Dihydrokainate-sensitive neuronal glutamate transport is required for protection of rat cortical neurons in culture against synaptically released glutamate

Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, L-serine-O-sulphate, but not by L-alpha-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20-24 h exposure to 1 mM dihydrokainate reduced cell survival to only 14.8 +/- 9.8% in neuronal cultures (P < 0.001; n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05; n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 +/- 4.9 mu m, compared to 1.2 +/= 0.3 mu M in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 mu M MK-801, 10 mu M tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mM) inhibited the (45)calcium uptake stimulated by 30 mu M N-methyl-D-aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mM dihydrokainate produced approximate to 25% inhibition of 30 mu M NMDA-induced current in cortical neurons. Dihydrokainate (1 mM) alone generated a small current (17% of the current produced by 30 mu M NMDA) that was blocked by 30 mu M 5,7-dichlorokynurenate and only weakly by 10 mu M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.