Journal: Frontiers in Cellular Neuroscience

Article Title: Aquaporin 4-Mediated Glutamate-Induced Astrocyte Swelling Is Partially Mediated through Metabotropic Glutamate Receptor 5 Activation

doi: 10.3389/fncel.2017.00116

Figure Lengend Snippet: Primer sequences of metabotropic glutamate receptors (mGluRs) .

Article Snippet: The selective mGluR5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 μM, TOCRIS, Bristol, UK), the selective mGlu5 antagonist fenobam (300 μM, TOCRIS) and the non-selective glutamate transporter inhibitor DL-threo-β-benzyloxyaspartic acid (DL-TBOA, 1 mM, TOCRIS) were added for 48 h with or without 1 mM glutamate to investigate their effect on AQP4 expression and astrocyte swelling.

Techniques: Sequencing

Journal: Frontiers in Cellular Neuroscience

Article Title: Aquaporin 4-Mediated Glutamate-Induced Astrocyte Swelling Is Partially Mediated through Metabotropic Glutamate Receptor 5 Activation

doi: 10.3389/fncel.2017.00116

Figure Lengend Snippet: mGlu5 mediated glutamate-induced astrocyte swelling and changes in AQP4 expression. (A) Expression of metabotropic glutamate receptor (mGluR) in cultured astrocytes, as measured by RT-PCR. (B) Quantitative analysis of data from (A) . Results showed that mGluR1, 3, 5, 7 and 8 were expressed in astrocytes, and mGluR5 expression was the highest. (C) Co-expression of AQP4 and mGluR5 in cultured astrocytes, as measured by double-immunofluorescence staining. (D) Quantitative analysis of cell perimeters after GFAP immunofluorescence staining. Compared to the control, the cell perimeters of astrocytes in the Glu and DHPG groups were larger, whereas the DMSO group showed no difference compared to the control. Cell perimeters in the Glu + Fenobam group were smaller than in the Glu group, and likewise smaller in the DHPG + Fenobam group vs. the DHPG group. However, there were no cell perimeter differences between the Glu + DL-TBOA and Glu groups. (E) Quantitative analysis of qRT-PCR results showed that AQP4 mRNA expression in the Glu and DHPG group was higher than in the control group, whereas the DMSO group did not differ from control. Compared to the Glu group, expression was lower in the Glu + Fenobam group, but the level in the Glu + DL-TBOA group did not differ. The level of AQP4 mRNA was lower in the DHPG + Fenobam group compared to the DHPG group. * P < 0.05, compared to the control group; # P < 0.05, compared to the Glu group; & P < 0.05, compared to the DHPG group; n = 9; graphed data show mean ± SD. DHPG, (S)-3,5-dihydroxyphenylglycine, an mGluR5 agonist; DL-TBOA, DL-threo-β-benzyloxyaspartic acid, a glutamate transporter-1 (GLT-1) inhibitor.

Article Snippet: The selective mGluR5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 μM, TOCRIS, Bristol, UK), the selective mGlu5 antagonist fenobam (300 μM, TOCRIS) and the non-selective glutamate transporter inhibitor DL-threo-β-benzyloxyaspartic acid (DL-TBOA, 1 mM, TOCRIS) were added for 48 h with or without 1 mM glutamate to investigate their effect on AQP4 expression and astrocyte swelling.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Double Immunofluorescence Staining, Immunofluorescence, Staining, Control, Quantitative RT-PCR

