Journal: bioRxiv

Article Title: Microglia undergo transcriptional, translational and functional adaptations to dark and light phases in laboratory mice

doi: 10.1101/2023.11.17.567571

Figure Lengend Snippet: Microglia display increased synaptic pruning during the active phase In the adult hippocampus. A Schematic representation of the experimental setup. Whole hippocampi were retrieved from perfused adult male mice either 4h post lights-off (active phase) or 4h post lights-on (sleep phase) for CD11b cell enrichment and subsequent flow cytometry analysis of vGlut1-posive cells. B Heatmap showing the differential expression (in average transcripts per million, TPM), for selected genes associated with microglial phagocytic functions from the total-RNA-seq of microglia 4h post lights-off or 4h post lights-on ( , 1 a.m. versus 1 p.m., Only genes that were significantly differentially expressed are represented, adj. p -value < 0.05) C Scatter plot showing the difference in percentage of vGlut1-positive microglia cells between the active and sleep phase, 4h post lights-off and on respectively, (unpaired t-test, t(5.84) = 4.84, p = 0.0031, N = 6/group). D Histogram showing the microglial vGlut1-PE mean fluorescence intensity normalized to spleen cells as negative control (lower panel). E Scatter plot showing the area under the curve (Au) for the microglial vGlut1 mean fluorescence intensity during the active (4h after light-off) and sleep (4h post light-on) phases, normalized to spleen cells as negative controls (unpaired t-test, t(10) = 2.98, p = 0.014, N = 6/group). F Heatmap showing differential expression (in average transcripts per million, TPM), for selected genes coding for chemokines (from the total-RNA-seq of microglia 1 p.m. versus 1 a.m.). Only genes that were significantly differentially expressed are represented (adj. p -value < 0.05). G Scatter plot comparing the percentage of CD45 hi cell population in the hippocampi of adult mice 4h after light-off (N = 17) and 4h post light-on (N = 13, unpaired t-test, t(28) = 3.08, p = 0.0046). Error bars represent the mean ± standard deviation. * p < 0.05, ** p < 0.01. Images created with Biorender.com.

Article Snippet: Following fixation, intracellular staining for vGLUT1 (1/200, Miltenyi Biotec, #130-120-764, 1h in 1x BD Permeabilization Buffer) was immediately performed.

Techniques: Flow Cytometry, Quantitative Proteomics, RNA Sequencing, Fluorescence, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: Microglia undergo transcriptional, translational and functional adaptations to dark and light phases in laboratory mice

doi: 10.1101/2023.11.17.567571

Figure Lengend Snippet: Gating strategy for the flow cytometry analysis of vGLUT1- positive inclusions within hippocampal microglia cells sorted during wither the active or the sleep phase. Supplementary Figure S6: A -log(FDR) values for biological processes associated with the genes deregulated in response to LPS in function of time of injection. B Unbiased Tmod enrichment analysis for reactome gene sets for the LPS response genes regulated by time of injection.

Article Snippet: Following fixation, intracellular staining for vGLUT1 (1/200, Miltenyi Biotec, #130-120-764, 1h in 1x BD Permeabilization Buffer) was immediately performed.

Techniques: Flow Cytometry, Injection

Journal: Neuropsychopharmacology Reports

Article Title: Preliminary analysis of hippocampus synaptic apoptosis and microglial phagocytosis induced by severe restraint stress

doi: 10.1002/npr2.12298

Figure Lengend Snippet: We acquired images from CA3 stratum lucidum region in control and stressed male C‐57 BL6 mice using confocal laser scanning microscopy, subsequently for analysis converted and binarize them using image converter/analyzer. Scale bar, 5 μm. (A) representative converted (left side) and binarized (right side) images in control and stressed mice. (B) ratios of the area where VGLUT1 co‐localizes with caspase 3 to the VGLUT1+ area.10‐d water immersion restraint stress significantly increase the value (control: n = 4 animals, stress: n = 3 animals, t5 = 3.0265, P = .0292). Error bars, SD

Article Snippet: For evaluating local synaptic apoptosis, sections were double stained with rabbit anti‐cleaved caspse‐3 (1:300; Cell Signaling, #9664) and guinea pig anti‐ vesicular glutamate transporter1 (VGLUT1) antibody (1:2000; synaptic systems, #135304) for 6 days at 4°C.

