Hippocampal neurons were isolated from embryonic day (E) 17 mice (male and female), according to a previously described protocol [40 (link)]. Cytosine
β-d-arabinofuranoside hydrochloride (Sigma-Aldrich, St. Louis, MO, USA; catalog no. 855855) was added 1 d after plating, to eliminate proliferating, undifferentiated, and glial cells. To
inhibit DNMT activity, the neurons were treated with 100 μM RG108 (Wako, Tokyo, Japan; catalog no. 041-30101) overnight. To suppress spontaneous neuronal activation, the neurons were treated
with 1 μM tetrodotoxin (Abcam, Cambridge, UK; catalog no. ab120055), from 1 to 10 DIV. To induce neuronal activation, 10 DIV neurons were treated with 50 µM bicuculline (Wako; catalog no.
026-16131) and 200 μM 4-aminopyridine (Sigma-Aldrich; catalog no. 275875) for 3 or 6 h. We confirmed the cell viability after neuronal activation by observing the rare presence of active
caspase 3-positive cells (Supplementary Fig. 10 ). Lentiviruses were produced as described previously [41 (link)]. The virus was introduced into the neurons at 6 DIV.
Mouse neuroblastoma Neuro2a cells were cultured in DMEM (Nacalai Tesque, Kyoto, Japan; catalog no. 08458-45) supplemented with 10% FBS (Sigma-Aldrich; catalog no. F7524) and 20 µM retinoic
acid (Sigma-Aldrich; catalog no. R2625).
β-d-arabinofuranoside hydrochloride (Sigma-Aldrich, St. Louis, MO, USA; catalog no. 855855) was added 1 d after plating, to eliminate proliferating, undifferentiated, and glial cells. To
inhibit DNMT activity, the neurons were treated with 100 μM RG108 (Wako, Tokyo, Japan; catalog no. 041-30101) overnight. To suppress spontaneous neuronal activation, the neurons were treated
with 1 μM tetrodotoxin (Abcam, Cambridge, UK; catalog no. ab120055), from 1 to 10 DIV. To induce neuronal activation, 10 DIV neurons were treated with 50 µM bicuculline (Wako; catalog no.
026-16131) and 200 μM 4-aminopyridine (Sigma-Aldrich; catalog no. 275875) for 3 or 6 h. We confirmed the cell viability after neuronal activation by observing the rare presence of active
caspase 3-positive cells (
Mouse neuroblastoma Neuro2a cells were cultured in DMEM (Nacalai Tesque, Kyoto, Japan; catalog no. 08458-45) supplemented with 10% FBS (Sigma-Aldrich; catalog no. F7524) and 20 µM retinoic
acid (Sigma-Aldrich; catalog no. R2625).