Primary cortical GABAergic interneuron and excitatory neuron cultures were prepared from embryonic day 15 (E15) embryos (vaginal plug = E0) according to standard protocols. Briefly, Atg7flox/flox or WT embryos were dissected from pregnant dam, with cortex and MGE subdissected and pooled in ice-cold Hanks’ balanced salt solution (HBSS). Dissected tissues were washed three times with ice-cold HBSS, followed by trypsinization for 15 min at 37°C. Trypsinization was terminated by the addition of equal volume of 10% fetal bovine serum (FBS)–Dulbecco’s modified Eagle’s medium (DMEM), and dissected tissues were triturated gently. Cell suspension was filtered through a 40-μm cell strainer to remove undigested tissues and plated onto polyethylenimine-coated cell culture plates or glass coverslips. Primary neuronal cultures were maintained in MACS Neuro Medium (130-093-570, Miltenyi Biotec Inc.) supplemented with MACS NeuroBrew-21 (130-093-566, Miltenyi Biotec Inc.), 0.5 mM GlutaMAX (35050061, Thermo Fisher Scientific Inc.), and 1% penicillin/streptomycin mix (09367-34, Nacalai Tesque Inc.) in a 37°C humidified incubator. All experiments were performed after 15 days in vitro (DIV15), with lentiviral infection performed on DIV0 for Atg7 deletion by Cre recombinase expression or GABARAPL2 ΔN overexpression, DIV5 for p62 overexpression or Tsc2 knockdown, and DIV10 for GABARAPL2 knockdown. For biochemical experiments, cells were plated on coated six-well plates at a density of 2.0 × 106 cells per well and infected with 20 to 25 μl of lentivirus per well. For immunofluorescence and electrophysiology experiments, cells were plated on coated glass coverslips at a density of 4.0 × 104 cells per well, maintained in 24-well plates, and infected with 1 μl of lentivirus per well.

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