Cao Zhengyu, Zou Xiaohan, Cui Yanjun, Hulsizer Susan, Lein Pamela J, Wulff Heike, Pessah Isaac N
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.
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
Mol Pharmacol. 2015 Apr;87(4):595-605. doi: 10.1124/mol.114.096701. Epub 2015 Jan 12.
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.
原代培养的海马神经元（HN）形成显示同步钙（Ca²⁺）振荡（SCOs）的功能网络，其模式影响可塑性。使用加载钙指示剂fluo-4-AM的小鼠HN研究了具有不同致痫机制的化学物质是否差异改变SCO模式。使用荧光成像酶标仪实时同时记录96孔中的细胞内Ca²⁺动力学。尽管在体外4天（DIV）时静止，但HN在16 DIV成熟形成广泛的树突网络时获得了独特的SCO模式。用红藻氨酸受体（KAR）激动剂红藻氨酸、钾（K⁺）通道阻滞剂4-氨基吡啶（4-AP）或毒蕈碱型乙酰胆碱受体激动剂毛果芸香碱进行刺激，导致SCO动力学发生明显变化。<1 μM红藻氨酸使基线Ca²⁺迅速升高（I期反应），伴有高频和低幅度SCOs（II期反应），而>1 μM红藻氨酸则完全抑制SCOs。KAR竞争性拮抗剂CNQX [6-氰基-7-硝基喹喔啉-2,3-二酮]（1 - 10 μM）使Ca²⁺动力学恢复到红藻氨酸处理前的模式。毛果芸香碱缺乏I期活性，但使II期SCOs延长7倍，而不改变其频率或幅度，阿托品（0.3 - 1 μM）可使该效应恢复正常。4-AP（1 - 30 μM）引发延迟的I期反应，伴有持续的高频、低幅度SCOs，用钾钙激活剂SKA-31 [萘并[1,2-d]噻唑-2-胺]预处理可减轻这些干扰。与其抗癫痫和神经保护活性一致，非选择性电压门控钠（Na⁺）和钙（Ca²⁺）通道阻滞剂拉莫三嗪部分解决了红藻氨酸和毛果芸香碱诱导的Ca²⁺失调。这种快速通量方法可以区分改变神经元网络中Ca²⁺动力学的不同致痫机制，可能有助于筛选抗癫痫药物候选物。
