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Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window

Abstract

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (0.8–12 mm2). The surgical procedure can be completed within 1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed.

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Figure 1: Schematic view of experimental preparations for in vivo imaging.
Figure 2: In vivo images of pyramidal neurons in the somatosensory cortex of YFP-H and GFP-M transgenic mice.
Figure 3: Chronic cranial window surgical procedure.
Figure 4: Long-term imaging of GFP-expressing pyramidal neurons in the adult neocortex.
Figure 5: Examples of spine and bouton scoring criteria.
Figure 6: Examples of dendritic spines that have been inconsistently scored by different observers.
Figure 7: Quantification of layer 5 B (L5B) pyramidal cell dendritic spine density and turnover.
Figure 8: Quantification of L6 terminaux bouton density and turnover.
Figure 9: Qualitative assessment of the ultrastructure under a chronic cranial window.
Figure 10: Immunohistochemical staining of glial markers under a chronic cranial window.

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Acknowledgements

We thank K. Masback, X. Zhang and A. Canty for helping with data analysis, and S. Song and T. O'Conner for writing image analysis software. This work was supported by HHMI, NIH, the IRP Foundation, the Swiss National Science Foundation, the Max Planck Society, the Larry L. Hillblom Foundation and the March of Dimes Foundation.

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Correspondence to Anthony Holtmaat, Vincenzo De Paola, Mark Hübener, Graham Knott, Elly Nedivi, Carlos Portera-Cailliau or Joshua T Trachtenberg.

Supplementary information

Supplementary Fig. 1

Long-term in vivo imaging of GFP-expressing L5B neuron in somatosensory cortex over > 6 months. (PDF 277 kb)

Supplementary Fig. 2

Spine densities and survival fractions in thinned skull preparations. (PDF 166 kb)

Supplementary Methods 1

Thinned skull preparation. A document describing the procedure for skull thinning. All experiments using animals should be carried out under institutional and national guidelines. (PDF 24 kb)

Supplementary Movie 1

Dendritic spines imaged with high resolution at depths of several hundred micrometers in XFP mice with sparse expression through a chronic cranial window. Stack of images from L1 to L6, encompassing the entire dendritic arbor of two L5B neurons in a GFP-M mouse (see Fig. 2b). The stamp at the top left corner indicates the depth of imaging in micrometers. White boxes indicate the location of three dendrite segments imaged at various depths including two apical oblique branches imaged at 410 μm and 615 μm from the dura, which correspond to the images shown in Figs. 2e-g. Increasing laser power was applied at greater depths. Images were obtained by RM and CP-C. All experiments using animals were carried out under institutional and national guidelines. (WMV 1961 kb)

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Holtmaat, A., Bonhoeffer, T., Chow, D. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 4, 1128–1144 (2009). https://doi.org/10.1038/nprot.2009.89

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