Mitochondrial nitric oxide mediates decreased vulnerability of hippocampal neurons from immature animals to NMDA

Abstract

Mitochondrial membrane potential (Δψm)-dependent Ca2+ uptake plays a central role in neurodegeneration after NMDA receptor activation. NMDA-induced Δψm dissipation increases during postnatal development, coincident with increasing vulnerability to NMDA. NMDA receptor activation also produces nitric oxide (NO), which can inhibit mitochondrial respiration, dissipating Δψm. Because Δψm dissipation reduces mitochondrial Ca2+ uptake, we hypothesized that NO mediates the NMDA-induced Δψ m dissipation in immature neurons, underlying their decreased vulnerability to excitotoxicity. Using hippocampal neurons cultured from 5- and 19-d-old rats, we measured NMDA-induced changes in [Ca2+] cytosol, Δψm, NO, and [Ca2+] mito. In postnatal day 5 (P5) neurons, NMDA mildly dissipated Δψm in a NO synthase (NOS)-dependent manner and increased NO. The NMDA-induced NO increase was abolished with carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone and regulated by [Ca2+] mito. Mitochondrial Ca2+ uptake inhibition prevented the NO increase, whereas inhibition of mitochondrial Ca2+ extrusion increased it. Consistent with this mitochondrial regulation, NOS and cytochrome oxidase immunoreactivity demonstrated mitochondrial localization of NOS. Furthermore, NOS blockade increased mitochondrial Ca2+ uptake during NMDA. Finally, at physiologic O2 tensions (3% O2), NMDA had little effect on survival of P5 neurons, but NOS blockade during NMDA markedly worsened survival, demonstrating marked neuroprotection by mitochondrial NO. In P19 neurons, NMDA dissipated Δψm in an NO-insensitive manner. NMDA-induced NO production was not regulated by Δψm, and NOS immunoreactivity was cytosolic, without mitochondrial localization. NOS blockade also protected P19 neurons from NMDA. These data demonstrate that mitochondrial NOS mediates much of the decreased vulnerability to NMDA in immature hippocampal neurons and that cytosolic NOS contributes to NMDA toxicity in mature neurons. Copyright © 2005 Society for Neuroscience.