AbstractCalcium 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.

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致心律失常性和致癫痫性。先前的研究表明，通过NMDA和烟碱乙酰胆碱受体（nAChRs）的Ca2+流入调节Ca2+振荡。然而，尚未全面研究缺血或nAChR阳性变构调节剂（PAM）药物对Ca2+振荡的影响，其水平将有助于高通量筛选。

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.

我们使用384孔板和FDSS-7000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Ca2+振荡的影响。缺血1小时和2小时导致Ca2+振荡频率增加，并且其振幅以时间依赖性方式降低。

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.

NMDA和AMPA受体抑制显着抑制Ca2+振荡。NR2A或NR2B的抑制对Ca振荡具有相反的作用。NMDA受体拮抗剂MK-801显着抑制缺血诱导的Ca2+振荡的增强，并且这些振荡的频率被NR2B抑制剂Ro-256981抑制。

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.

在3D神经球中，α7nAChR激动剂的应用增加了Ca2+振荡的频率，而α4β2的激活没有影响。尼古丁和PNU-120596（II型PAM）的组合以不同于I型PAM的方式影响Ca2+振荡的频率和幅度。

These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia..

这些系统不仅可用于检测致痫性，还可用于寻找抗脑缺血的神经保护剂。。

IntroductionCalcium (Ca2+) is a crucial intracellular second messenger that precisely regulates a multitude of physiological functions within cells, particularly in the brain1,2. The changes in intracellular Ca2+ levels play a pivotal role in neurotransmitter release and other synaptic processes, as well as in regulating cell life and death3,4.

引言钙（Ca2+）是一种关键的细胞内第二信使，可精确调节细胞内的多种生理功能，特别是在大脑中1,2。细胞内Ca2+水平的变化在神经递质释放和其他突触过程以及调节细胞生命和死亡中起关键作用3,4。

The alterations in intracellular Ca2+ levels have been demonstrated to serve as a crucial biomarker for the investigation of the effects of compounds on neuronal network activity5,6,7. The occurrence of Ca2+ oscillations in neuronal cultures correlates with the formation of new synapses and the development of neuronal networks8.

细胞内Ca2+水平的改变已被证明是研究化合物对神经元网络活性影响的关键生物标志物5,6,7。神经元培养物中Ca2+振荡的发生与新突触的形成和神经元网络的发展有关8。

These oscillations correspond to bursts of network activity and can be modulated by the application of excitatory and inhibitory compounds9. The removal of extracellular Ca2 + eliminates the spontaneous Ca2 oscillations, the removal of extracellular Ca2+, L-type Ca2+ channel blockers, and N-methyl-D-aspartate receptor (NMDAR) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonists eliminates L-type Ca2+ channel blockers and oscillations9,10,11.The measurement of Ca2+ transients in rat cortical neurons by high-throughput fluorescence techniques would be of significant value in the assessment of seizure risk of compounds, particularly at the early stages of drug discovery.

这些振荡对应于网络活动的爆发，可以通过应用兴奋性和抑制性化合物来调节9。去除细胞外Ca2+，消除自发的Ca2振荡，去除细胞外Ca2+，L型Ca2+通道阻滞剂和N-甲基-D-天冬氨酸受体（NMDAR）或α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPAR）拮抗剂消除L型Ca2+通道阻滞剂和振荡9,10,11。通过高通量荧光技术测量大鼠皮层神经元中的Ca2+瞬变对于评估化合物的癫痫发作风险具有重要价值，特别是在药物发现的早期阶段。

The 2D networks of primary cortical neurons have been employed in the assessment of potential neurotoxicity or epileptogenicity on multi-well microelectrode arrays12,13,14 and Ca2+ oscillation assays, utilizing a microplate reader with Fluo-3/AM or Fluo-4 Ca2+ indicators15,16. The cultures of human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes exhibit measurable spontaneous Ca2+ oscil.

原代皮层神经元的2D网络已被用于评估多孔微电极阵列12,13,14和Ca2+振荡测定的潜在神经毒性或致痫性，利用具有Fluo-3/AM或Fluo-4 Ca2+指示剂的酶标仪15,16。人诱导多能干细胞（iPSC）衍生的神经元和星形胶质细胞的培养物表现出可测量的自发Ca2+OSCI。

Data availability

数据可用性

The authors declare that all data supporting the findings of this study are available in the paper, as well as its Supplementary Information.

