Journal: Cells

Article Title: Early Age- and Sex-Dependent Regulation of Astrocyte-Mediated Glutamatergic Synapse Elimination in the Rat Prefrontal Cortex: Establishing an Organotypic Brain Slice Culture Investigating Tool

doi: 10.3390/cells12232761

Figure Lengend Snippet: Progressive expression of MEGF10 and synaptic markers during the cortical critical period in male and female littermates. ( A ) Representative graphs from the Rat Brain Atlas to show the areas of tissue dissected for Western blots. ( B , D , F ) For males, representative lanes were cropped from immunoblots to show changes in MEGF10 ( B ) PSD95 ( D ) and synaptophysin ( F ) from isolated cortices of male pups at postnatal days (P) 7, 14, 21 and 32. Full blots are shown in . ( C , E , G ) Quantitative analysis of total MEGF10 ( C ), PSD95 ( E ) and synaptophysin ( G ) protein expression normalized to β-actin (for MEGF10) and cofilin (for PSD95 and synaptophysin) in male pups at different developmental stages. Each dot represents one animal. Statistical analysis was performed via a one-way ANOVA and Tukey’s multiple comparison. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ( H , J , L ) For females, representative lanes were cropped from immunoblots of MEGF10 ( H ), PSD95 ( J ) and synaptophysin ( L ) from isolated PFC of female pups at postnatal days (P) 7, 14, 21 and 32. Full blots are shown in . ( I , K , M ) Quantitative analysis of total MEGF10 ( I ), PSD95 ( K ) and synaptophysin ( M ) protein expression normalized to β-actin (for MEGF10) and cofilin (for PSD95 and synaptophysin) in female pups at different developmental stages. Each dot represents one animal. Statistical analysis was performed via one-way ANOVA and Tukey’s multiple comparison. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All data are presented as the mean ± SD.

Article Snippet: Membranes were blocked with 5% milk or BSA in Tris-buffered saline + Tween-20 (TBST) and incubated overnight at 4 °C with primary antibodies as follows: anti-MEGF10 antibody (1:500, Thermofisher, Cat#PA5-76556, Waltham, MA, USA), anti-PSD95 antibody (1:2500, Sigma-Aldrich, Cat#MAB1596, St. Louis, MO, USA), anti-synaptophysin antibody (1:5000, Abcam, Cat#ab52636, Cambridge, UK), anti-β-actin antibody (1:10,000, Abcam, Cat#ab179467) and anti-cofilin antibody (1:1000, Cell Signaling, Cat#5175S, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Isolation, Comparison

Journal: Cells

Article Title: Early Age- and Sex-Dependent Regulation of Astrocyte-Mediated Glutamatergic Synapse Elimination in the Rat Prefrontal Cortex: Establishing an Organotypic Brain Slice Culture Investigating Tool

doi: 10.3390/cells12232761

Figure Lengend Snippet: Astrocyte-mediated synaptic pruning during developmental stages in male and female infralimbic/prelimbic areas of the PFC. ( A ) Representative graphs from the Rat Brain Atlas (46) to show areas of tissue labelled for immunofluorescent immunohistochemistry and confocal imaging. ( B ) Representative confocal images of brain slices from male littermates labelled with GFAP/S100ß (astrocytes, magenta), synaptophysin (green) and LAMP1 (blue) at P7, 14, 21 and 32. Scale bar 10 µm. ( C ) Representative confocal (upper panels) and Imaris surface-rendered (lower panels) images of analyzed male astrocytes (insets from ( B )). In the 3D reconstructions, only LAMP1+ and synaptophysin+ spots inside the astrocyte volume are rendered. Scale bar 2 µm. ( D ) Quantification of the engulfed synaptophysin spots within LAMP1 spots in astrocytes of the PFC normalized to the astrocyte volume at different postnatal developmental stages in male littermates. Each dot represents the average data of 6 analyzed astrocytes from each animal: n = 4 animals. One-way ANOVA, Tukey’s multiple comparison, * p < 0.05; ** p < 0.01; **** p < 0.0001. ( E ) Representative confocal images of brain slices from female littermates labelled with GFAP/S100ß (astrocytes, magenta), synaptophysin (green) and LAMP1 (blue) at P7, 14, 21 and 32. Scale bar 10 µm. ( F ) Representative confocal (upper panels) and Imaris surface-rendered (lower panels) images of analyzed female astrocytes (insets from ( E )). In the 3D reconstructions, only LAMP1+ and synaptophysin+ spots inside the astrocyte volume are rendered. Scale bar 2 µm. ( G ) Quantification of the engulfed synaptophysin spots within LAMP1 spots in astrocytes of the PFC normalized to the astrocyte volume at different postnatal developmental stages of female littermates. Each dot represents the average data of six analyzed astrocytes from each animal: n = 4 animals. One-way ANOVA, Tukey’s multiple comparison, ** p < 0.01. All data are presented as the mean ± SD.

