Fig. 1.

Copper protects hippocampal neurons from NMDA receptor-mediated excitotoxicity. (A) Rat hippocampal neurons were cultured and stimulated (300 μM glutamate/30 μM glycine/10 μM CNQX in ECS) or mock-treated (ECS with 2 mM MgCl₂) for 5 min, followed by incubation in conditioned media for 24 hr after insult. Neurons were then incubated with 2 μM calcein AM for 30 min at 37°C, washed in dye-free ECS with 2 mM MgCl₂, and assayed for viability via epifluorescent microscopy as described in Materials and Methods. In some experiments, neurons were preexposed to 200 μM BCS for 24 hr (BCS) or treated with 200 μM CuCl₂ during the insult (Cu). Mock-treated coverslips were exposed to ECS with 2 mM MgCl₂. (B) Neuronal survival was quantitated after an insult of 300 μM glutamate plus 30 μM glycine with an additional 200 μM CuCl₂ (Glu/Gly & CNQX) as described above. In some experiments, neurons were preexposed to 200 μM BCS for 24 hr (BCS), treated with 200 μM CuCl₂ during the insult (Cu), preexposed to 200 μM BCS for 24 hr and treated with 200 μM CuCl₂ during the insult (BCS + Cu), or pretreated with 200 μM CuCl₂ for 5 min before the insult (Cu 5′). (C) Rat hippocampal neurons were cultured and stimulated with Glu/Gly plus CNQX alone (upper trace) or with 200 μM CuCl₂ (lower trace) for the times indicated, and Ca²⁺ influx was assayed as described in Materials and Methods. (D) Rat hippocampal neurons were cultured and stimulated with Glu/Gly plus CNQX alone (Control) or with 200 μM CuCl₂ (Cu), and the relative Ca²⁺ influx at 5 min was determined as described. (∗, P < 0.05.)