µm. d Fraction of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). e Mean log D eff per neuron of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). f Relative frequency distributions of D effs of individual synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. g Fraction of perisynaptic SMTs that stay within perisynaptic/synaptic region once entered (captured), exited at least once, but end up staying inside the perisynaptic/synaptic region (returned) and perisynaptic tracks that escaped the perisynaptic/synaptic region (escaped) ( n = 25 neurons). h Mean MSD curve over time of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Widefield of dendrite expressing Homer1c-mCherry and SEP-mGluR5. Scale bar, 5 µm. b Single-molecule trajectories (SMTs) of mGluR5 (each trajectory is assigned a random color) relative to the Homer1c PSD mask (gray) in the same dendrite as shown in a. Scale bar, 2 µm. c Example PSDs (gray) with their perisynaptic zone (orange ring) and mGluR5 SMTs color-coded for their subsynaptic localization. Scale bar, 1 µm. d Fraction of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). e Mean log D eff per neuron of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs ( n = 25 neurons). f Relative frequency distributions of D effs of individual synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. g Fraction of perisynaptic SMTs that stay within perisynaptic/synaptic region once entered (captured), exited at least once, but end up staying inside the perisynaptic/synaptic region (returned) and perisynaptic tracks that escaped the perisynaptic/synaptic region (escaped) ( n = 25 neurons). h Mean MSD curve over time of synaptic, perisynaptic, and transient perisynaptic mGluR5 SMTs. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Expressing

    a Widefield image of a dendritic spine expressing SEP-mGluR5 (cyan) and Homer1c-mCherry (red). (larger ROI shown in Fig. ). Scale bar, 1 µm. b The same spine as in a showing immobile (black) and mobile (red) SMTs of mGluR5 relative to the Homer1c PSD mask (larger ROI shown in Fig. ). Scale bar, 500 nm. c Transient confinement zones (red circles) of the mobile trajectories (random colors) shown in B (larger ROI shown in Fig. ). Scale bar, 500 nm. d Relative frequency plots of the dwell time (s) and e average radius of confinement zones for mGluR5 SMTs. f Example trajectory (assigned to perisynaptic fraction) that undergoes transient confinement in the perisynaptic zone, color-coded for entering the synapse (black) and perisynaptic zone (orange) and the confinement zones (red). The trajectory starts (green dot) and ends (red dot) in perisynaptic transient confinement zones. Scale bar, 100 nm. g The diffusion coefficient and h confinement index L over time for the trajectory shown in f , using the same color-coding. i The same example synapse as in a – c with hotspots of transient confinement zones, immobile tracks, and both images combined, color-coded for the frequency of confinement zones and/or immobile tracks (larger ROI shown in Fig. ). Scale bar, 500 nm. j Relative frequency distribution of the distance of confinement zones of mGluR5 and k center of immobile mGluR5 trajectories to the border of the PSD ( = 0 and indicated by dashed line). Data in this figure is the same dataset as used in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Widefield image of a dendritic spine expressing SEP-mGluR5 (cyan) and Homer1c-mCherry (red). (larger ROI shown in Fig. ). Scale bar, 1 µm. b The same spine as in a showing immobile (black) and mobile (red) SMTs of mGluR5 relative to the Homer1c PSD mask (larger ROI shown in Fig. ). Scale bar, 500 nm. c Transient confinement zones (red circles) of the mobile trajectories (random colors) shown in B (larger ROI shown in Fig. ). Scale bar, 500 nm. d Relative frequency plots of the dwell time (s) and e average radius of confinement zones for mGluR5 SMTs. f Example trajectory (assigned to perisynaptic fraction) that undergoes transient confinement in the perisynaptic zone, color-coded for entering the synapse (black) and perisynaptic zone (orange) and the confinement zones (red). The trajectory starts (green dot) and ends (red dot) in perisynaptic transient confinement zones. Scale bar, 100 nm. g The diffusion coefficient and h confinement index L over time for the trajectory shown in f , using the same color-coding. i The same example synapse as in a – c with hotspots of transient confinement zones, immobile tracks, and both images combined, color-coded for the frequency of confinement zones and/or immobile tracks (larger ROI shown in Fig. ). Scale bar, 500 nm. j Relative frequency distribution of the distance of confinement zones of mGluR5 and k center of immobile mGluR5 trajectories to the border of the PSD ( = 0 and indicated by dashed line). Data in this figure is the same dataset as used in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Expressing, Diffusion-based Assay

