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LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Abstract

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5–6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.

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Figure 1: AOD-based high-speed two-photon microscope.
Figure 2: Mouse surgery for the craniotomy preparation.
Figure 3: Craniotomy preparation before imaging.
Figure 4: Reduction of heartbeat-induced noise by agarose application.
Figure 5: Flowchart for the entire experimental procedure.
Figure 6: Filtering procedure for spine calcium signals.

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Acknowledgements

We thank J. Lou for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (IRTG 1373), the European Research Area (ERA)-Net Program and the Schiedel Foundation. A.K. is a Carl von Linde Senior Fellow of the Institute for Advanced Study of the Technische Universität München.

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X.C., U.L., Z.V., H.J., D.D., N.L.R. and A.K. performed the experiments and the analysis. X.C. and A.K. wrote the paper together with all authors.

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Correspondence to Arthur Konnerth.

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The authors declare no competing financial interests.

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Chen, X., Leischner, U., Varga, Z. et al. LOTOS-based two-photon calcium imaging of dendritic spines in vivo. Nat Protoc 7, 1818–1829 (2012). https://doi.org/10.1038/nprot.2012.106

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