Abstract
Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.
Highlights
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS)
Our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca2؉]i with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron
This increase could be completely blocked by 0.3 M MK-801 (Fig. 1C), a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist, which suggested a direct neurotoxicity evoked by the overactivation of NMDARs in our system
Summary
Western Blot Analysis of NMDAR Expression and MAPK Activation—For assessment of MAPK activation in cultured hippocampal neurons, on different days in vitro, neurons were starved for 6 h in DMEM without any supplement and stimulated with NMDA or not in Locke’s solution for 15 min as was done in the in vitro excitotoxic experiments (antagonists were pretreated for 20 min). To examine the activation of MAPKs in vivo, tissue lysates of P0 or young adult (3 month) (n ϭ 6) hippocampi were obtained by RIPA (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM PMSF, 1ϫ Roche Applied Science complete mini protease inhibitor) lysis buffer 30 min after i.c.v. injection of NMDA (60 mM, 1 l) or PBS as done in in vivo excitotoxic experiments. Data are presented as mean Ϯ S.E. from at least three independent experiments
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