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[Preprint]. 2023 Feb 2:rs.3.rs-2501039. [Version 1] doi: 10.21203/rs.3.rs-2501039/v1

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Fig. 3 — a Developmental formation and maturation of neurons, astrocytes, and synapses (top) (adapted and modified from [46]). Confocal micrographs of WFA (yellow) and AldheGFP (green) immunofluorescence in mouse cerebral cortex in postnatal days 10 (P10), 20 (P20) and 28 (P28). High magnification 3D volume images of PNNs (3^rd row from top) and astrocytes (4^th row from top) in different postnatal ages showing concurrent maturation. Scale bar 50μm in top two rows, 2μm in bottom 3 rows.

b Top, confocal micrograph of developing PNN (WFA - yellow) showing astrocytic processes (AldheGFP - green) and excitatory synaptic terminals (vGlut1 - red). The rectangular area (white) is magnified in the bottom panel images. line intensity profile of PNN (white dotted line in bottom right image) shows peaks of vGlut1 and AldheGFP in the PNN holes (marked by arrows). Gray area represents the WFA threshold. Scale 5μm (top main image) and 2μm (bottom panel images).

c Representative confocal micrographs showing NeuN (red), AldheGFP (green), and WFA (yellow) fluorescence in cortical neurons without (left panel) and with PNN (middle panel). Bottom images show the binarized pericellular astrocytic coverage area (green) of the cell (pink) of interest. Right panel shows confocal micrographs of PV (red), AldheGFP (green), and WFA (yellow) in the PV neurons with and without PNN. Bottom images show binarized pericellular astrocytic coverage areas (green) in PV+ neurons with (white arrow) and without (yellow arrow) a PNN. Scale 5μm.

d Top graph showing a significantly lower pericellular AldheGFP area (normalized to cell perimeter) in NeuN PNN+ neurons (0.27 ± 0.01) compared to the NeuN+ PNN− neurons (0.49 ± 0.08). n = 22c/3m (NeuN PNN+); n = 28c/5m (NeuN PNN⁻). Bottom graph shows a significantly lower pericellular AldheGFP area (normalized to cell perimeter) of PV+ PNN+ neurons (0.35 ± 0.1) compared to the PV+ PNN⁻ neurons (0.46 ± 0.12). n = 20c/4m (PV+ PNN+); n = 34c/7m (PV+ PNN-).

e Representative confocal micrographs showing NeuN (red), AldheGFP (green), and WFA (yellow) fluorescence in hippocampal CA1 (left), CA2 (middle), and CA3 (right) stratum pyramidale. Scale 5μm.

f Bar graph representing the mean total coverage area of AldheGFP (green) and WFA (red) in CA1, CA2, and CA3 areas. Due to PNNs, WFA covered area in CA2 is significantly higher (893.23 ± 383.56) compared to CA1 (65.16 ± 112.02) and CA3 (82.03 ± 71.57); however, AldheGFP covered area in CA2 (900.02 ± 268.61) remains statistically indifferent from CA1 (1014.81 ± 594.63) and CA3 (740.86 ± 251.12). n = 9s/3m in CA1 group; 12s/4m in CA2 and CA3 groups. Red and green lines show comparisons between red (WFA) bars and green bars (AldheGFP) respectively.

g Graphs show non-significant total astrocytic area (AldheGFP) normalized to neuronal area (NeuN) in CA1 (0.55 ± 0.35) CA2 (0.45 ± 0.15) and CA3 (0.35 ± 0.11) regions. n = 9s/3m in CA1; 10s/4m in CA2 and CA3.

h Graphs showing statistically non-significant total astrocytic area (Kir4.1) normalized to neuronal area (NeuN) in CA1 (0.63 ± 0.22) CA2 (0.45 ± 0.26) and CA3 (0.60 ± 0.28) regions. n = 9s / 3m in CA1; 12s / 4m in CA2 and 13s/4m CA3.

c, s, and m indicate the number of cells, sections, and mice respectively. Bar data are expressed as mean±SD; dots on the bars represent the individual data points. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns = P > 0.05. unpaired two-tailed t-test with welch correction in d; One-way ANOVA, Tukey’s post-hoc test in f, g, and h.