Modulation of Ca²⁺ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca²⁺ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca²⁺ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca²⁺ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca²⁺ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca²⁺ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca²⁺ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca²⁺ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca²⁺ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca²⁺ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.