Journal: Glia

Article Title: Persisting Region‐Specific Microglia Activation and Synaptic Imbalance During Remission in a DSS ‐Induced Colitis Model

doi: 10.1002/glia.70208

Figure Lengend Snippet: Divergent microglial polarization associates with region‐specific synaptic remodeling, cortical excitatory‐inhibitory imbalance and hippocampal adaptive plasticity. (a) Representative confocal IF images of cortical microglia showing Iba1 (gray), iNOS (green), and merged channels (right). Individual microglial cell showing co‐localization of Iba1 and iNOS. Scale bar = 10 μm. (b, c) Cortical microglial polarization. Mean fluorescence intensity (MFI) of iNOS and ARG1 in Iba1 + microglia from (b) prefrontal cortex (PFC) and (c) anterior cingulate cortex (ACC). (d) Synaptosomes were first identified by size (300–1000 nm, Suppl. Fig. 4f) and FM4‐64FX + membrane staining (left panel), then selected for co‐expression of the pan‐synaptic vesicle markers synaptophysin and synaptobrevin‐2 (Syp/SB2 + ). From the Syp/SB2 + population, excitatory and inhibitory postsynaptic densities were discriminated using the postsynaptic receptor subunit GluR1 (AMPA receptor subunit 1) versus the inhibitory postsynaptic scaffold proteins NLG2 (neuroligin‐2), gephyrin, and glycine receptor, Suppl. Fig.4f; within the GluR1 + population, the presynaptic vesicular glutamate transporter VGLUT1 was quantified. In parallel, the presynaptic vesicular GABA transporter VGAT was quantified directly from the Syp/SB2 + population. (e) Frequency of VGLUT1 + glutamatergic synaptosomes and GluR1 + postsynaptic densities. (f) Frequency of VGAT + GABAergic presynaptic vesicles and inhibitory postsynaptic scaffolds (NLG2, gephyrin, glycine receptor). Cortical gene expression of (g) Chil3 (Ym‐1, alternative activation marker), (h) Slc17a7 (VGLUT1), and (i) Slc32a1 (VGAT) fold change in mRNA levels normalized to Hprt . (j–m) Frequency of cortical c‐Fos immunofluorescence co‐labeled with NeuN from (k) ACC, (l) motor cortex 1 (M1), and (m) PFC. (n) Representative hippocampal microglial showing Iba1 (gray), ARG1 (yellow) and merged channels. Scale bar = 10 μm. MFI of iNOS and ARG1 in (o) dentate gyrus (DG), (p) CA1, and (q) CA3. (r, s) Hippocampal synaptosome profiling. (t–v) Hippocampal gene expression. (w–z) Hippocampal c‐Fos immunofluorescence co‐labeled with NeuN. Individual values shown in the graphs represent mean ± SEM. Statistical analysis was performed using one‐way ANOVA with Tukey's multiple comparisons test or Kruskal–Wallis test with Dunn's post hoc test as appropriate. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: Primary antibodies against synaptic proteins were incubated for 45 min at 4°C: Synaptophysin (#101308, Synaptic Systems), Synaptobrevin2 (#104318, Synaptic Systems), GluR1 (#13185, Cell Signaling Technology; #ABN421, Sigma‐Aldrich), Neuroligin2‐AF546 (#129511, Synaptic Systems), Gephyrin‐AF546 (#147011BT, Synaptic Systems), Glycine receptor‐AF546 (#146011BT, Synaptic Systems), VGLUT1 (#135011BT, Synaptic Systems), VGAT (#131011BT, Synaptic Systems).

Techniques: Fluorescence, Membrane, Staining, Expressing, Gene Expression, Activation Assay, Marker, Immunofluorescence, Labeling