Document Type : Original Article
Authors
1
Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2
Department of Pharmacology and Toxicology, School of Pharmacy, Iran University of Medical Sciences, Tehran, Iran.
3
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
4
Division of Neurobiology, Faculty of Biology, Ludwig-Maximilians-Universität München, München, Germany.
5
Pain Research Center, Neuroscience Institute, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran.
6
Department of Pharmacology and Toxicology, School of Pharmacy, Iran University of Medical Sciences, Tehran, Iran.
7
Iranian National Center for Addiction Studies (INCAS), Tehran University of Medical Sciences, Tehran, Iran.
8
Electrophysiology Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran.
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.
Keywords