Rapid throughput analysis demonstrates that chemicals with distinct seizurogenic mechanisms differentially alter Ca2+ dynamics in networks formed by hippocampal neurons in culture

Abstract

Primary cultured hippocampal neurons (HN) form functional networks displaying synchronous Ca2+ oscillations (SCOs) whose patterns influence plasticity. Whether chemicals with distinct seizurogenic mechanisms differentially alter SCO patterns was investigated using mouse HN loaded with the Ca2+ indicator fluo-4-AM. Intracellular Ca2+ 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 Ca2+ (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 mM) normalized Ca2+ 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 voltagegated Na+ and Ca2+ channel blocker lamotrigine partially resolved kainate- and pilocarpine-induced Ca2+ dysregulation. This rapid throughput approach can discriminate among distinct seizurogenic mechanisms that alter Ca2+ dynamics in neuronal networks and may be useful in screening antiepileptic drug candidates.