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Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo

Nature Methods volume 1, pages 31–37 (2004)Cite this article

Abstract

Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.

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Figure 1: In vivo staining pattern of neocortical cells after application of SR101.
Figure 2: Gap junctions permit rapid spread of SR101.
Figure 3: SR101-labeled cells are immunopositive for S-100β protein, but not for either the neuron-specific nuclear protein NeuN or, in the vast majority of cases, the enzyme CNPase in vitro.
Figure 4: In vivo colabeling in neocortex using SR101.
Figure 5: Simultaneous calcium imaging of neuronal and glial networks in vivo.

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Acknowledgements

We thank J.R. Wolff for comments on the manuscript, S. Jung and D.R. Littman for providing the green fluorescent microglial mouse line, M. Kaiser for expert technical assistance, V. Grinevich for help with the antibody staining and B. Sakmann for generous support. A.N. was supported by a predoctoral fellowship of the Boehringer Ingelheim Fonds.

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Authors and Affiliations

  1. Abteilung Zellphysiologie, Max-Planck Institut für medizinische Forschung, Jahnstr. 29, Heidelberg, 69120, Germany

    Axel Nimmerjahn, Jason N D Kerr & Fritjof Helmchen

  2. Abteilung Neurogenetik, Max-Planck Institut für experimentelle Medizin, Hermann-Rein-Str. 3, Göttingen, 37075, Germany

    Frank Kirchhoff

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Supplementary information

Supplementary Fig. 1

SR101 staining shows no signs of acute or long-term phototoxicity and can be applied repeatedly. (PDF 1896 kb)

Supplementary Table 1

Quantitative comparison between the Texas Red-hydrazide (SR101-fixable analog) and the counter-immunostains in vitro. (PDF 17 kb)

Supplementary Table 2

Quantitative comparison between SR101-labeled and EGFP-expressing cells in the transgenic mice in vivo. (PDF 15 kb)

Supplementary Methods (PDF 22 kb)

Supplementary Video 1

Three-dimensional distribution of SR101 labeled cells in mouse neocortex in vivo. The images are maximum-intensity side-projections from a stack of fluorescence images recorded approximately 30 minutes after dye application. Individual focal planes were recorded in 3 μm steps starting from the pia surface down to 705 μm depth. (AVI 480 kb)

Supplementary Video 2

SR101 uptake in EGFP-expressing astrocytes in TgN(GFAP-EGFP) mice. The focus series is an overlay of the simultaneously recorded green and red fluorescence images of EGFP-expressing astrocytes and SR101 labeled cells, respectively. Images are 103 μm on side and were recorded approximately 310 μm below the pia with 1.0 μm axial spacing. (AVI 336 kb)

Supplementary Video 3

SR101 is not taken up by EGFP-expressing microglial cells in CX3CR1-deficient mice. The focus series is an overlay of the simultaneously recorded green and red fluorescence images showing no overlap of microglial and SR101-labeled cells. Images are 103 μm on side and were recorded approximately 140 μm below the pia with 1.0 μm axial spacing. (AVI 690 kb)

Supplementary Video 4

Focus series demonstrating the close association between SR101 labeled cells (shown in green) and the cortical microvasculature (shown in red). Blood plasma was stained using a tail vein injection of FITC-labeled dextran. SR101 labeling was achieved using surface application of the dye. The endothelial sheet surrounding the blood vessels is visible as a dark gap (unstained area) between end feet and labeled vessel lumen. Images are 87 μm on side and were recorded starting from 110 μm below the pia in axial steps of 1.0 μm. (AVI 543 kb)

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Nimmerjahn, A., Kirchhoff, F., Kerr, J. et al. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1, 31–37 (2004). https://doi.org/10.1038/nmeth706

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