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In vivo dendritic calcium imaging with a fiberoptic periscope system

Nature Protocols volume 4, pages 1551–1559 (2009)Cite this article

Abstract

Dendritic recordings in freely moving animals present great challenges using the current approaches. Here we present in detail a microendoscopic technique (the 'periscope' method) for measuring intracellular calcium activity directly from the apical dendrites of L5 pyramidal neurons from the pia down to depths of ∼700 μm in anesthetized and freely moving rats. This method gives high signal-to-noise dendritic fluorescence responses to sensory stimuli, and has been proven to be inexpensive, straightforward and reliable, allowing essentially unrestricted behavior. We describe refinements and practical optimizations of procedures aimed at achieving dendritic Ca2+ imaging in freely moving animals. The periscope imaging technique presented here is also ideal for combining with other in vivo recording techniques. The protocol, from the beginning of anesthesia to starting dendritic imaging, can be completed in 5 h.

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Figure 1: Example of a gradient index (GRIN) lens.
Figure 2: Fiberoptic systems for recording dendritic Ca2+ activity.
Figure 3: The epifluorescence microscope attached to the fiberoptic system for in vivo imaging.
Figure 4: Structure of the periscope.
Figure 5: Bolus loading of Ca2+-sensitive dye into layer 5.
Figure 6: Headmount for awake experiments.
Figure 7: Example of dendritic Ca2+ imaging under anesthesia.
Figure 8: Example of dendritic Ca2+ imaging in a freely moving rat.

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Acknowledgements

We thank H.-R. Lüscher and J.J. Letzkus for their helpful comments on the manuscript, and D. Limoges and J. Burkhalter for their expert technical support. This work was supported by the Swiss National Science Foundation (Grant Nr. PP00A-102721/1) and SystemsX.ch (NEUROCHOICE).

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  1. Physiologisches Institut, Universität Bern, Bern, Switzerland

    Masanori Murayama & Matthew E Larkum

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  1. Masanori Murayama
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  2. Matthew E Larkum
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Correspondence to Matthew E Larkum.

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Murayama, M., Larkum, M. In vivo dendritic calcium imaging with a fiberoptic periscope system. Nat Protoc 4, 1551–1559 (2009). https://doi.org/10.1038/nprot.2009.142

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