This article describes a method for inducing neuronal excitotoxicity in mouse primary cerebellar granule neurons using NMDA and glycine. The process leads to significant cellular damage and neuronal death, allowing for further analysis of excitotoxicity mechanisms.
Begin with a culture of mouse primary cerebellar granule neurons.
Add high concentrations of N-methyl-D-aspartate, or NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. Incubate the culture.
NMDA and glycine bind to their respective sites on the NMDA receptors, leading to overstimulation and causing a massive influx of calcium ions.
The increased intracellular calcium activates calcium-dependent phospholipases, which degrade cell membrane phospholipids, resulting in membrane damage.
Additionally, intracellular calcium activates proteases that degrade cellular proteins.
The elevated calcium levels also induce mitochondrial stress and fragmentation, leading to the release of reactive oxygen species, or ROS, and causing oxidative stress.
Furthermore, intracellular calcium activates DNases, which, together with ROS, cause DNA damage, ultimately resulting in neuronal death.
Replace the medium with fresh medium without NMDA and glycine to halt the signaling cascade.
The NMDA-induced neuronal injury model is ready for further analysis.
To induce neuronal excitotoxicity with NMDA, following seven days in vitro, treat the cerebellar granule neurons with 100 micromolar NMDA and 10 micromolar glycine for one hour. Then, replace the medium with conditioned medium from the parallel cultures with no treatment. This concentration results in 50% cell death at 24 hours post-treatment.
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