    a Schematic of monomeric full-length mGluR5WT (top) and mGluR5∆C (bottom) lacking its C-terminal tail. b Representative gSTED images of dendrite expressing SEP-mGluR5WT and SEP-mGluR5∆C, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16) and mGluR5∆C (orange; n = 14) localization in spines: (1) synaptic enrichment ( p = 0.1565), (2) synaptic + perisynaptic enrichment ( p = 0.1488), (3) perisynaptic enrichment (0.3696) and (4) homogeneous distribution ( p = 0.0204; two-sided unpaired t test for each category). On top are representative images of the different categories of mGluR5 localization (cyan), relative to Homer1c (red), at spines. Scale bar, 1 µm. e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11) and mGluR5∆C ( n = 13, p < 0.0001; unpaired t test). f Fraction of synaptic ( p = 0.3529), perisynaptic ( p = 0.0218) and transient perisynaptic trajectories ( p = 0.0254) of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). g Mean log D eff per neuron of synaptic ( p = 0.5923), perisynaptic ( p = 0.0008), and transient perisynaptic ( p = 0.0025) trajectories of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). h Mean log D eff per neuron of trajectories inside confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11, p = 0.0053; two-sided unpaired t test). i Relative frequency distribution of the distance of confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11) to the border of the PSD (=0 and indicated by dashed line) (two-way repeated measures ANOVA with Bonferroni’s multiple comparisons test, at 25 nm distance from PSD border: p = 0.0003). j Example synapses of mGluR5WT and mGluR5∆C with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), k for being mobile (red) and immobile (black), l for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and m hotspots of immobile tracks (shown in k ) and confinement zones (shown in l ), color-coded for their frequency. Scale bars, 500 nm. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Schematic of monomeric full-length mGluR5WT (top) and mGluR5∆C (bottom) lacking its C-terminal tail. b Representative gSTED images of dendrite expressing SEP-mGluR5WT and SEP-mGluR5∆C, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16) and mGluR5∆C (orange; n = 14) localization in spines: (1) synaptic enrichment ( p = 0.1565), (2) synaptic + perisynaptic enrichment ( p = 0.1488), (3) perisynaptic enrichment (0.3696) and (4) homogeneous distribution ( p = 0.0204; two-sided unpaired t test for each category). On top are representative images of the different categories of mGluR5 localization (cyan), relative to Homer1c (red), at spines. Scale bar, 1 µm. e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11) and mGluR5∆C ( n = 13, p < 0.0001; unpaired t test). f Fraction of synaptic ( p = 0.3529), perisynaptic ( p = 0.0218) and transient perisynaptic trajectories ( p = 0.0254) of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). g Mean log D eff per neuron of synaptic ( p = 0.5923), perisynaptic ( p = 0.0008), and transient perisynaptic ( p = 0.0025) trajectories of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11; two-sided unpaired t test for each category). h Mean log D eff per neuron of trajectories inside confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11, p = 0.0053; two-sided unpaired t test). i Relative frequency distribution of the distance of confinement zones of mGluR5WT ( n = 8) and mGluR5∆C ( n = 11) to the border of the PSD (=0 and indicated by dashed line) (two-way repeated measures ANOVA with Bonferroni’s multiple comparisons test, at 25 nm distance from PSD border: p = 0.0003). j Example synapses of mGluR5WT and mGluR5∆C with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), k for being mobile (red) and immobile (black), l for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and m hotspots of immobile tracks (shown in k ) and confinement zones (shown in l ), color-coded for their frequency. Scale bars, 500 nm. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Expressing, Labeling

