title: Rapid throughput analysis demonstrates that chemicals with distinct seizurogenic mechanisms differentially alter Ca2+ dynamics in networks formed by hippocampal neurons in culture.
abstract: Primary cultured hippocampal neurons (HN) form functional networks displaying synchronous Ca(2+) oscillations (SCOs) whose patterns influence plasticity. Whether chemicals with distinct seizurogenic mechanisms differentially alter SCO patterns was investigated using mouse HN loaded with the Ca(2+) indicator fluo-4-AM. Intracellular Ca(2+) 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 Ca(2+) (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 Ca(2+) 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 Ca(2+) channel blocker lamotrigine partially resolved kainate- and pilocarpine-induced Ca(2+) dysregulation. This rapid throughput approach can discriminate among distinct seizurogenic mechanisms that alter Ca(2+) dynamics in neuronal networks and may be useful in screening antiepileptic drug candidates.
authors.affiliation: 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.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.affiliation: 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.
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Zou
authors.affiliation: 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.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Cui
authors.affiliation: 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.
authors.forename: Susan
authors.initials: S
authors.lastname: Hulsizer
authors.affiliation: 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.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Lein
authors.affiliation: 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.
authors.forename: Heike
authors.initials: H
authors.lastname: Wulff
authors.affiliation: 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 inpessah@ucdavis.edu.
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Pessah
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D000927:Anticonvulsants / Q000737:chemistry / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D057166:High-Throughput Screening Assays; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D007608:Kainic Acid / Q000494:pharmacology; D000077213:Lamotrigine; D008810:Mice, Inbred C57BL; D018721:Muscarinic Agonists / Q000494:pharmacology; D009415:Nerve Net; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D018092:Kainic Acid Receptors / Q000819:agonists; D012640:Seizures / Q000378:metabolism*; D014227:Triazines / Q000494:pharmacology
chemical_list: D015761:4-Aminopyridine; D000927:Anticonvulsants; D002118:Calcium; D007608:Kainic Acid; D000077213:Lamotrigine; D018721:Muscarinic Agonists; D026902:Potassium Channel Blockers; D018092:Kainic Acid Receptors; D014227:Triazines