Evaluating Pilocarpine Using Multiple Neuronal Cell Types in an in vitro Model for Detection of Drug-induced Neurotoxicity and Seizurogenic Response

Pilocarpine is a well known muscarinic receptor agonist which is used to treat glaucoma and dry mouth.  When administered at high concentrations, pilocarpine is used to induce seizures and produce an epileptic phenotype in the rat.

Our research at Cyprotex has focused on pilocarpine as a reference neurotoxin and we have used this drug to evaluate different in vitro cell-based models of seizurogenic neurotoxicity using a microelectrode array (MEA) platform. Three different cell types were assessed:

  • Co-culture of human iPSC-derived glutamatergic neurons and astrocytes (Cellular Dynamics iCell)
  • Cryopreserved rat cortical neurons
  • Cryopreserved rat hippocampal neurons

The co-culture of human iPSC-derived glutamatergic neurons with astrocytes demonstrated significant sensitivity to pilocarpine-induced CNS liability when tested at 14 days in vitro (DIV). Activity rates were significantly reduced, burst structure was significantly deteriorated, and a decrease in network synchrony was observed at concentrations as low as 3µM. The effects detected were indicative of a possible seizurogenic response or, at the very least, a highly disruptive neural response. However, neither the rat cortical neurons nor the rat hippocampal neurons produced a typical seizurogenic or neurotoxic response at 14 DIV at the concentration range tested. When tested again at 21 DIV, the rat cortical neurons appeared to respond similarly to the human iPSC co-culture.  Although effects on activity rates were not as pronounced, the effects on burst organization and network synchrony were quite similar with significant reductions in the organization of burst structure and synchrony across the network. When tested at 21 DIV, the rat hippocampal neurons had a completely different response than either the human iPSC co-culture or the rat cortical neurons.  The rat hippocampal neurons demonstrated a significant increase in the regulation and the length of burst duration and an increase in spikes that occurred in bursts.  The activity rates remained unchanged while the regulation of burst and network organization were substantially effected.   These results suggest a maturation dependent effect in primary rat neuronal cultures.  Rat cortical neurons behave much like the human iPSC-derived glutaneurons while the rat hippocampal cells have a completely different response.  The hippocampus is where the initial response to pilocarpine originates in the rat so this differential response may be important.

This research was presented at the Safety Pharmacology Society Annual Meeting in Washington DC from Sept 30-Oct 3, 2018.

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