A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice.
A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice.
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DOI：
10.1016/j.neuron.2018.07.020
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发表时间：
2018-08-22
期刊：
影响因子：
16.2
通讯作者：
Zhong H
中科院分区：
文献类型：
--
作者：
Ma L;Jongbloets BC;Xiong WH;Melander JB;Qin M;Lameyer TJ;Harrison MF;Zemelman BV;Mao T;Zhong H
神经调节对脑功能施加强有力的控制，并且cAMP依赖性蛋白激酶（PKA）是多种神经调节剂的中心下游介质。虽然已经开发了基因编码的PKA传感器，但尚未建立活小鼠中PKA活性的单细胞成像。在这里，我们使用双光子荧光寿命成像显微镜（2pFLIM）来可视化遗传编码的PKA传感器对神经调节剂去甲肾上腺素和多巴胺的反应。我们筛选了2pFLIM的PKA传感器，并进一步开发了一种具有更高灵敏度和更宽动态范围的变体（命名为tAKARα）。该传感器允许检测PKA激活去甲肾上腺素在生理相关的浓度和动力学和光遗传释放多巴胺。tAKARα的体内纵向2pFLIM成像追踪了清醒小鼠个体神经元中的双向PKA活性，并揭示了与觉醒、药理学操作和运动相关的神经调节PKA事件。这种新的传感器与2pFLIM相结合，将能够在清醒的动物中询问神经调节诱导的PKA信号。它仍然具有挑战性的可视化神经调制，神经元通信的主要模式，在体内。Ma等人证明，改进的遗传编码传感器的双光子荧光寿命成像能够在行为小鼠中以单细胞分辨率监测神经调节PKA信号传导。
Neuromodulation imposes powerful control over brain function, and cAMP-dependent protein kinase (PKA) is a central downstream mediator of multiple neuromodulators. Although genetically-encoded PKA sensors have been developed, single-cell imaging of PKA activity in living mice has not been established. Here, we used two-photon fluorescence lifetime imaging microscopy (2pFLIM) to visualize genetically-encoded PKA sensors in response to the neuromodulators norepinephrine and dopamine. We screened available PKA sensors for 2pFLIM and further developed a variant (named tAKARα) with increased sensitivity and a broadened dynamic range. This sensor allowed detection of PKA activation by norepinephrine at physiologically-relevant concentrations and kinetics and by optogenetically released dopamine. In vivo longitudinal 2pFLIM imaging of tAKARα tracked bidirectional PKA activities in individual neurons in awake mice, and revealed neuromodulatory PKA events that were associated with wakefulness, pharmacological manipulation, and locomotion. This new sensor combined with 2pFLIM will enable interrogation of neuromodulation-induced PKA signaling in awake animals. It remains challenging to visualize neuromodulation, a major mode of neuronal communication, in vivo. Ma et al. demonstrate that two-photon fluorescence lifetime imaging of an improved genetically-encoded sensor enables monitoring of neuromodulatory PKA signaling with single-cell resolution in behaving mice.
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影响因子：
16.2
作者：
Chen Y;Granger AJ;Tran T;Saulnier JL;Kirkwood A;Sabatini BL
通讯作者：
Sabatini BL
影响因子：
4.8
作者：
Choi, J;Ko, J;Kim, E
通讯作者：
Kim, E
影响因子：
64.8
作者：
Dodge-Kafka, KL;Soughayer, J;Scott, JD
通讯作者：
Scott, JD
影响因子：
--
作者：
Depry, Charlene;Allen, Michael D.;Zhang, Jin
通讯作者：
Zhang, Jin
DOI：
10.1523/jneurosci.2246-11.2011
发表时间：
2011-07-27
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Adamantidis AR;Tsai HC;Boutrel B;Zhang F;Stuber GD;Budygin EA;Touriño C;Bonci A;Deisseroth K;de Lecea L
通讯作者：
de Lecea L