Pharmacological inhibition of PLC and PKC triggers epileptiform activity in hippocampal neurons

Calcium signaling pathways play a crucial role in neuronal and glial function, yet the effects of inhibiting specific components of these pathways remain poorly understood. Here, we investigated how various inhibitors affect calcium dynamics in neurons and astrocytes within hippocampal co-cultures under normal conditions and during bicuculline-induced epileptiform activity. We found that phospholipase C (PLC) inhibitor U73122 and protein kinase C (PKC) inhibitors BIM IX and Go 6976 independently induced epileptiform activity in neurons, characterized by synchronized calcium oscillations similar to those caused by bicuculline. Notably, these inhibitors did not affect astrocytic calcium dynamics. In contrast, IP3 receptor inhibitor 2-APB and calmodulin inhibitor calmidazolium triggered significant calcium responses in both neurons and astrocytes. The 2-APB application led to an immediate increase in neuronal calcium levels and cessation of calcium oscillations, while also inducing varied calcium responses in astrocytes. Calmidazolium caused elevated calcium levels in both cell types, with neurons maintaining calcium oscillations at increased baseline levels. Interestingly, PI3 kinase inhibitor AS-605240 and ryanodine receptor inhibitor dantrolene showed no significant effects on calcium dynamics in either cell type. Electrophysiological recordings confirmed that both PLC and PKC inhibition induced paroxysmal depolarization shifts similar to those observed during bicuculline-induced epileptiform activity. These findings reveal previously unknown effects of commonly used signaling pathway inhibitors on neuronal excitability and calcium homeostasis, which should be considered when designing experiments and interpreting results involving these compounds. Our results also suggest a potential role for PLC and PKC in maintaining the excitation-inhibition balance in neuronal networks.