Immunofluorescence GABA and NeuN-stained tissue sections were imaged using a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss AG) with a 40× oil immersion objective with a numerical aperture (NA) of 1.4. Images were taken as z-stacks (five slices, 1-μm intervals) with a scan zoom of 1× for CA3 area and 0.5× for the CA1 area and an image size of 1024 × 1024 pixels.

For GABAergic synaptic cluster analysis, vesicular GABA transporter (VGAT), gephyrin and GABAARγ2 subunits were stained, and sections were imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss AG). For glutamatergic inputs, vesicular glutamate transporter (vglut)1-2 and PV cells stained sections were imaged. A 63× oil immersed objective with a NA of 1.4 was used for synapse analysis. Images were taken as z-stacks (five slices, 0.2-μm intervals) with a scan zoom of 1.5× and an image size of 1024 × 1024 pixels. Imaging parameters were kept constant over all conditions. After acquisition, images were processed using 2D super resolution Airy Scan processing run in automated mode. image acquisition was conducted in the stratum pyramidale and stratum radiatum of the CA1 and CA3 region.

For analysis of PNNs around PV+ interneurons, double-stained sections were imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss AG) with a 10× objective (NA 0.45) and taking image stacks composed of 22 slices at a z-intervals of 0.4 μm. Image acquisition was performed in the CA1 and the CA3 area of the hippocampus and image settings were kept constant between genotypes within every age.

Samples were imaged with a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss AG). Image stacks (six optical sections, 0.5-μm step size) from CA1 and CA3 stratum pyramidale and radiatum were acquired with at 40× objective, N.A. 1.4.

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