Journal: Frontiers in Cellular Neuroscience

Article Title: Aquaporin 4-Mediated Glutamate-Induced Astrocyte Swelling Is Partially Mediated through Metabotropic Glutamate Receptor 5 Activation

doi: 10.3389/fncel.2017.00116

Figure Lengend Snippet: Increased co-expression of mGluR5 and AQP4 in a rat model of transient middle cerebral artery occlusion (tMCAO). (A) Hematoxylin and eosin (HE) staining showed that neuronal cells in the sham group were arranged regularly, and glial cells and capillary morphogenesis were normal. After tMCAO, most cell arrangement was disordered, with pyknotic or severely shrunken nuclei; in certain areas, hypervacuolization and cellular edema were found. (B) Double immunofluorescence staining showed the expression of GFAP (red) and AQP4 (green) in astrocytes, which increased after tMCAO. (C) Double immunofluorescence staining showed that mGluR5 (red) and AQP4 (green) were rarely co-expressed in pericapillary areas in the normal brain, but co-expression was increased in the pericapillary area and cell membranes of astrocytes after tMCAO. Scale bar shows 20 μm. (D) Statistical analysis of chart (B) . (E) Colocalization of AQP4 and mGluR5 was determined using ImageJ software. Three independent fields containing more than three randomly selected images per condition in three different experiments were assessed and analyzed, and the average Mander’s overlap coefficient was calculated. * P < 0.05, compared to the sham group, graphed data show mean ± SD.

Article Snippet: The selective mGluR5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 μM, TOCRIS, Bristol, UK), the selective mGlu5 antagonist fenobam (300 μM, TOCRIS) and the non-selective glutamate transporter inhibitor DL-threo-β-benzyloxyaspartic acid (DL-TBOA, 1 mM, TOCRIS) were added for 48 h with or without 1 mM glutamate to investigate their effect on AQP4 expression and astrocyte swelling.

Techniques: Expressing, Staining, Double Immunofluorescence Staining, Software

Journal: bioRxiv

Article Title: SRC family kinase inhibition rescues molecular and behavioral phenotypes, but not protein interaction network dynamics, in a mouse model of Fragile X syndrome

doi: 10.1101/2023.06.20.545800

Figure Lengend Snippet: A) Experimental design. B) Principal component graph of median fluorescent intensity (MFI) matrices from N=4 cultures of FMR1 -/y or WT neurons treated with aCSF, NMDA or DHPG. Each colored dot represents an independent agonist-treated cell culture. C) Topological overlap matrix (TOM) plot showing clustering of individual protein interactions into modules. Each point on the dendrograms represents an individual interaction, each pixel of the plot indicates the correlation between the interaction on the X and Y axis (dark = higher correlation coefficient), and the colored boxes outline each module of interest. D) Module-trait correlation table shows the correlation coefficient (top number) and p-value (bottom number) of the correlation between the eigenvector describing each module (indicated by colored bars at left), and the binary-coded hypothesis listed below the table. E) Interactions that were both individually statistically significant by a Bonferroni-corrected adaptive non-parametric test corrected for multiple comparisons comparing treatment groups (ANC, see methods), and were a member of a module of interest, are represented by a row-scaled heatmap (blue-low abundance, orange-high abundance). Interaction labels are colored based on their assigned module. F) Example of an interaction in the NMDA-responsive blue module, Homer1_SAPAP. MFI, median fluorescent intensity. Asterisk represents statistically significant difference measured by ANC. G) Averaged scaled value of all interactions in the NMDA-responsive blue module. Asterisks represent statistically significant difference by one-way ANOVA followed by Sidak’s multiple comparison test, p<0.05. H) Example of an interaction in the DHPG-responsive brown module, CAMKII_Fyn. Asterisk represents statistically significant difference by ANC, p<0.05. I) Averaged scaled value of all interactions in the DHPG-responsive brown module. Asterisks represent statistically significant difference by one-way ANOVA followed by Sidak’s multiple comparison test, p<0.05. J) Example of an interaction in the genotype-correlated yellow module, PI3K_PI3K. Asterisk represents statistically significant difference by ANC, p<0.05. K) Averaged scaled value of all interactions in the genotype-correlated yellow module. Asterisks represent statistically significant difference by one-way ANOVA followed by Sidak’s multiple comparison test, p<0.05. N = 4 cultures from 4 different mice per condition.

