Glyoxal

Primary cultures of dissociated hippocampal neurons were prepared basically as previously described (Ichinose et al., 2015; Kaech and Banker, 2006) . The dissociated hippocampal neurons at E17.5 were plated onto eight-well chambered cover glasses (Nunc) precoated with polyethyleneimine and poly-L-lysine at a density of 1.5 3 10 4 cells/well in 0.3 mL of MEM (GIBCO) supplemented with 10% horse serum, 1 mM pyruvate, 0.6% glucose, and 2 mM GlutaMAX (GIBCO) in a 5% CO 2 atmosphere. After 3-4 h of incubation, the culture medium was substituted with MEM supplemented with 1 mM pyruvic acid, 0.6% glucose, 2 mM GlutaMAX, and 2% B27 (GIBCO). The pyramidal neurons were selected based on their morphology and by negative anti-Prox1 immunostaining and used for the analyses.
The primary culture of cortical neurons was performed as previously described (Ichinose et al., 2019) .
For pharmacological treatment, 500 mM betaine (Wako), 500 mM pyridoxamine (Sigma-Aldrich), and 200 mM glyoxal (Wako) were added to the culture media during the first 24-h period after plating.