Article Snippet: Membranes were blocked with 5% milk or BSA in Tris-buffered saline + Tween-20 (TBST) and incubated overnight at 4 °C with primary antibodies as follows: anti-MEGF10 antibody (1:500, Thermofisher, Cat#PA5-76556, Waltham, MA, USA), anti-PSD95 antibody (1:2500, Sigma-Aldrich, Cat#MAB1596, St. Louis, MO, USA), anti-synaptophysin antibody (1:5000, Abcam, Cat#ab52636, Cambridge, UK), anti-β-actin antibody (1:10,000, Abcam, Cat#ab179467) and anti-cofilin antibody (1:1000, Cell Signaling, Cat#5175S, Danvers, MA, USA).

Techniques: Immunohistochemistry, Imaging, Comparison

Journal: Cells

Article Title: Early Age- and Sex-Dependent Regulation of Astrocyte-Mediated Glutamatergic Synapse Elimination in the Rat Prefrontal Cortex: Establishing an Organotypic Brain Slice Culture Investigating Tool

doi: 10.3390/cells12232761

Figure Lengend Snippet: MEGF10 expression in the cortex and astrocyte phagocytic capacity in infralimbic/prelimbic areas of the PFC of male and female OBSCs. ( A ) Representative lanes cropped from immunoblots show MEGF10 protein expression in the cortex isolated from male-derived OBSCs at DIV7, 14 and 21. Full blots are shown in . ( B ) Quantitative analysis of total MEGF10 protein expression normalized to β-actin in male-derived OBSCs at different timepoints. Each dot represents one animal, n = 6–7 animals. Statistical analysis was performed via one-way ANOVA repeated measures, with Tukey’s multiple comparison, ns, not significant. ( C ) Representative confocal images of OBSCs labelled with GFAP (astrocytes, magenta), synaptophysin (green) and LAMP1 (blue) at DIV7, 14 and 21 in male-derived OBSCs. Scale bar 25 µm. ( D ) Quantification of co-localized voxels positive for LAMP1, synaptophysin and GFAP. The phagocytic index is expressed as the ratio of “synphys+/LAMP1+/GFAP+” to “synphys+/LAMP1+” colocalized voxels. Each dot represents the average data of five pictures from one animal, n = 3–5 animals. Statistical analysis was performed via one-way ANOVA repeated measures (mixed effects) and Tukey’s multiple comparison, * p < 0.05; ** p < 0.01. All data are presented as the mean ± SD. ( E ) Representative lanes cropped from immunoblots show MEGF10 protein expression in the cortex isolated from female-derived OBSCs at DIV7, 14 and 21. Full blots are shown in . ( F ) Quantitative analysis of total MEGF10 protein expression normalized to β-actin in female-derived OBSCs at different timepoints. Each dot represents one animal, n = 5–7 animals. Statistical analysis was performed via one-way ANOVA repeated measures and Tukey’s multiple comparison, ns, not significant. ( G ) Representative confocal images of OBSCs labelled with GFAP (astrocytes, magenta), synaptophysin (green) and LAMP1 (blue) at DIV7, 14 and 21 in female-derived OBSCs. Scale bar 25 µm. ( H ) Quantification of co-localized voxels positive for LAMP1, synaptophysin and GFAP. The phagocytic index is expressed as the ratio of “synphys+/LAMP1+/GFAP+” to “synphys+/LAMP1+” colocalized voxels. Each dot represents the average data of five pictures from each animal, n = 4 animals. Statistical analysis was performed via one-way ANOVA repeated measures and Tukey’s multiple comparison, * p < 0.05; trend, p = 0.0795. All data are presented as the mean ± SD.

Article Snippet: Membranes were blocked with 5% milk or BSA in Tris-buffered saline + Tween-20 (TBST) and incubated overnight at 4 °C with primary antibodies as follows: anti-MEGF10 antibody (1:500, Thermofisher, Cat#PA5-76556, Waltham, MA, USA), anti-PSD95 antibody (1:2500, Sigma-Aldrich, Cat#MAB1596, St. Louis, MO, USA), anti-synaptophysin antibody (1:5000, Abcam, Cat#ab52636, Cambridge, UK), anti-β-actin antibody (1:10,000, Abcam, Cat#ab179467) and anti-cofilin antibody (1:1000, Cell Signaling, Cat#5175S, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Isolation, Derivative Assay, Comparison