Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping.
Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping.
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DOI：
10.3389/fncir.2014.00138
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发表时间：
2014
影响因子：
3.5
通讯作者：
Burdine RD
中科院分区：
文献类型：
--
作者：
Kim CK;Miri A;Leung LC;Berndt A;Mourrain P;Tank DW;Burdine RD
斑马鱼幼体提供了对整个中枢神经系统的神经活动进行大规模光学成像的潜力；然而，有几个障碍挑战了它们的实用性。首先，到目前为止，只有受精后7天(DPF)的早期幼虫才有泛神经元探针的表达，这排除了在电路更成熟的以后时间点的成像。其次，基因编码的钙指示剂(GECI)的核排斥限制了成像过程中收集的功能荧光信号的分辨率。在这里，我们报告了利用前面描述的优化的Gal4-UAS系统创建的转基因斑马鱼品系，该品系显示出GCaMP3在大脑中至少14DPF的强健的核靶向表达。我们在体外证实了修饰的探针的核靶向性和功能性，并测量了其对动作电位(AP)的反应动力学。然后，我们通过识别自发眼球运动期间尾部后脑神经元中眼睛位置敏感的荧光波动，在转基因斑马鱼品系中展示了核定位的GCaMP3的活体功能。我们的方法论方法将通过提高功能钙信号的分辨率并允许改进的GECI或潜在的任何探针在大脑中进一步表达来促进斑马鱼幼体电路的研究。
Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. First, ~panneuronal probe expression has to date only been demonstrated at early larval stages up to 7 days post-fertilization (dpf), precluding imaging at later time points when circuits are more mature. Second, nuclear exclusion of genetically-encoded calcium indicators (GECIs) limits the resolution of functional fluorescence signals collected during imaging. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf utilizing a previously described optimized Gal4-UAS system. We confirmed both nuclear targeting and functionality of the modified probe in vitro and measured its kinetics in response to action potentials (APs). We then demonstrated in vivo functionality of nuclear-localized GCaMP3 in transgenic zebrafish strains by identifying eye position-sensitive fluorescence fluctuations in caudal hindbrain neurons during spontaneous eye movements. Our methodological approach will facilitate studies of larval zebrafish circuitry by both improving resolution of functional Ca2+ signals and by allowing brain-wide expression of improved GECIs, or potentially any probe, further into development.
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影响因子：
14.9
作者：
Baron, U;Gossen, M;Bujard, H
通讯作者：
Bujard, H
影响因子：
4.6
作者：
Kimura, Yukiko;Satou, Chie;Higashijima, Shin-ichi
通讯作者：
Higashijima, Shin-ichi
DOI：
10.1073/pnas.1012185108
发表时间：
2011-01-18
影响因子：
11.1
作者：
Kinkhabwala, Amina;Riley, Michael;Fetcho, Joseph
通讯作者：
Fetcho, Joseph
影响因子：
3.4
作者：
Bengtson, C. Peter;Freitag, H. Eckehard;Bading, Hilmar
通讯作者：
Bading, Hilmar
影响因子：
2.5
作者：
Fujimoto, Esther;Gaynes, Brooke;Brimley, Cameron J.;Chien, Chi-Bin;Bonkowsky, Joshua L.
通讯作者：
Bonkowsky, Joshua L.