Spontaneous Ca2+ events are linked to the development of neuronal firing during maturation in mice primary hippocampal culture cells
2022
Sokolov, Rostislav A. | Mukhina, Irina V.
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 and 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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