Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays
Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays
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DOI：
10.1152/jn.01122.2015
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发表时间：
2016-06-01
影响因子：
2.5
通讯作者：
Graef, John D.
中科院分区：
文献类型：
--
作者：
Newberry, Kim;Wang, Shuya;Graef, John D.
感觉神经元活性的体外表型分析是鉴定潜在止痛化合物的重要工具。这些分析的典型特征是过度兴奋和/或异常的自发活跃的细胞。虽然人工电生理学实验提供了高分辨率的生物物理数据来表征体外模型和潜在的治疗模式(例如，动作电位特征、特定离子通道的作用和受体)，但这些技术因其低吞吐量而受到阻碍。我们使用多孔多电极阵列(MEAs)建立了一个自发活动的背根节(DRG)平台，大大提高了在细胞网络的背景下评估多种化合物和条件对DRG兴奋性的影响的能力。我们发现选择性Na+和Ca~(2+)通道阻滞剂可减弱DRG自发放电，K~+通道阻滞剂可增强自发性DRG放电。此外，瞬时受体电位阳离子通道亚家族V成员1激动剂辣椒素和缓激肽均可增强自发活动，并可被神经激肽受体拮抗剂完全阻断。最后，我们通过证明常用的神经病理性疼痛疗法抑制DRG自发活动来验证该方法的使用。总体而言，我们优化了多孔MEA平台上的原代大鼠DRG细胞，以生成并鉴定自发活性培养物，这些培养物有可能用作体外表型分析，以评估啮齿动物疼痛模型的潜在治疗方法。
In vitro phenotypic assays of sensory neuron activity are important tools for identifying potential analgesic compounds. These assays are typically characterized by hyperexcitable and/or abnormally, spontaneously active cells. Whereas manual electrophysiology experiments provide high-resolution biophysical data to characterize both in vitro models and potential therapeutic modalities (e.g., action potential characteristics, the role of specific ion channels, and receptors), these techniques are hampered by their low throughput. We have established a spontaneously active dorsal root ganglia (DRG) platform using multiwell multielectrode arrays (MEAs) that greatly increase the ability to evaluate the effects of multiple compounds and conditions on DRG excitability within the context of a cellular network. We show that spontaneous DRG firing can be attenuated with selective Na+ and Ca2+ channel blockers, as well as enhanced with K+ channel blockers. In addition, spontaneous activity can be augmented with both the transient receptor potential cation channel subfamily V member 1 agonist capsaicin and the peptide bradykinin and completely blocked with neurokinin receptor antagonists. Finally, we validated the use of this assay by demonstrating that commonly used neuropathic pain therapeutics suppress DRG spontaneous activity. Overall, we have optimized primary rat DRG cells on a multiwell MEA platform to generate and characterize spontaneously active cultures that have the potential to be used as an in vitro phenotypic assay to evaluate potential therapeutics in rodent models of pain.