Pilocarpine is a known muscarinic receptor agonist developed to treat dry mouth and glaucoma. It is also used as a model for rat epilepsy, where it induces status epilepticus at relatively high doses (380 mg/kg) resulting in brain injury and neuronal loss, thus producing an epileptic phenotype.
Cyprotex has focused our research on pilocarpine as a reference neurotoxin, using the drug to examine several in vitro cell-based models of seizurogenic neurotoxicity. Using a microelectrode array (MEA) platform, in 3 different cell types:
- Cryopreserved rat cortical neurons
- Cryopreserved rat hippocampal neurons
- Co-cultured human iPSC-derived iCell glutamatergic neurons and astrocytes
After 14 days in vitro (DIV), the human co-culture showed significant sensitivity to pilocarpine-induced CNS liability: activity rates and network synchrony decreased, and a significant deterioration in burst structure was observed.
Neither the rat cortical neurons nor the hippocampal neurons produced a seizurogenic phenotype when tested at 14 DIV. However, further testing at 21 DIV demonstrated significant increased effects on these cell types.
Investigations into muscarinic acetylcholine receptor expression levels (M1, M2, M3 and M4) were performed at 7 and 14 DIV for all three cell types, and at 21 and 28 DIV in the rat neurons:
- Rat cortical neurons express M2 at 14 DIV, but do not express M3 and M4 until day 21
- Rat hippocampal neurons express M2 at 14 DIV, but did not express M3 and M4 until day 21
- iCell glutaneurons express M2, M3 and M4 at 14 DIV
The results of this study have suggested that there is a maturation-dependent effect in primary rat neuronal cultures and that response to pilocarpine is mediated by M3 or M4.
This research was presented at the 58th annual SOT meeting and ToxExpo, Baltimore, March 10-14 2019.