Dose-Dependent Modulation of NMDA Receptors: Neuroprotective Mechanisms against Oxidative Stress in Hippocampal Neurons.

Abstract

N-Methyl-D-Aspartate (NMDA) receptors are involved in synaptic plasticity and neuronal communication. They have various responses to oxidative stress based on the dosage of agonists or antagonists that may be applied. This study focuses on modulation of NMDA receptors in primary hippocampal neurons in oxidative stress condition to understand the effects of NMDA receptor activation and inhibition. In our experiments, primary hippocampal neurons were treated with NMDA and MK-801 to assess their effect on cell viability and apoptosis. Oxidative stress was induced at different concentrations, to evaluate NMDA receptor activity and the neuroprotective effects of MK-801. Apoptosis rates were specified by applying flow cytometry, and assaying caspase-3 activity. Intracellular calcium levels were monitored using fluorescent dye Fura-2 AM. NMDA at 200 μM significantly prevented the cytotoxic effect induced by H₂O₂ (P<0.001). MK-801 with concentrations of 5 to 20 μM, could reverse the cytotoxic effect of H₂O₂. As a result, it significantly inhibited the toxicity of H₂O₂ on neuronal cells (P<0.001), while 40 μM could not reverse its effects. NMDA (200 μM) increased neuronal survival to 88.3% in the presence of H₂O₂ and prevented apoptosis. MK-801 (5 μM) also elevated cell survival to 87.2%. Treatment with NMDA (200 µM) + H₂O₂ also did not show any changes in the Fura-2AM fluorescence compared to the H₂O₂ group (P>0.05). However, MK-801+ H₂O₂ reduced the effects of H₂O₂ on the fluorescence ratio and calcium influx considerably in comparison with the H₂O₂ group (P<0.01). Treatment with MK-801 (5 μM) effectively mitigated the effects of H₂O₂ on caspase-3 activity compared to the H₂O₂ group (P<0.001). Importantly, the dose-dependent effects of NMDA receptors offer a new path into finding therapeutic strategies for neurodegenerative diseases .