Article Snippet: Type I mGluR agonist DHPG (Tocris, 0805; 100 µM) was dissolved in HEPES-aCSF in the presence of non-mGluR glutamate receptor blockers (CNQX, Tocris, 0190, 40 µM; Nimodipine, Tocris, 0600, 1 µM; D(-)-2-Amino-5 phosphonopentanoic acid (APV), Sigma, A5282, 50 µM).

Techniques: Cell Culture, Comparison

Journal: bioRxiv

Article Title: SRC family kinase inhibition rescues molecular and behavioral phenotypes, but not protein interaction network dynamics, in a mouse model of Fragile X syndrome

doi: 10.1101/2023.06.20.545800

Figure Lengend Snippet: A) Experimental design. B-G) Average scaled values of protein interaction modules that respond to NMDA (blue, B-D) and DHPG (brown, E-G) in WT and FMR1 -/y brain slices at P7 (B,E), P17 (C,F) and P60 (D,G). Asterisks represent statistically significant difference by one-way ANOVA followed by Sidak’s multiple comparison test, p<0.05. H-J) Node-edge diagrams show protein interactions that changed in response to DHPG stimulation (compared to aCSF) in WT (H) or FMR1 -/y slices (I) at P17, or interactions that differed comparing FMR1 -/y _aCSF vs. WT_aCSF at p17 (J). Nodes represent proteins, edges represent protein-protein interactions that were significantly different in the given comparison; red = increased, blue = decreased. The relative magnitude of the change is reflected in edge thickness. All interactions shown were both statistically significant by a Bonferroni-corrected adaptive non-parametric test corrected for multiple comparisons (ANC), and were a member of a significant module. N = 4 animals per age/condition.

Article Snippet: Type I mGluR agonist DHPG (Tocris, 0805; 100 µM) was dissolved in HEPES-aCSF in the presence of non-mGluR glutamate receptor blockers (CNQX, Tocris, 0190, 40 µM; Nimodipine, Tocris, 0600, 1 µM; D(-)-2-Amino-5 phosphonopentanoic acid (APV), Sigma, A5282, 50 µM).

Techniques: Comparison

Journal: bioRxiv

Article Title: SRC family kinase inhibition rescues molecular and behavioral phenotypes, but not protein interaction network dynamics, in a mouse model of Fragile X syndrome

doi: 10.1101/2023.06.20.545800

Figure Lengend Snippet: A) Experimental design. B) The averaged scaled value of the NMDA-responsive blue module was significantly reduced following NMDA in all groups. FMR1 -/y showed significant module pre-activation, which was significantly exacerbated by SCB. C) The averaged scaled value of the DHPG-responsive brown module was significantly increased following DHPG in WT slices. In FMR1 -/y slices, the module did not respond to DHPG with or without SCB pretreatment. D) Experimental design. E) Interactions that were both individually statistically significant by a Bonferroni-corrected adaptive non-parametric test corrected for multiple comparisons (ANC) comparing treatment groups, and were a member of a CNA module that correlated with genotype, are represented by a row-scaled heatmap (blue-low abundance, orange-high abundance). Dotted lines divide interactions that were abnormal in FMR1 -/y slices and returned to WT levels by SCB and MPEP (top), those that were changed by SCB and MPEP (middle), and those that remained abnormal despite SCB/MPEP treatment (bottom). F,G) Two interactions were ‘normalized’ by SCB and MPEP treatment, including NMDAR2A_Shank3 (F) and SynGAP_Shank1 (G). MFI, median fluorescent intensity. Asterisks represents statistically significant difference by ANC, p<0.05. N = 4 WT and 4 FMR1 -/y animals; slices from each FMR1 -/y animal were treated with aCSF, SCB and MPEP to directly compare the effects of treatment.

Article Snippet: Type I mGluR agonist DHPG (Tocris, 0805; 100 µM) was dissolved in HEPES-aCSF in the presence of non-mGluR glutamate receptor blockers (CNQX, Tocris, 0190, 40 µM; Nimodipine, Tocris, 0600, 1 µM; D(-)-2-Amino-5 phosphonopentanoic acid (APV), Sigma, A5282, 50 µM).

