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

• Published in: Scientific reports Vol. 14; no. 1; pp. 27667 - 18
• Main Authors: Sasaki, Tsutomu, Hisada, Sunao, Kanki, Hideaki, Nunomura, Kazuto, Lin, Bangzhong, Nishiyama, Kumiko, Kawano, Tomohito, Matsumura, Shigenobu, Mochizuki, Hideki
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.11.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/154; 631/154/1438; 631/154/570; 631/378/1689; 631/378/340; 631/378/87; Acetylcholine receptors (nicotinic); Agonists; Allosteric properties; Ca2+ oscillations; Calcium antagonists; Calcium influx; Calcium signalling; Dizocilpine; Drug development; Drug screening; Glutamate receptors; Glutamic acid receptors (ionotropic); High-throughput screening; Humanities and Social Sciences; Inhibitory postsynaptic potentials; Ischemia; multidisciplinary; N-Methyl-D-aspartic acid receptors; nAChR; Neuromodulation; Neuroprotection; Neuroprotective agents; PAM; Science; Science (multidisciplinary); α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors; High-throughput screening; Ischemia; oscillations; PAM; nAChR; Ca
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-77882-w

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca 2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca 2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca 2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca 2+ 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 Ca 2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca 2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.