*4.1. Organotypic Hippocampal Slices Cultures (OHSCs)*

Animal experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University Health System (permit no.: 2018-0095, approval date: 8 May 2018). Postnatal Sprague-Dawley rats (6–7 d) were used according to the method of Stoppini et al. [35]. We used 45 rat pups, and 40 hippocampal slices were used in each group (one batch contained five hippocampal slices, *n* = 8 batches). Hippocampi were dissected and placed in Gey's salt solution (Sigma, Saint Louis, MO, USA) with glucose (6.5 mg/mL). Slices (350 μm thick) were cut parallel to the transverse axis of the hippocampus using a chopper (McIlwain tissue chopper; Mickle Laboratory Engineering Ltd., Surrey, UK). Millicell culture inserts (Millipore, Billerica, MA, USA) containing five slices were cultured in 6-well plates with medium (50% opti-MEM, 25% HBSS, 25% horse serum, 6.5 mg/mL glucose, pH adjusted to 7.2) for 3 w or 9 w.

### *4.2. Propidium Iodide (PI) Staining*

To induce oxidative injury, 5 μM KA (Sigma) was applied for 18 h and was included with fresh medium in 3 w or 9 w after hippocampal slice culture (KA + vehicle). AA with medium was replaced for 24 h with 5 ug/mL of propidium iodide (PI) (5 μg/mL, Sigma), which was added to the culture medium (KA + AA). In chronic AA treatment, OHSCs were maintained in culture medium with 500 μM AA for 6 w before KA exposure (9 w-daily). As previously described [36], neuronal death was assessed by quantifying the fluorescence intensity of PI. This was based on the principle that PI is impermeable to normal plasma membranes and cells, and when cells are damaged, they migrate to the nucleus, where they form a complex with DNA, making the nucleus fluoresce. PI uptake images were captured with a fluorescence microscope digital camera (BX-51, Olympus, Tokyo, Japan) and quantified with the MetaMorph Imaging System (Universal Image Co, Downingtown, PA, USA). PI uptake was measured before (Pre) and 24 h after the application of drugs, and the value of the measured fluorescence area was expressed [(24 h − Pre/full kill − Pre) × 100] as the % PI uptake. N-methyl-D-aspartate (NMDA) (100 μM, Sigma) was applied to induce fulminant death of pyramidal neurons (full kill) at the termination of each experiment [37,38]. AA treatment alone (500 μM) showed no toxic effect in KA-untreated normal OHSCs (Normal group) at 3 w or 9 w.
