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Updated: Aug 18, 2026
Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Published on: June 9, 2017
Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression
Sabine Golde1, Siddharthan Chandran, Guy C Brown
1Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, UK. sg274@cus.cam.ac.uk
Abstract:
Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.
Insights
Nitric oxide (NO) from activated microglia causes neuronal death. This neurotoxin acts directly on immature neurons but indirectly via NMDA receptors on mature neurons, indicating complex brain injury mechanisms.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Activated microglia are implicated in neurodegeneration following brain injury.
- Microglia release neurotoxins like nitric oxide (NO), but their precise role is unclear.
- Understanding NO's contribution to neurotoxicity is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the mechanisms of microglia-induced neurodegeneration.
- To determine the role of nitric oxide (NO) in neuronal death mediated by activated microglia.
- To explore the differential effects of NO on neurons of varying maturity.
Main Methods:
- Co-culture system using rat microglia and embryonic cortical neurons.
- Activation of microglia using interferon gamma (IFNγ) and lipopolysaccharide (LPS).
- Assessment of neuronal survival following NO exposure, with and without NMDA receptor blockade (MK801).
Main Results:
- Inducible NO synthase (iNOS)-derived NO from activated microglia is solely responsible for neuronal death.
- Immature neurons are sensitive to direct NO toxicity, unaffected by NMDA receptor blockade.
- Mature neurons (12 days in culture) show 60-70% rescue with MK801, indicating NO-elicited excitotoxicity via glutamate release.
- Neuronal NMDA receptor expression increases with maturation, correlating with excitotoxin susceptibility.
Conclusions:
- Nitric oxide (NO) induces neuronal death through two distinct pathways: direct toxicity to immature neurons and indirect, NMDA receptor-mediated excitotoxicity in mature neurons.
- These findings highlight a complex interplay between inflammatory and excitotoxic mechanisms in mature neuronal injury.
- The study elucidates NO's dual role in neurodegeneration, dependent on neuronal maturation state.