    a Schematic of mGluR5-STGtail (top) and mGluR5∆C-STGtail (bottom). b Representative gSTED images of dendrite expressing SEP-mGluR5-STGtail and SEP-mGluR5∆C-STGtail, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16), mGluR5-STGtail (blue; n = 11), and mGluR5∆C-STGtail (green; n = 25) localization in spines: (1) synaptic enrichment, (2) synaptic + perisynaptic enrichment, (3) perisynaptic enrichment and (4) homogeneous distribution ( p < 0.0001, Kruskal–Wallis test for each category with Dunn’s multiple comparisons test: p = 0.5398 for mGluR5WT vs. mGluR5-STGtail, p < 0.0001 for mGluR5WT vs. mGluR5∆C-STGtail and p = 0.0011 for mGluR5-STGtail vs. mGluR5∆C-STGtail). e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11), mGluR5-STGtail ( n = 12) and mGluR5∆C-STGtail ( n = 27; p < 0.0001, one-way ANOVA with Dunnet’s multiple comparisons test: compared to mGluR5WT p = 0.4700 for mGluR5-STGtail and p < 0.0001 for mGluR5∆C-STGtail). f Example synapses of mGluR5-STGtail and mGluR5∆C-STGtail with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), g for being mobile (red) and immobile (black), h for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and i hotspots of immobile tracks (shown in g ) and confinement zones (shown in h ), color-coded for their frequency. Scale bar, 500 nm. The mGluR5WT dataset shown in d and e is also shown in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Schematic of mGluR5-STGtail (top) and mGluR5∆C-STGtail (bottom). b Representative gSTED images of dendrite expressing SEP-mGluR5-STGtail and SEP-mGluR5∆C-STGtail, additionally labeled with an anti-GFP nanobody Atto647N (cyan), and Homer1c-mCherry (red; confocal). Scale bar, 2 µm. c Zooms of spines indicated in b with asterisks. Scale bar, 500 nm. d Quantification of mGluR5WT (black; n = 16), mGluR5-STGtail (blue; n = 11), and mGluR5∆C-STGtail (green; n = 25) localization in spines: (1) synaptic enrichment, (2) synaptic + perisynaptic enrichment, (3) perisynaptic enrichment and (4) homogeneous distribution ( p < 0.0001, Kruskal–Wallis test for each category with Dunn’s multiple comparisons test: p = 0.5398 for mGluR5WT vs. mGluR5-STGtail, p < 0.0001 for mGluR5WT vs. mGluR5∆C-STGtail and p = 0.0011 for mGluR5-STGtail vs. mGluR5∆C-STGtail). e Quantification of the ratio of spine over dendrite intensity of mGluR5WT ( n = 11), mGluR5-STGtail ( n = 12) and mGluR5∆C-STGtail ( n = 27; p < 0.0001, one-way ANOVA with Dunnet’s multiple comparisons test: compared to mGluR5WT p = 0.4700 for mGluR5-STGtail and p < 0.0001 for mGluR5∆C-STGtail). f Example synapses of mGluR5-STGtail and mGluR5∆C-STGtail with trajectories color-coded for being synaptic (black), perisynaptic (orange), and transient perisynaptic (blue), g for being mobile (red) and immobile (black), h for being transiently confined trajectories (random colors) with corresponding confinement zones (red circles), and i hotspots of immobile tracks (shown in g ) and confinement zones (shown in h ), color-coded for their frequency. Scale bar, 500 nm. The mGluR5WT dataset shown in d and e is also shown in Fig. , as these figures show different aspects of the same experiment. Data are represented as means ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Expressing, Labeling

    a Schematic of the FKBP-rapalog-FRB heterodimerization system (left) and how this system is used to recruit mGluR5 to the PSD (right). b Live-cell time-lapse images of SEP-mGluR5-FRB before (0’) and 20 and 30 min after rapalog application. The dendrites are color-coded for the fluorescence intensity of SEP-mGluR5-FRB. Scale bar, 2 µm. c Live-cell time-lapse images of the same dendrite as shown in b showing the relative localization SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) before (0’) and 20 and 30 min after rapalog application. Scale bar, 2 µm. d Zoom of spine indicated in c with asterisk before (0’) and 20 min after rapalog application. Scale bar, 500 nm. e Line profile of the spine in d , indicated with dotted line, showing the localization of mGluR5 (cyan) relative to Homer1c (red) before (0’) and 20 min after rapalog application. f Quantification of SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) intensity in spines over time upon rapalog application at t = 0 ( n = 17). Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Schematic of the FKBP-rapalog-FRB heterodimerization system (left) and how this system is used to recruit mGluR5 to the PSD (right). b Live-cell time-lapse images of SEP-mGluR5-FRB before (0’) and 20 and 30 min after rapalog application. The dendrites are color-coded for the fluorescence intensity of SEP-mGluR5-FRB. Scale bar, 2 µm. c Live-cell time-lapse images of the same dendrite as shown in b showing the relative localization SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) before (0’) and 20 and 30 min after rapalog application. Scale bar, 2 µm. d Zoom of spine indicated in c with asterisk before (0’) and 20 min after rapalog application. Scale bar, 500 nm. e Line profile of the spine in d , indicated with dotted line, showing the localization of mGluR5 (cyan) relative to Homer1c (red) before (0’) and 20 min after rapalog application. f Quantification of SEP-mGluR5-FRB (cyan) and 2xFKBP-Homer1c-mCherry (red) intensity in spines over time upon rapalog application at t = 0 ( n = 17). Data are represented as means ± SEM. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Fluorescence

