In Vitro Neurotoxicity Assay

Hippocampal neurons were subjected to neurotoxicity as previously described¹⁴. At DIV13, primary hippocampal cells cultured in glass chamber slides (Lab-Tek) were transfected with DsRed and pRK5, SEP-GluN2A, SEP-GluN2B, or SEP-GluN2C. At DIV15, the medium was replaced with standard HEPES-buffered saline solution (7.4 pH) containing (in mM): 116 NaCl, 5.4 KCl, 0.80 MgSO₄, 1.01 NaH₂PO₄, 25 NaHCO₃, 12 HEPES, 5.5 D-glucose, 1.8 CaCl₂. Time-lapse imaging was performed for 25 min following 25–200 μM NMDA (Tocris Bioscience) treatment and images collected every 2.5 min. Confocal images were acquired using either 40× or 63× objective. Serial optical sections were obtained at 0.5–1 μm intervals. Neurotoxicity was determined by cell morphology (cell body swelling and dendritic varicosities) visualized by DsRed fluorescence. Quantification of dendritic varicosities was performed on three dendrites at least 10 μm away from the soma that were randomly chosen, and the number of beaded structures along the same 10 μm length of dendrite was counted. The data were expressed as the number of varicosities per 10 μm of dendrite. Receptor spine/dendritic segment enrichment was determined as previously described¹⁸. Enrichment of receptors on spines is defined as: (spine green/spine red fluorescence)/(dendrite green/dendrite red fluorescence).