Journal: bioRxiv

Article Title: Astrocyte-derived TNF and glutamate critically modulate microglia reactivity by methamphetamine

doi: 10.1101/2021.02.22.432170

Figure Lengend Snippet: A: Primary cortical microglia expressing the ROS FRET biosensor (HSP) were incubated with ACM CT (left panel) and then exposed to ACM Meth (right panel). Time-lapses of CFP/FRET ratio changes for the HSP biosensor (normalized at 0 min) are shown according to the scale (n=4 cells pooled across two independent experiments). Scale bars, 10μm. B: Fluorescence imaging of primary cortical microglia immunolabeled for iNOS (green) and F-actin (grey; labeled with Alexa Fluor 647 Phalloidin obtained from Thermo Scientific (MA, USA)) and treated with ACM CT or ACM Meth for 24h (n=3 independent experiments). Graph (means and SEM) displays iNOS intensity normalized to the ACM CT. *p<0.05 (unpaired t test). Scale bar, 10μm. C: Primary cortical astrocytes expressing the glutamate release FRET biosensor (FLIPE) were exposed to TNF (50nM). Time-lapses of CFP/FRET ratio changes for the FLIPE biosensor (normalized at 0 min) are shown according to the scale (n=6 cells pooled across two independent experiments). Scale bars, 10μm. D: qRT-PCR for TNF, IL-1β and IL-6 from the striatum or hippocampus of mice administered with saline or binge Meth and sacrificed 24h after (n=4-5 mice per group). Graphs (means and SEM) display the fold change of indicated transcripts. *p<0.05 and **p<0.01 (unpaired t test). E: Primary cortical astrocytes expressing the endoplasmic reticulum calcium release FRET biosensor (D1ER) were exposed to Meth (100µM) (upper panels; blue circles) or TNF (50nM) (bottom panels; red circles). Time-lapses of CFP/FRET ratio changes for the D1ER biosensor (normalized at 0 min) are shown according to the scale (n=3-4 cells pooled across 2-3 independent experiments). Scale bars, 10μm. F: Primary cortical astrocytes expressing the glutamate release FRET biosensor (FLIPE) were exposed to TNF (50nM) (upper panels; black circles) or XestosponginC (500nM) + TNF (50nM) (bottom panels; lilac circles). Time-lapses of CFP/FRET ratio changes for the FLIPE biosensor (normalized at 0 min) are shown according to the scale (n=4 cells pooled across two independent experiments). Scale bars, 20μm.

Article Snippet: Primary microglia or astrocyte were plated on plastic-bottom culture dishes μ-Dish35mm (iBidi, Martinsried, DE) and transfected with FRET biosensor for glutamate (pDisplay FLIPE-600n, plasmid 13545), ROS (pFRET-HSP33 cys, plasmid 16076) or calcium (pcDNA-D1ER, plasmid 36325), all from Addgene (MA, USA) using jetPRIME ® from Polyplus (NY, USA).

Techniques: Expressing, Incubation, Fluorescence, Imaging, Immunolabeling, Labeling, Quantitative RT-PCR, Saline

Journal: bioRxiv

Article Title: Astrocyte-derived TNF and glutamate critically modulate microglia reactivity by methamphetamine

doi: 10.1101/2021.02.22.432170

Figure Lengend Snippet: A: Confocal imaging of hippocampal sections from mice treated with Meth or saline (CT) and immunostained for GFAP (green) and TNF (red). Graphs display the GFAP/TNF colocalization puncta (upper graph) or GFAP intensity (bottom graph) normalized to the CT values (3/4 sections per animal from n=3 mice). *p<0.05 (unpaired t test). Scale bars, 50μm B: Primary cortical astrocytes from WT or TNF KO mice expressing the glutamate release FRET biosensor (FLIPE) were exposed to Meth 100µM. Time-lapses of CFP/FRET ratio changes for the FLIPE biosensor (normalized at 0 min) shows the maximum effect of Meth in both genotypes and are coded according to the scale (n=3-8 cells pooled across 2-3 independent experiments). Scale bars, 10μm C: Primary cortical astrocytes expressing the glutamate release FRET biosensor (FLIPE) were exposed to Meth, BAPTA-AM (10µM) + Meth 100µM (upper panels), XestosponginC (XeC; 500nM) + Meth 100µM (middle panels) or Tetanus toxin (Tet; 500nM) + Meth (bottom panels). Time-lapses of CFP/FRET ratio changes for the FLIPE biosensor (normalized at 0 min) show the maximum effect of Meth and are coded according to the scale (n=5-7 cells pooled across 3 independent experiments). *p<0.05 (two-way ANOVA vs CT 0 min); # p<0.05 (two-way ANOVA vs CT Meth). Scale bars, 10μm. D: Fluorescence imaging of primary cortical microglia immunolabeled for iNOS and treated with glutamate 100µM (n=3 independent cultures). Graph (means and SEM) displays iNOS intensity normalized to the CT. *p<0.05 (Mann-Whitney test). Scale bar, 10μm. E: Primary cortical microglia expressing the ROS FRET biosensor (HSP) were exposed to glutamate 100µM. Time-lapses of CFP/FRET ratio changes for the HSP biosensor (normalized at 0 min) shows the maximum effect of Meth and are coded according to the scale (n=5 cells pooled across two independent experiments). *p<0.05. Scale bars, 10μm. F: Primary cortical microglia from WT or TNF KO mice expressing the ROS FRET biosensor HSP were incubated with ACM CT and then exposed to ACM Meth 100µM. Time-lapses of CFP/FRET ratio changes for the HSP biosensor (normalized at 0 min) show the maximum effect of Meth and are coded according to the scale (n=4 cells pooled across two independent experiments). *p<0.05, §non-significant. Scale bars, 10μm.

Article Snippet: Primary microglia or astrocyte were plated on plastic-bottom culture dishes μ-Dish35mm (iBidi, Martinsried, DE) and transfected with FRET biosensor for glutamate (pDisplay FLIPE-600n, plasmid 13545), ROS (pFRET-HSP33 cys, plasmid 16076) or calcium (pcDNA-D1ER, plasmid 36325), all from Addgene (MA, USA) using jetPRIME ® from Polyplus (NY, USA).

Techniques: Imaging, Saline, Expressing, Fluorescence, Immunolabeling, MANN-WHITNEY, Incubation