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Research ArticleArticle

Rapid Throughput Analysis Demonstrates that Chemicals with Distinct Seizurogenic Mechanisms Differentially Alter Ca2+ Dynamics in Networks Formed by Hippocampal Neurons in Culture

Zhengyu Cao, Xiaohan Zou, Yanjun Cui, Susan Hulsizer, Pamela J. Lein, Heike Wulff and Isaac N. Pessah
Molecular Pharmacology April 2015, 87 (4) 595-605; DOI: https://doi.org/10.1124/mol.114.096701
Zhengyu Cao
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Xiaohan Zou
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Yanjun Cui
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Susan Hulsizer
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Pamela J. Lein
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Heike Wulff
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Isaac N. Pessah
State Key Laboratory of Natural Medicines and Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing, P.R. China (Z.C., X.Z., Y.C.); Department of Molecular Biosciences, School of Veterinary Medicine (Z.C., Y.C., S.H., P.J.L., I.N.P.) and Department of Pharmacology, School of Medicine (H.W.),University of California, Davis, California
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Abstract

Primary cultured hippocampal neurons (HN) form functional networks displaying synchronous Ca2+ oscillations (SCOs) whose patterns influence plasticity. Whether chemicals with distinct seizurogenic mechanisms differentially alter SCO patterns was investigated using mouse HN loaded with the Ca2+ indicator fluo-4-AM. Intracellular Ca2+ dynamics were recorded from 96 wells simultaneously in real-time using fluorescent imaging plate reader. Although quiescent at 4 days in vitro (DIV), HN acquired distinctive SCO patterns as they matured to form extensive dendritic networks by 16 DIV. Challenge with kainate, a kainate receptor (KAR) agonist, 4-aminopyridine (4-AP), a K+ channel blocker, or pilocarpine, a muscarinic acetylcholine receptor agonist, caused distinct changes in SCO dynamics. Kainate at <1 µM produced a rapid rise in baseline Ca2+ (Phase I response) associated with high-frequency and low-amplitude SCOs (Phase II response), whereas SCOs were completely repressed with >1 µM kainate. KAR competitive antagonist CNQX [6-cyano-7-nitroquinoxaline-2,3-dione] (1-10 µM) normalized Ca2+ dynamics to the prekainate pattern. Pilocarpine lacked Phase I activity but caused a sevenfold prolongation of Phase II SCOs without altering either their frequency or amplitude, an effect normalized by atropine (0.3–1 µM). 4-AP (1–30 µM) elicited a delayed Phase I response associated with persistent high-frequency, low-amplitude SCOs, and these disturbances were mitigated by pretreatment with the KCa activator SKA-31 [naphtho[1,2-d]thiazol-2-ylamine]. Consistent with its antiepileptic and neuroprotective activities, nonselective voltage-gated Na+ and Ca2+ channel blocker lamotrigine partially resolved kainate- and pilocarpine-induced Ca2+ dysregulation. This rapid throughput approach can discriminate among distinct seizurogenic mechanisms that alter Ca2+ dynamics in neuronal networks and may be useful in screening antiepileptic drug candidates.

Footnotes

    • Received November 10, 2014.
    • Accepted January 12, 2015.
  • This research was supported by the CounterACT Program, National Institutes of Health Office of the Director and the National Institute of Neurological Disorders and Stroke [Grant U54-NS079202], the National Institute of Environmental Health Sciences [Grants 1R01-ES020392, 3P01-ES011269, and P42-ES04699], the Natural Science Foundation of China [Grant 81473539], the Jiangsu Provincial Natural Science Foundation [Grant BK20141357], and the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University [Grant SKLNMZZJQ201402].

  • dx.doi.org/10.1124/mol.114.096701.

  • Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 87 (4)
Molecular Pharmacology
Vol. 87, Issue 4
1 Apr 2015
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Research ArticleArticle

Rapid Throughput SCO Analysis of Seizurogenic Agents

Zhengyu Cao, Xiaohan Zou, Yanjun Cui, Susan Hulsizer, Pamela J. Lein, Heike Wulff and Isaac N. Pessah
Molecular Pharmacology April 1, 2015, 87 (4) 595-605; DOI: https://doi.org/10.1124/mol.114.096701

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Research ArticleArticle

Rapid Throughput SCO Analysis of Seizurogenic Agents

Zhengyu Cao, Xiaohan Zou, Yanjun Cui, Susan Hulsizer, Pamela J. Lein, Heike Wulff and Isaac N. Pessah
Molecular Pharmacology April 1, 2015, 87 (4) 595-605; DOI: https://doi.org/10.1124/mol.114.096701
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