    a Maximum projections of the GCaMP6f stream (50 s) in a representative dendrite before (baseline) and after 30 min rapalog application. Scale bar, 5 µm. b Zoom of spine 2 indicated in a with asterisk, expressing SNAP-mGluR5-FRB labeled with the cell-impermeable SNAPdye JF646 (cyan) and 2xFKBP-Homer1c-mCherry (red) before and 30 min after rapalog-induced recruitment. Scale bar, 1 µm. c ∆F/F 0 traces of GCaMP6f signal from two spines indicated in a with asterisks before and after 30 min of rapalog-induced recruitment of mGluR5 to Homer1c. d Quantification of mSCT frequencies upon application of rapalog in neurons expressing SNAP-mGluR5-FRB and 2xFKBP-Homer1c-mCherry ( n = 43 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test) and e in neurons expressing SNAP-mGluR5 and 2xFKBP-Homer1c-mCherry (control; n = 37 neurons, p = 0.5819, two-sided Wilcoxon matched-pairs signed rank test). f Average traces of all mSCTs per neuron (gray) and average mSCT trace of all neurons (red) before and after 30 min of rapalog. g Quantification of mSCT decay tau times (s) upon application of rapalog ( n = 37 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test). h Model of deregulated calcium signaling upon mGluR5 recruitment to the synapse during spontaneous synaptic activity. Medians are indicated by the red lines. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

    doi: 10.1038/s41467-022-35680-w

    Figure Lengend Snippet: a Maximum projections of the GCaMP6f stream (50 s) in a representative dendrite before (baseline) and after 30 min rapalog application. Scale bar, 5 µm. b Zoom of spine 2 indicated in a with asterisk, expressing SNAP-mGluR5-FRB labeled with the cell-impermeable SNAPdye JF646 (cyan) and 2xFKBP-Homer1c-mCherry (red) before and 30 min after rapalog-induced recruitment. Scale bar, 1 µm. c ∆F/F 0 traces of GCaMP6f signal from two spines indicated in a with asterisks before and after 30 min of rapalog-induced recruitment of mGluR5 to Homer1c. d Quantification of mSCT frequencies upon application of rapalog in neurons expressing SNAP-mGluR5-FRB and 2xFKBP-Homer1c-mCherry ( n = 43 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test) and e in neurons expressing SNAP-mGluR5 and 2xFKBP-Homer1c-mCherry (control; n = 37 neurons, p = 0.5819, two-sided Wilcoxon matched-pairs signed rank test). f Average traces of all mSCTs per neuron (gray) and average mSCT trace of all neurons (red) before and after 30 min of rapalog. g Quantification of mSCT decay tau times (s) upon application of rapalog ( n = 37 neurons, p < 0.0001, two-sided Wilcoxon matched-pairs signed rank test). h Model of deregulated calcium signaling upon mGluR5 recruitment to the synapse during spontaneous synaptic activity. Medians are indicated by the red lines. Source data are provided as a Source Data file.

    Article Snippet: Then neurons were incubated with rabbit anti-mGluR5 (Alomone Labs) diluted 1:50 and mouse anti-PSD-95 (Neuromab) diluted 1:300 in 0.1% Triton X-100 and 5% NGS in PBS/Gly for 2 h at RT.

    Techniques: Expressing, Labeling, Activity Assay

    Transcripts encoding metabotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Metabotropic glutamate receptor types are listed in the tables on the left. In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+

    Journal: Biology of Reproduction

    Article Title: Glutamate can act as a signaling molecule in mouse preimplantation embryos

    doi: 10.1093/biolre/ioac126

    Figure Lengend Snippet: Transcripts encoding metabotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Metabotropic glutamate receptor types are listed in the tables on the left. In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+" means upregulation and “−" means downregulation in blastocysts compared to oocytes) and corresponding P -values are shown. Transcripts were detected by RT-PCR and representative agarose gels with separated PCR products are shown in the panels on the right. Lanes: Grm1 , Glutamate receptor, metabotropic 1; Grm2 , Glutamate receptor, metabotropic 2; Grm3 , Glutamate receptor, metabotropic 3; Grm4 , Glutamate receptor, metabotropic 4; Grm5 , Glutamate receptor, metabotropic 5; Grm6 , Glutamate receptor, metabotropic 6; Grm7 , Glutamate receptor, metabotropic 7; Grm8 , Glutamate receptor, metabotropic 8; MW, molecular weight markers; A, positive control tissue; B, ovulated oocytes; C, blastocysts. The MWs in base pairs (bp) are indicated to the right of the panels. * Primers for Grm5 receptor type were designed in this study.

