Optical measurement of neuronal activity in the developing cerebellum of zebrafish using voltage-sensitive dye imaging
Optical measurement of neuronal activity in the developing cerebellum of zebrafish using voltage-sensitive dye imaging
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DOI：
10.1097/wnr.0000000000001113
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发表时间：
2018-11-07
期刊：
影响因子：
1.7
通讯作者：
Tsuda, Sachiko
中科院分区：
文献类型：
--
作者：
Okumura, Kanoko;Kakinuma, Hisaya;Tsuda, Sachiko
电压敏感染料（VSD）成像能够快速、直接和同时检测形成神经元回路的神经元群的膜电位。这使得检测到超极化和去极化，它们的平衡在许多大脑区域的功能中起着关键作用。其中包括小脑，它含有大量的抑制性神经元。然而，功能发展的机制尚不清楚。本研究采用研究神经发生的理想模型系统，对斑马鱼幼体小脑进行VSD成像，分析发育中的小脑的神经元活动，重点关注兴奋和抑制。我们对斑马鱼整个小脑进行了体内高速成像，使用广泛使用的VSD -4- anepps进行了染色。为了检验这种VSD能否检测到斑马鱼小脑的神经元活动，我们在VSD成像时施加电刺激，结果显示刺激后小脑广泛检测到去极化。这些反应在河豚毒素处理后大部分消失，表明Di-4-ANEPPS能够光学测量斑马鱼发育中的小脑中的神经元活动。此外，在刺激时也检测到超极化信号，但这些信号在使用微毒素（一种GABAa受体抑制剂）处理后显着减少，表明这些反应代表抑制信号。该方法将详细分析小脑在发育阶段的兴奋和抑制的时空动态，从而更深入地了解脊椎动物小脑的功能发育。
Voltage-sensitive dye (VSD) imaging enables fast, direct, and simultaneous detection of membrane potentials from a population of neurons forming neuronal circuits. This enables the detection of hyperpolarization together with depolarization, whose balance plays a pivotal role in the function of many brain regions. Among these is the cerebellum, which contains a significant number of inhibitory neurons. However, the mechanism underlying the functional development remains unclear. In this study, we used a model system ideal to study neurogenesis by applying VSD imaging to the cerebellum of zebrafish larvae to analyze the neuronal activity of the developing cerebellum, focusing on both excitation and inhibition. We performed in-vivo high-speed imaging of the entire cerebellum of the zebrafish, which was stained using Di-4-ANEPPS, a widely used VSD. To examine whether neuronal activity in the zebrafish cerebellum could be detected by this VSD, we applied electrical stimulation during VSD imaging, which showed that depolarization was detected widely in the cerebellum upon stimulation. These responses mostly disappeared following treatment with tetrodotoxin, indicating that Di-4-ANEPPS enabled optical measurement of neuronal activity in the developing cerebellum of zebrafish. Moreover, hyperpolarizing signals were also detected upon stimulation, but these were significantly reduced by treatment with picrotoxin, a GABAa receptor inhibitor, indicating that these responses represent inhibitory signals. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum along its developmental stages, leading to a deeper understanding of the functional development of the cerebellum in vertebrates.