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Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with <t>control</t> <t>hGFAP-tdTomato</t> astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.
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Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with <t>control</t> <t>hGFAP-tdTomato</t> astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.
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Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with <t>control</t> <t>hGFAP-tdTomato</t> astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.
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Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with <t>control</t> <t>hGFAP-tdTomato</t> astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.
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Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with <t>control</t> <t>hGFAP-tdTomato</t> astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.
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Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with <t>control</t> <t>hGFAP-tdTomato</t> astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.
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Image Search Results


Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with control hGFAP-tdTomato astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.

Journal: Cell reports

Article Title: Mitochondrial biogenesis in developing astrocytes regulates astrocyte maturation and synapse formation.

doi: 10.1016/j.celrep.2021.108952

Figure Lengend Snippet: Figure 5. Mitochondrial content and distribution are disrupted in mGluR5-deficient astrocytes (A and B) Confocal sections show mito in L5 cortical astrocytes on P17 visualized with IUE of mitoYFP and mTAGBFP2 cytoplasmic filler. Scale bar: 10 mm. (C) Rendering of the two reconstructed astrocytes and mito networks shown in (A) and (B). Red boxed regions highlight the significant depletion of mito in small processes of amGluR5cKO astrocytes in comparison with control hGFAP-tdTomato astrocytes. (D and E) Single confocal planes of the images shown in (A) and (B). Arrowheads indicate the network of mito in the main processes. Scale bar: 5 mm. (F) Rendering of two reconstructed astrocytes and mito networks shown in (A) and (B). Arrowheads indicate some of the fragmented mito (0.3–3 mm3) highlighted in (H). (G) Quantification of mito occupancy of P17 and P50 tdTomato+ astrocytes. *p < 0.05, **p < 0.01 (n = 3–4 mice in each group; unpaired Student’s t test). (H) Pseudocolor-coded representation of volume/sphericity of the two reconstructed mito networks shown in (A) and (B). (I) Quantification of the distribution of mito morphology. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3–4 mice in each group; one-way ANOVA followed by Bonferroni’s post hoc correction). (J) Confocal sections of mito in cultured astrocytes as visualized with MitoTracker (mito = cyan, tdTomato+ astrocytes = magenta). The dotted lines show astrocyte boundaries. Scale bar: 20 mm and 5 mm. (K) Quantification of the volume of astrocytes occupied by mito on DIV10. ***p < 0.001 (n = 3 mice in each group; unpaired Student’s t test). All error bars represent mean values ± SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 5-bromo-20-deoxyuridine (BrdU) Sigma-Aldrich Cat# HY-15910 N-[2-[[[3-(4-Chlorophenyl)-2propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)4-methoxybenzenesulphonamide (KN93) Sigma-Aldrich Cat# 422711 Rapamycin Sigma-Aldrich Cat# R0395 (RS)-3,5-Dihydroxyphenylglycine (DHPG) R&D Systems Cat# 0342 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA-AM) Sigma-Aldrich Cat# 11696 Critical commercial assays ATP determination Kit ThermoFisher Cat# A22066 L-lactate assay kit Sigma Cat# MAK065 Seahorse XFp Mito stress test kit Seahorse Bioscience, Bucher Biotec AG Cat# 103010-100 Seahorse XFp Fluxpak kit Seahorse Bioscience, Bucher Biotec AG Cat# 103022-100 Deposited data No deposited data N/A N/A Experimental models: Cell lines No cell lines used N/A N/A Experimental models: Organisms/strains Mouse: B6N.129(FVB)-Ppargc1a < tm2.1Brsp > /J Case: PGC1aLox/Lox The Jackson Laboratory JAX stock #SN9666 Mouse: B6N.129(FVB)-Ppargc1a < tm2.1Brsp > /J Case x Tg(GFAP-cre/ERT2)1Fki x B6.Cg-Gt(ROSA)26Sortm14(CAGtdTomato)Hze/J: aPGC1acKO-tdTomato This paper N/A Mouse: Tg(GFAP-cre/ERT2)1Fki x B6.CgGt(ROSA)26Sortm14(CAG-tdTomato)Hze/J X mGluR5lox: amGluR5cKO-tdTomato This paper N/A Mouse: mGluR5Lox/Lox Laboratory of Margarita Behrens at Salk institute for Biological Studies Barnes et al., 2015 Mouse: Tg(GFAP-cre/ERT2)1Fki xB6.CgGt(ROSA)26Sortm14(CAG-tdTomato)Hze/J: hGFAP-tdTomato This paper N/A Mouse: TgN (hGFAP-ECFP)-GCED: GFAP-ECFP Franck Kirchhoff Lab Hirrlinger et al., 2005 Mouse: Gt(ROSA)26Sor m14(CAG-tdTomato)Hze/J: tdTomato The Jackson Laboratory JAX Stock #007914 Oligonucleotides Primers for genotyping: See Table S1 N/A Primers for semiquantitative RT-PCR: See Table S2 N/A Recombinant DNA Plasmid: PiggyBac transposase Laboratory of Franck Polleux at Columbia University N/A Plasmid: PBmTAGBFP2_P2a_mtYFP Laboratory of Franck Polleux at Columbia University N/A Software and algorithms IMARIS Bitplane AG https://imaris.oxinst.com GraphPad Prism 8.0.2 GraphPad Software LLC https://www.graphpad.com Leica LAS X Leica systems https://www.leica-microsystems.com Fiji/ImageJ NIH https://imagej.nih.gov Adobe Photoshop CS5 Adobe System Incorporated N/A Cell Reports 35, 108952, April 13, 2021 e2

Techniques: Comparison, Control, Cell Culture