Removable cranial windows for long-term imaging in awake mice

Abstract

Cranial window implants in head-fixed rodents are becoming a preparation of choice for stable optical access to large areas of the cortex over extended periods of time. Here we provide a highly detailed and reliable surgical protocol for a cranial window implantation procedure for chronic wide-field and cellular imaging in awake, head-fixed mice, which enables subsequent window removal and replacement in the weeks and months after the initial craniotomy. This protocol has facilitated awake, chronic imaging in adolescent and adult mice over several months from a large number of cortical brain regions; targeted virus and tracer injections from data obtained using prior awake functional mapping; and functionally targeted two-photon imaging across all cortical layers in awake mice using a microprism attachment to the cranial window. Collectively, these procedures extend the reach of chronic imaging of cortical function and dysfunction in behaving animals.

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Figure 1: Experimental paradigm and surgical procedures involved in chronic imaging in awake mice.
Figure 2: Illustration of chronic window implants for awake imaging in adolescent and adult mice.
Figure 3: Placement in the stereotax and initial skin incision.
Figure 4: Implanting the headpost.
Figure 5: Placing the mouse into the headpost clamps and drilling the craniotomy.
Figure 6: Cranial window placement.
Figure 7: Cranial window implantation and placement of the imaging well.
Figure 8: Cranial window removal and cleaning.
Figure 9: Targeted GCaMP3 expression and subcellular imaging in awake mice.
Figure 10: Long-term clarity of cranial windows and window replacements.
Figure 11: Chronic functional imaging across cortical layers using a microprism.

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Acknowledgements

The authors thank W.-C. Lee, M. Histed, D. Smith, K. Kuchibhotla and M. Henry for assistance in developing the initial window implant and headpost design; A. Vagodny, J. Curry, C. Holland, K. Levandowski and D. Woloszyn for technical assistance; and J. Maunsell for sharing his surgical rig during initial stages of development of these procedures. M. Kirk performed the window implant shown in Figure 9. We also thank members of the Reid and Andermann laboratories for useful discussions. We thank L.L. Looger, Janelia Farm Research Campus, Howard Hughes Medical Institute for provision of AAV-GCaMP3 (ref. 21) via the University of Pennsylvania Vector Core. We thank V. Jayaraman, R.A. Kerr, D.S. Kim, L.L. Looger and K. Svoboda from the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project, Janelia Farm Research Campus, Howard Hughes Medical Institute, for provision of AAV-GCaMP6 (ref. 22) via the University of Pennsylvania Vector Core. The writing of this protocol was supported by grants from the Smith Family Foundation, the Pew Scholars Program in the Biomedical Sciences, the Klarman Family Foundation, the Boston Nutrition and Obesity Research Center, the Association for Aging Research New Investigator Award in Alzheimer's Disease and the Harvard–Massachusetts Institute of Technology (MIT) Joint Research Grants Program (M.L.A.).

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Authors

Contributions

The procedures were developed by G.J.G., D.K.R., M.L.A. and A.M.K., and refined by L.L.G. and V.B., together with assistance from R.C.R. The figures and schematics were done by G.J.G. and M.L.A. The associated surgeries were done by G.J.G. (except for the surgery shown in Fig. 9). Analyses of glial activation were carried out by D.P.S. Finally, G.J.G. and M.L.A. wrote the manuscript with assistance from D.K.R., A.M.K., L.L.G., V.B. and R.C.R.

Corresponding author

Correspondence to Mark L Andermann.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 No significant glial cell activation under cranial window.

(a-b), Representative images of Iba-1 immunohistochemistry (Wako 019-19741, rabbit anti-Iba1, 1:500) in V1 contralateral (a) and ipsilateral (b) to the cranial window implanted >10 days before. Schematic in lower left of b represents location of the cranial window. Scale bar: 100 μm. (c-d) Quantification of microglia activation. There is no significant difference in CD68 immunoreactivity (c; Serotec MCA1957, rat anti-CD68, 1:200), a marker of microglial activation or numbers of Iba-1+ cells (d) in V1 contralateral or ipsilateral to the cranial window. N = 4 mice. Error bars = SEM. (e-f) Representative images of GFAP immunohistochemistry in V1 contralateral (e) and ipsilateral (f) to the cranial window implanted >10 days before. Arrow in f indicates approximate center of the implant, enlarged in H. Scale bar: 100 μm. (g) Quantification of reactive astrocytes as measured by GFAP levels (Sigma G9269, rabbit anti-GFAP, 1:200). There is no significant difference in GFAP immunoreactivity in V1 contralateral or ipsilateral to the cranial window. N = 3 mice. Error bars = SEM. (h) Enlarged region from f, arrow indicates approximate center of the implant. Some strong GFAP immunoreactivity is observed at the pial surface but reactivity is minimal throughout the cortex.

Supplementary Figure 2 Description of titanium headpost and surgical rig.

(a) Schematic illustrating how the custom materials involved in the headpost and cranial window implant (see PROCEDURE Steps 1-77) are combined. (b) Image of the mouse loaded in the stereotax after the headpost has been implanted. (c) Image of the titanium headpost (left) and a schematic drawn to scale (right). (d) Side-on image of the headpost clamp. (e) Image illustrating how to calibrate the planar alignment of the headpost clamps. Appropriately leveled clamping of a glass slide (black arrow) should not cause the glass to crack following secure tightening of both Altos clamps. (f) Close up of Altos clamp illustrating placement on headpost arm on lower jaw of clamp (black arrow). (g) Image of customized stereotaxic setup, facilitating the transition between earbar and headpost clamp configurations prior to drilling the craniotomy.

Supplementary Figure 3 Marking the center of the craniotomy and retraction of the temporalis muscle.

(a) Left: Image with periosteum removed and skull cleaned (PROCEDURE Steps 26-27). The center of the craniotomy (dashed circle) is etched into the skull (PROCEDURE Steps 28-29; using an FG1/4 carbide) at 3.10 mm lateral to lambda and 1.64 mm anterior to the lambdoid suture (our standard coordinates for implant of a 5 mm diameter cranial window over area V1 and neighboring areas of posterior visual cortex. