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.

Ca2+ oscillations; High-throughput screening; Ischemia; PAM; nAChR.