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Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo

Nature Protocols volume 1, pages 380–386 (2006)Cite this article

Abstract

One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 μm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is ∼2 h. Thereafter, imaging experiments might be performed for at least 6 h.

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Figure 1: Experimental arrangement for in vivo staining of neuronal populations with Ca2+-indicator dyes.
Figure 2: MCBL allows staining of cortical tissue at various developmental stages.
Figure 3: Assessment of the staining quality.
Figure 4: Spontaneous Ca2+ transients in the visual cortex of an adult mouse.
Figure 5: Ca2+ transients in the individual cortical neurons evoked by sensory stimulation.
Figure 6: Double staining and depth profile.
Figure 7: MCBL using a low concentration of the indicator dye.

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Acknowledgements

We thank M.A. Busche for help with experiments shown in Figure 6b. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 391 and SFB 596) and the Bundesministerium für Bildung und Forschung (NGFN-2).

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Authors and Affiliations

  1. Institut für Physiologie, Ludwig-Maximilians Universität München, Pettenkoferstrasse 12, München, 80336, Germany

    Olga Garaschuk

  2. Institut für Neurowissenschaften, Technische Universität München, Biedersteinerstrasse 29, München, 80802, Germany

    Ruxandra-Iulia Milos & Arthur Konnerth

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  1. Olga Garaschuk
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  2. Ruxandra-Iulia Milos
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  3. Arthur Konnerth
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Correspondence to Arthur Konnerth.

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Garaschuk, O., Milos, RI. & Konnerth, A. Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1, 380–386 (2006). https://doi.org/10.1038/nprot.2006.58

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