    Article Snippet: Anti-mGluR5 Antibody (Alomone Labs, Cat# AGC-007, dilution 1:50), Anti-GRIK3 (GluK3) Antibody (Alomone Labs, Cat# AGC-040, dilution 1:100), Anti-GRIK4 (KA1) Antibody (Alomone Labs, Cat# AGC-041, dilution 1:50), Anti-GRIK5 (GluK5) Antibody (Alomone Labs, Cat# AGC-042, dilution 1:100), GRIA3 Antibody (LifeSpan Biosciences, Cat# LS-C331307, dilution 1:50), and Anti-GluR4 (GluA4) Antibody (Alomone Labs, Cat# AGC-019, dilution 1:50).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Positive Control

    Glutamate effects are blocked with AMPA/kainate and GRM5 receptor antagonists. Cell numbers and proportions of dead cells in blastocysts pretreated with the mixture of AMPA, kainate, and GRM5 receptor antagonists (CNQX and MPEP) prior to L-glutamic acid exposure. GlAc 5 mM, blastocysts incubated with L-glutamic acid (at 5 mM final concentration) for 24 h; CNQX + MPEP, blastocysts incubated with CNQX and MPEP (at 300 μM and 10 μM final concentrations, respectively) for 24 h; GlAc 5 mM + (CNQX + MPEP), blastocysts incubated with CNQX + MPEP antagonists for 20 min prior to addition of L-glutamic acid (for the following 24 h incubation). TE, trophectoderm, ICM, inner cell mass. The number of blastocysts in the groups ( n ): Control, n = 32; GlAc 5 mM, n = 33; CNQX + MPEP, n = 28; GlAc 5 mM + (CNQX + MPEP), n = 34. The values are arithmetical mean + SEM. Statistical significance of differences: * P < 0.05, * * P < 0.01, * * * P < 0.001.

    Journal: Biology of Reproduction

    Article Title: Glutamate can act as a signaling molecule in mouse preimplantation embryos

    doi: 10.1093/biolre/ioac126

    Figure Lengend Snippet: Glutamate effects are blocked with AMPA/kainate and GRM5 receptor antagonists. Cell numbers and proportions of dead cells in blastocysts pretreated with the mixture of AMPA, kainate, and GRM5 receptor antagonists (CNQX and MPEP) prior to L-glutamic acid exposure. GlAc 5 mM, blastocysts incubated with L-glutamic acid (at 5 mM final concentration) for 24 h; CNQX + MPEP, blastocysts incubated with CNQX and MPEP (at 300 μM and 10 μM final concentrations, respectively) for 24 h; GlAc 5 mM + (CNQX + MPEP), blastocysts incubated with CNQX + MPEP antagonists for 20 min prior to addition of L-glutamic acid (for the following 24 h incubation). TE, trophectoderm, ICM, inner cell mass. The number of blastocysts in the groups ( n ): Control, n = 32; GlAc 5 mM, n = 33; CNQX + MPEP, n = 28; GlAc 5 mM + (CNQX + MPEP), n = 34. The values are arithmetical mean + SEM. Statistical significance of differences: * P < 0.05, * * P < 0.01, * * * P < 0.001.

    Article Snippet: Anti-mGluR5 Antibody (Alomone Labs, Cat# AGC-007, dilution 1:50), Anti-GRIK3 (GluK3) Antibody (Alomone Labs, Cat# AGC-040, dilution 1:100), Anti-GRIK4 (KA1) Antibody (Alomone Labs, Cat# AGC-041, dilution 1:50), Anti-GRIK5 (GluK5) Antibody (Alomone Labs, Cat# AGC-042, dilution 1:100), GRIA3 Antibody (LifeSpan Biosciences, Cat# LS-C331307, dilution 1:50), and Anti-GluR4 (GluA4) Antibody (Alomone Labs, Cat# AGC-019, dilution 1:50).

    Techniques: Incubation, Concentration Assay

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Alzheimer’s vulnerable brain region relies on a distinct retromer core dedicated to endosomal recycling

    doi: 10.1016/j.celrep.2021.110182

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-mGluR5 , Alomones lab , Cat#AGC-007; RRID: AB_2039991.

    Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Construct, Plasmid Preparation, Software

    List of TRPV1 antibodies tested on sheep DRGs

    Journal: eNeuro

    Article Title: Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect

    doi: 10.1523/ENEURO.0237-21.2021

    Figure Lengend Snippet: List of TRPV1 antibodies tested on sheep DRGs

    Article Snippet: Anti-TRPV1, guinea pig polyclonal , Primary , Alomone , AGP-118.

    Techniques: