Rapid Throughput Analysis Demonstrates that Chemicals with Distinct Seizurogenic Mechanisms Differentially Alter Ca2+ Dynamics in Networks Formed by Hippocampal Neurons in Culture

Primary cultured hippocampal neurons (HN) form functional networks displaying synchronous Ca(2+) oscillations (SCOs) whose patterns influence plasticity. Whether chemicals with distinct seizurogenic mechanisms differentially alter SCO patterns was investigated using mouse HN loaded with the Ca(2+) indicator fluo-4-AM. Intracellular Ca(2+) dynamics were recorded from 96 wells simultaneously in real-time using fluorescent imaging plate reader. Although quiescent at 4 days in vitro (DIV), HN acquired distinctive SCO patterns as they matured to form extensive dendritic networks by 16 DIV. Challenge with kainate, a kainate receptor (KAR) agonist, 4-aminopyridine (4-AP), a K(+) channel blocker, or pilocarpine, a muscarinic acetylcholine receptor agonist, caused distinct changes in SCO dynamics. Kainate at <1 µM produced a rapid rise in baseline Ca(2+) (Phase I response) associated with high-frequency and low-amplitude SCOs (Phase II response), whereas SCOs were completely repressed with >1 µM kainate. KAR competitive antagonist CNQX [6-cyano-7-nitroquinoxaline-2,3-dione] (1-10 µM) normalized Ca(2+) dynamics to the prekainate pattern. Pilocarpine lacked Phase I activity but caused a sevenfold prolongation of Phase II SCOs without altering either their frequency or amplitude, an effect normalized by atropine (0.3-1 µM). 4-AP (1-30 µM) elicited a delayed Phase I response associated with persistent high-frequency, low-amplitude SCOs, and these disturbances were mitigated by pretreatment with the KCa activator SKA-31 [naphtho[1,2-d]thiazol-2-ylamine]. Consistent with its antiepileptic and neuroprotective activities, nonselective voltage-gated Na(+) and Ca(2+) channel blocker lamotrigine partially resolved kainate- and pilocarpine-induced Ca(2+) dysregulation. This rapid throughput approach can discriminate among distinct seizurogenic mechanisms that alter Ca(2+) dynamics in neuronal networks and may be useful in screening antiepileptic drug candidates.