Techniques: Activation Assay

Journal: Heliyon

Article Title: The protective effects of methylene blue on astrocytic swelling after cerebral ischemia-reperfusion injuries are mediated by Aquaporin-4 and metabotropic glutamate receptor 5 activation

doi: 10.1016/j.heliyon.2024.e29483

Figure Lengend Snippet: Co-expression of AQP4 and mGluR5 in astrocytes. (A) Immunofluorescence double staining of AQP4 and mGluR5 in cultured astrocytes. (B) GFAP and AQP4 double immunofluorescence staining in the brain tissues of each group. Scale bars show 20 μm.

Article Snippet: Twenty-four h prior to OGD, 100 μM (S)-3,5-dihydroxyphenylglycine (DHPG, selective mGluR5 agonist, Cat.# 0805, TOCRIS, Bristol, UK) and 300 μM fenobam (selective mGlu5 antagonist, Cat.# 2386, TOCRIS) were administered to examine their effects on AST swelling and AQP4 expression.

Techniques: Expressing, Immunofluorescence, Double Staining, Cell Culture, Double Immunofluorescence Staining

Journal: Heliyon

Article Title: The protective effects of methylene blue on astrocytic swelling after cerebral ischemia-reperfusion injuries are mediated by Aquaporin-4 and metabotropic glutamate receptor 5 activation

doi: 10.1016/j.heliyon.2024.e29483

Figure Lengend Snippet: Effect of MB on the expression of mGluR5 and AQP4 in tMCAO brain tissue. (A). The expression of AQP4 and mGluR5 in cultured astrocytes by Western blotting and its subsequent analysis using ImageJ software (B). Double immunofluorescence staining showing the expression of mGluR5 and AQP4 in pericapillary areas in the normal brain and cell membranes of astrocytes after tMCAO, and MB treatment. Scale bars show 20 μm. All results are obtained on the basis of 3 repeated independent samples which were repeated for 3 times. Values are shown as the mean ± SD, ***p < 0.001 vs. control; ##p < 0.01, ###p < 0.001 vs. OGD/R group.

Article Snippet: Twenty-four h prior to OGD, 100 μM (S)-3,5-dihydroxyphenylglycine (DHPG, selective mGluR5 agonist, Cat.# 0805, TOCRIS, Bristol, UK) and 300 μM fenobam (selective mGlu5 antagonist, Cat.# 2386, TOCRIS) were administered to examine their effects on AST swelling and AQP4 expression.

Techniques: Expressing, Cell Culture, Western Blot, Software, Double Immunofluorescence Staining, Control

Journal: Heliyon

Article Title: The protective effects of methylene blue on astrocytic swelling after cerebral ischemia-reperfusion injuries are mediated by Aquaporin-4 and metabotropic glutamate receptor 5 activation

doi: 10.1016/j.heliyon.2024.e29483

Figure Lengend Snippet: The effect of MB on astrocyte swelling and AQP4 (regulation of mGluR5). (A). immunofluorescence staining for GFAP in cultured astrocytes. Scale bars show 50 μm. (B). Astrocyte perimeters in each group measured according to Panel (A). Ten cells in each field under high magnification (200 × ) were randomly selected. Three fields in three wells were measured in every group, and the values of nine fields were calculated as the cell perimeter of each group. (C). AQP4 protein expression in each group determined through Western blotting. (D). Quantitative analysis of AQP4 protein expression in each group. All results are obtained on the basis of 3 repeated independent samples which were repeated for 3 times. All data are shown as the mean ± SD, *p < 0.05 vs. control.

Article Snippet: Twenty-four h prior to OGD, 100 μM (S)-3,5-dihydroxyphenylglycine (DHPG, selective mGluR5 agonist, Cat.# 0805, TOCRIS, Bristol, UK) and 300 μM fenobam (selective mGlu5 antagonist, Cat.# 2386, TOCRIS) were administered to examine their effects on AST swelling and AQP4 expression.

Techniques: Immunofluorescence, Staining, Cell Culture, Expressing, Western Blot, Control