Dendrite arbor reconstructions and spine density analysis

P1 pups were ICV-injected with 3 x 10⁸ pAAV-FLEX-tdTomato viral particles (Addgene #28306-AAV9) combined with 40 µg of ASOs, as detailed above. At P18, mice were anesthetized with ketamine/xylazine and transcardially perfused with ice-cold 4% paraformaldehyde (PFA). Brains were removed and post-fixed overnight in 4% PFA at 4 °C with gentle agitation. For dendritic reconstructions, 250 µm coronal slices were sectioned using a Leica VT1000S vibratome. Slices containing the hippocampus were incubated with a 1:800 Hoechst 33342 solution in PBS for 10 min at RT under gentle agitation and then washed 3X with PBS. Slices were subsequently mounted onto glass slides with Fluoromount-G. CA1 pyramidal tdTomato⁺ neurons were identified based on the cell body location relative to other hippocampal areas. Full z-stack images were acquired on a Nikon A1R confocal Ti2-E microscope. Dendritic arbors were traced using the Simple Neurite Tracer (SNT) plugin of the Fiji software, as previously described⁶⁵. Sholl analysis, total dendrite length, and branching point number were subsequently quantified using the same software. For spine density analysis, 50 µm coronal slices were acquired on a vibratome. Sections containing the hippocampus were blocked for 1 hr in 5% normal goat serum (NGS), 1% BSA, and 0.3% Triton-X100 in PBS at RT. Sections were then incubated overnight at 4 °C with rabbit anti-RFP. Sections were washed three times for 10 min and incubated for 90 min with goat anti-rabbit Alexa Fluor 488. Brain sections were washed 3 times and counterstained with Hoechst 33342. Spines on secondary apical dendrites of CA1 hippocampal pyramidal neurons were imaged and analyzed with NeuroLucida360 and the dendritic length was calculated using NeuroExplorer 360 as described⁶⁶.