Techniques: Control, Confocal Laser Scanning Microscopy

Journal: Neuropsychopharmacology Reports

Article Title: Preliminary analysis of hippocampus synaptic apoptosis and microglial phagocytosis induced by severe restraint stress

doi: 10.1002/npr2.12298

Figure Lengend Snippet: (A) Representative 3D reconstruction images obtained from CA3 stratum lucidum region in control and stressed male C‐57 BL6 mice. Iba1+ microglia and LAMP1+ structures are visualized at left side. Iba1+ microglia, LAMP1+ structures and VGLUT1+ structures are visualized at right side. Scale bar, 5 μm. (B) Ratios of the volume of the LAMP1+ structures contained in the Iba1+ microglia to the volume of the Iba1+ microglia. There was no significant difference between control and stressed groups in the ratio (control: n = 4 animals, stress: n = 3 animals, t5 = 0.8946, P = .412). Error bars, SD . (C) Ratios of the volume of engulfed VGLUT1 within LAMP1+ structures contained in the Iba1+ microglia to the volume of the Iba1+ microglia. There was no significant difference between control and stressed groups in the ratio (control: n = 4 animals, stress: n = 3 animals, t5 = 0.4414, P = .6773). Error bars, SD

Article Snippet: For evaluating local synaptic apoptosis, sections were double stained with rabbit anti‐cleaved caspse‐3 (1:300; Cell Signaling, #9664) and guinea pig anti‐ vesicular glutamate transporter1 (VGLUT1) antibody (1:2000; synaptic systems, #135304) for 6 days at 4°C.

Techniques: Control

Journal: Cell Death and Differentiation

Article Title: Kidins220/ARMS controls astrocyte calcium signaling and neuron–astrocyte communication

doi: 10.1038/s41418-019-0431-5

Figure Lengend Snippet: Kidins220 expression in astrocytes is required for proper neuronal development. a Wild-type neurons were plated on confluent wild-type (+/+) or Kidins220−/− astrocyte cultures, fixed and stained with anti-β tubulin III antibodies after 3 DIV to visualize, and quantify network development. Left: representative images of neuron–astrocyte cocultures. Scale bar, 25 µm. Middle: Sholl analysis of wild-type neurons grown on either wild-type or Kidins220−/− astrocytes for 3 DIV. Genotype effect: p = 0.03; **p < 0.01, repeated measures ANOVA followed by the Bonferroni’s multiple comparison test (n = 5 for both wild-type and Kidins220−/− cultures). Right: total dendritic length of wild-type neurons grown on either wild-type or Kidins220−/− astrocytes for 3 DIV. **p < 0.01, unpaired Student’s t test (n = 22 wild-type and Kidins220−/− cells from five independent cultures). Upper panels: representative images of wild-type neurons plated on confluent wild-type or Kidins220−/− astrocyte cultures stained with anti-VGLUT1 (b) or anti-VGAT (c) antibodies at 5, 7, and 10 DIV. Scale bars, 5 µm. Lower panels: quantification of the density of VGLUT1 and VGAT-positive boutons under the various experimental conditions. *p < 0.05, unpaired Student’s t test (n = 20 wild-type and Kidins220−/− cells from five independent cultures). Values are expressed as means ± S.E.M. in all panels

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Kidins220 (GSC16, #AB34790, Abcam, Cambridge, UK), rabbit monoclonal anti-GAPDH (14C10, #2118, Cell signaling, Leiden, The Netherlands), rabbit polyclonal anti-active caspase 3 (#AF835, R&D Systems, Minneapolis, MN, USA), rabbit anti-β tubulin III (#T2200, Sigma-Aldrich, Milan, Italy), guinea pig polyclonal anti-vesicular glutamate transporter-1 (VGLUT1, #AB5905, Merck-Millipore, Darmstadt, Germany), rabbit polyclonal anti-vesicular GABA transporter (VGAT, #131003, Synaptic System, Goettingen, Germany), mouse monoclonal anti-glial fibrillary acidic protein (GFAP, #G3893, Sigma-Aldrich), rabbit polyclonal anti-TRPV4 (#ab39260, Abcam), mouse anti-neuronal nuclei (NeuN, #MAB377, Merck-Millipore), chicken anti-NeuN (#266006, Synaptic Systems), guinea pig anti-Iba1 (#234004, Synaptic Systems), and rabbit anti-Olig2 (#AB9610, Merck-Millipore).

Techniques: Expressing, Staining