"In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging
"In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging
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DOI：
10.1007/s00424-003-1138-4
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发表时间：
2003-09-01
影响因子：
4.5
通讯作者：
Konnerth, A
中科院分区：
文献类型：
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作者：
Brustein, E;Marandi, N;Konnerth, A
斑马鱼幼虫是分析早期发育阶段行为和潜在神经元网络活动的强大模型。在此，我们在此制备过程中采用了“体内”Ca2+ 成像的新方法。我们证明，将膜渗透性 Ca2+ 指示剂染料快速注射到斑马鱼幼虫的脊髓中，会导致整个脊髓快速染色。使用双光子成像，我们可以以单细胞分辨率同时监测来自大量脊髓神经元的 Ca2+ 信号。为了测试该方法，通过离子电渗应用谷氨酸和 GABA 或甘氨酸（首次在活体制剂中观察到）产生 Ca2+ 瞬变。甘氨酸诱发的 Ca2+ 瞬变被士的宁阻断。触发移动斑马鱼逃避反射的感觉刺激在脊髓网络的不同神经元中引起 Ca2+ 瞬变。此外，长期记录显示单个脊髓神经元中存在自发的 Ca2+ 瞬变。通常，这种活动在网络中的许多神经元之间同步发生。总之，新方法可以对发育中的神经网络的功能组织进行单细胞分辨率的可靠分析。
The zebrafish larva is a powerful model for the analysis of behaviour and the underlying neuronal network activity during early stages of development. Here we employ a new approach of 'in vivo' Ca2+ imaging in this preparation. We demonstrate that bolus injection of membrane-permeable Ca2+ indicator dyes into the spinal cord of zebrafish larvae results in rapid staining of essentially the entire spinal cord. Using two-photon imaging, we could monitor Ca2+ signals simultaneously from a large population of spinal neurons with single-cell resolution. To test the method, Ca2+ transients were produced by iontophoretic application of glutamate and, as observed for the first time in a living preparation, of GABA or glycine. Glycine-evoked Ca2+ transients were blocked by the application of strychnine. Sensory stimuli that trigger escape reflexes in mobile zebrafish evoked Ca2+ transients in distinct neurons of the spinal network. Moreover, long-term recordings revealed spontaneous Ca2+ transients in individual spinal neurons. Frequently, this activity occurred synchronously among many neurons in the network. In conclusion, the new approach permits a reliable analysis with single-cell resolution of the functional organisation of developing neuronal networks.