Related Experiment Video
Updated: Aug 9, 2026
Primary Culture of Mouse Dopaminergic Neurons
Published on: September 8, 2014
Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice
Hiroshi Katsuki1, Mitsugi Okawara, Haruki Shibata
1Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Abstract:
Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.
Insights
Thrombin triggers microglial activation, leading to nitric oxide (NO) production and dopaminergic neuron death in the midbrain. Inhibiting NO production or mitogen-activated protein kinases (MAPKs) protects these neurons.
Area of Science:
- Neuroscience
- Cell Biology
- Toxicology
Background:
- Activated microglia play a role in neurodegenerative diseases.
- Thrombin, a protease, activates microglia and is implicated in neuronal damage.
Purpose of the Study:
- To investigate the mechanisms of thrombin-induced neurotoxicity in midbrain dopaminergic neurons.
- To determine the role of microglia and nitric oxide (NO) in this process.
Main Methods:
- Organotypic midbrain slice cultures were treated with thrombin.
- Measurements included dopaminergic neuron count, NO production, lactate dehydrogenase release, and propidium iodide uptake.
- Inhibitors of inducible NO synthase (iNOS) and mitogen-activated protein kinases (MAPKs) were used.
- Microglia depletion and antioxidative drugs were also employed.
Main Results:
- Thrombin caused dopaminergic neuron loss, increased NO production, and tissue injury.
- Inhibition of iNOS protected dopaminergic neurons but not overall tissue.
- MAPK inhibitors (ERK, p38, JNK) reduced iNOS induction and dopaminergic cell death.
- Microglia depletion abrogated thrombin-induced NO production and dopaminergic cell death.
- Antioxidative drugs protected dopaminergic neurons but not tissue.
Conclusions:
- NO production from MAPK-dependent microglial iNOS induction is critical for thrombin-induced dopaminergic neurodegeneration.
- Thrombin-induced damage to other midbrain cells is MAPK-dependent but NO-independent.