作者声明，支持本研究结果的所有数据及其补充信息均可在本文中找到。

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Download referencesFundingThis work was supported by the following grants: JSPS KAKENHI Grant number 21K07414 (to T.S.) and Smoking Research Foundation (to T.S.)Author informationAuthors and AffiliationsDepartment of Neurology, Graduate School of Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka, 565-0871, JapanTsutomu Sasaki, Sunao Hisada, Hideaki Kanki, Kumiko Nishiyama, Tomohito Kawano & Hideki MochizukiStemRIM Institute of Regeneration-Inducing Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka, 565-0871, JapanTsutomu SasakiCenter for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Science, Osaka University, 1‑6 Yamadaoka, Suita, Osaka, 565‑0871, JapanKazuto Nunomura & Bangzhong LinGraduate School of Comprehensive Rehabilitation, Osaka Prefecture University, Osaka, 583-8555, JapanShigenobu MatsumuraAuthorsTsutomu SasakiView author publicationsYou can also search for this author in.

下载参考文献资助这项工作得到了以下资助：JSPS KAKENHI资助号21K07414（授予T.S.）和吸烟研究基金会（授予T.S.）作者信息作者和附属机构大阪大学医学研究生院神经病学系，山冈2-2，大阪，565-0871，日本佐佐木，久田秀男，菅木秀子，西山Kumiko，川野友仁和Hideki MochizukiStemRIM大阪大学再生诱导医学研究所，山冈2-2，大阪，565-0871，大阪大学药物科学研究生院日本佐佐木支持药物发现和生命科学研究中心，1-6 Yamadaoka，Suita，Osaka，565-0871，JapanKazuto Nunomura＆Bangzhong LinGraduate School of Comprehensive Recoveration，Osaka Prefessional University，Osaka，583-8555，Japanshinobu MatsumuraAuthors Tsutomu SasakiView Author Publications你也可以在中搜索这位作者。

PubMed Google ScholarSunao HisadaView author publicationsYou can also search for this author in

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PubMed Google ScholarHideaki KankiView作者出版物您也可以在

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PubMed Google ScholarContributionsTS, SH, KN and BL planned the study. TS, SH, HK, KN, BL, KN, SM, and TK performed the experiments. TS and SH wrote the manuscript. TS, SH, HK, TK, KT, SM, and HM reviewed and revised the manuscript. All authors read and approved the manuscript.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献者，SH，KN和BL计划了这项研究。TS，SH，HK，KN，BL，KN，SM和TK进行了实验。TS和SH写了手稿。TS，SH，HK，TK，KT，SM和HM审查并修订了手稿。所有作者都阅读并批准了手稿。对应作者对应。

Tsutomu Sasaki.Ethics declarations

佐佐木通。道德宣言

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Ethics approval

道德认可

All experiments were performed following the Use of Laboratory Animals and ARRIVE guidelines. The Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine approved all animal protocols and experiments (Permission number: 30-091-001, 05-078-0000).

所有实验均在使用实验动物和ARRIVE指南后进行。大阪大学医学研究生院机构动物护理和使用委员会批准了所有动物方案和实验（许可号：30-091-00105-078-0000）。

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所有作者均同意并批准发表本手稿。

Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic Supplementary MaterialBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Rights and permissions.

Additional informationPublisher的noteSpringer Nature在已发布地图和机构隶属关系中的管辖权主张方面保持中立。电子补充材料流是指向电子补充材料的链接。补充材料1补充材料2补充材料3权利和许可。

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

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You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

。如果材料未包含在文章的知识共享许可中，并且您的预期用途不受法律法规的许可或超出许可用途，则您需要直接获得版权所有者的许可。

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Reprints and permissionsAbout this articleCite this articleSasaki, T., Hisada, S., Kanki, H. et al. Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis.

转载和许可本文引用本文Sasaki，T.，Hisada，S.，Kanki，H。等人通过高通量分析调节原代皮层神经元缺血后的Ca2+振荡和烟碱乙酰胆碱受体。

Sci Rep 14, 27667 (2024). https://doi.org/10.1038/s41598-024-77882-wDownload citationReceived: 27 July 2024Accepted: 25 October 2024Published: 12 November 2024DOI: https://doi.org/10.1038/s41598-024-77882-wShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

科学报告1427667（2024）。https://doi.org/10.1038/s41598-024-77882-wDownload引文收到日期：2024年7月27日接受日期：2024年10月25日发布日期：2024年11月12日OI：https://doi.org/10.1038/s41598-024-77882-wShare本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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KeywordsCa2+ oscillationsHigh-throughput screeningIschemianAChRPAM

关键词Ca2+振荡高通量筛选缺血ACHRPM

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Cellular neuroscienceDiseases of the nervous systemDrug discoveryDrug safetyMolecular neuroscienceToxicology

细胞神经科学神经系统疾病药物发现药物安全性分子神经科学毒理学