Perez-Alvarez Alberto, Huhn Florian, Dürst Céline D, Franzelin Andreas, Lamothe-Molina Paul J, Oertner Thomas G
Institute for Synaptic Physiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Rapp OptoElectronic GmbH, Wedel, Germany.
Front Mol Neurosci. 2021 Mar 4;14:635820. doi: 10.3389/fnmol.2021.635820. eCollection 2021.
The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.
神经元广泛的树突分支被认为积极参与信息处理。树突含有丰富多样的配体激活和电压激活离子通道以及代谢型受体。此外，它们能够从细胞内储存库释放钙。在特定条件下，大型神经元会产生局部局限于树突段的钙峰。为了研究树突中的钙信号传导，我们引入了TubuTag，一种锚定在细胞骨架上的基因编码比率型钙传感器。当用紫光照射时，TubuTag通过从绿色荧光到红色荧光的不可逆光转换来整合细胞质钙信号。我们使用具有大视野的定制双光子显微镜以亚细胞分辨率对CA1区的锥体神经元进行成像。光转换在顶端树突的最远端部分最强，这表明树突钙信号的幅度存在梯度。由于荧光读出可以在光转换后数小时进行，TubuTag将有助于研究大量神经元中的树突信号整合和钙稳态。
