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Spontaneous Ca 2+ events are linked to the development of neuronal firing during maturation in mice primary hippocampal culture cells.
Archives of Biochemistry and Biophysics 2022 June 22
Calcium is one of the most vital intracellular secondary messengers that tightly regulates a variety of cell physiology processes, especially in the brain. Using a fluorescent Ca2+ -sensitive Oregon Green probe, we revealed three different amplitude distributions of spontaneous Ca2+ events (SCEs) in neurons between 15-26 days in vitro (DIV) culture maturation. We detected a series of amplitude events: micro amplitude SCE (microSCE) 25% increase from the baseline, intermediate amplitude SCE (interSCE) as 25-75%, and macro amplitude SCE (macroSCE) - over 75%. The SCEs were fully dependent on extracellular Ca2+ and neuronal network activity and vanished in the Ca2+ -free solution, 10 mM Mg2+ -block, or in the presence of voltage-gated Na+ -channel blocker, tetrodotoxin. Combined patch-clamp and Ca2+ -imaging techniques revealed that microSCE match single action potential (AP), interSCE - burst of 3-12 APs, and macroSCE - 'superburst' of 10+ APs. MicroSCEs were blocked by a common α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) receptor antagonist, CNQX. The γ-aminobutyric acid (GABA) A-type receptor (GABAAR) picrotoxin blockade and L-type voltage-dependent Ca2+ -channel inhibitor diltiazem significantly reduced microSCE frequency. InterSCEs were inhibited by CNQX, but picrotoxin treatment significantly increased its amplitude. The N-methyl-d-aspartate (NMDA) receptor antagonist, D-APV, voltage-gated K+ -channel blocker, tetraethylammonium, noticeably suppressed interSCE amplitude. We also demonstrate that macroSCEs were AMPA/KA receptor-independent.
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