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通过高通量分析研究原代皮质神经元缺血后钙振荡和烟碱型乙酰胆碱受体的调节。

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

机构信息

Department of Neurology, Graduate School of Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka, 565-0871, Japan.

StemRIM Institute of Regeneration-Inducing Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka, 565-0871, Japan.

出版信息

Sci Rep. 2024 Nov 12;14(1):27667. doi: 10.1038/s41598-024-77882-w.

DOI:10.1038/s41598-024-77882-w
PMID:39532929
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11557898/
Abstract

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 influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca 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 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 oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca 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 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 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.

摘要

原代神经元培养物和 iPSC 中的钙振荡已被用于研究药物开发中的心律失常和致痫性。以前的研究表明，通过 NMDA 和烟碱型乙酰胆碱受体 (nAChR) 的 Ca 内流调节 Ca 振荡。然而，迄今为止，还没有全面研究缺血或 nAChR 阳性变构调节剂 (PAM) 药物对 Ca 振荡的影响，而这种影响需要在高通量筛选中得到促进。我们使用 FDSS-7000 在 384 孔板中研究了缺血和 nAChR 亚型或 nAChR PAM 激动剂对高密度 2D 和 3D 球体原代神经元培养物中 Ca 振荡的影响。缺血 1 和 2 小时会导致 Ca 振荡的频率增加，幅度呈时间依赖性降低。NMDA 和 AMPA 受体抑制显著抑制 Ca 振荡。NR2A 或 NR2B 的抑制对 Ca 振荡产生相反的影响。NMDA 受体拮抗剂 MK-801 显著抑制缺血诱导的 Ca 振荡增强，而 NR2B 抑制剂 Ro-256981 抑制这些振荡的频率。在 3D 神经球中，α7nAChR 激动剂的应用增加了 Ca 振荡的频率，而 α4β2 的激活没有影响。尼古丁和 PNU-120596（II 型 PAM）的联合作用以不同于 I 型 PAM 的方式影响 Ca 振荡的频率和幅度。这些系统不仅可用于检测致痫性，还可用于寻找针对脑缺血的神经保护剂。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b186/11557898/d4c716f1085b/41598_2024_77882_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b186/11557898/08af3e7f433d/41598_2024_77882_Fig1_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b186/11557898/d4c716f1085b/41598_2024_77882_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b186/11557898/08af3e7f433d/41598_2024_77882_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b186/11557898/a40cbf347cb5/41598_2024_77882_Fig2_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b186/11557898/d4c716f1085b/41598_2024_77882_Fig7_HTML.jpg

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