Fig 2. Loss of astrocyte-synapse interactions and increased loss of excitatory post-synaptic marker Homer1 near hippocampal NL-F KI bulbous astrocytes.

A) Schematic of astrocytic-synapse interactions. B-F) Representative images and quantification of astrocytic EAAT1-, excitatory post-synaptic Homer1- and pre-synaptic Synaptophysin-immunoreactive puncta in the 6-mo hippocampal CA1 SR using super-resolution Airyscan confocal microscopy. B) Quantification of the number of colocalized Homer1- and Synaptophysin-immunoreactive spots shown as density per μm³ using Imaris. Transparent points = individual ROIs (3 ROIs per mouse; 15 WT and 21 NL-F KI ROIs sampled in total); full points = mouse average of ROIs (n=5–7 male mice per genotype). Unpaired two-tailed student’s t-test on mouse average. C) Quantification of the number of EAAT1-immunoreactive spots shown as density per μm³ using Imaris. Transparent points = individual ROIs (3 ROIs per mouse; 15 WT and 21 NL-F KI ROIs sampled in total); full points = mouse average of ROIs (n=5–7 male mice per genotype). Unpaired two-tailed student’s t-test on mouse average. D) Quantification of the number of colocalized EAAT1, Homer1 and Synaptophysin spots divided by the number of colocalized Homer1 and Synaptophysin spots shown as %. Transparent points = individual ROIs (3 ROIs per mouse; 15 WT and 21 NL-F KI ROIs sampled in total); full points = mouse average of ROIs (n=5–7 male mice per genotype). Unpaired two-tailed student’s t-test on mouse average. E) Quantification of the number of colocalized EAAT1, Homer1 and Synaptophysin spots divided by the number of EAAT1 spots using Imaris shown as %. Transparent points = individual ROIs (3 ROIs per mouse; 15 WT and 21 NL-F KI ROIs sampled in total); full points = mouse average of ROIs (n=5–7 male mice per genotype). Unpaired two-tailed student’s t-test on mouse average. F) Representative image of B-E for EAAT1 (blue), Homer1 (green) and Synaptophysin (red). Scale bar = 1 μm. Insets show regions of triple colocalization. G) Representative images of Ezrin (green) immunoreactivity in the 6-mo NL-F KI hippocampus near bushy or bulbous S100β⁺ astrocytes (magenta). Scale bar = 10 μm. Images are modified on ImageJ as shown by white ROI. H) Quantification of the total Ezrin spot volume on S100β⁺ astrocytes/S100β volume using Imaris 3D surface rendering and surface-spot colocalization. Transparent points = individual astrocytes (5–8 astrocytes per condition; 36 NL-F KI bushy and 30 NL-F KI bulbous astrocytes sampled in total); full points = mouse average of ROIs (n=5 male mice). Paired two-tailed student’s t-test on mouse average. I) Representative images for excitatory post-synaptic Homer1-immunoreactive puncta (green) in the 6-mo NL-F KI hippocampus near bushy or bulbous S100β⁺ astrocytes (magenta) using super-resolution Airyscan confocal microscopy. Scale bar = 1 μm. J) Quantification of the number of Homer1-immunoreactive puncta shown as density per μm³ using Imaris. Transparent points = individual ROIs (3 ROIs per condition; 18 ROIs sampled in total per condition), full points = mouse average of ROIs (n=6 male mice). Wilcoxon matched pairs signed rank test on mouse average. All data shown as mean ± SEM. p-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.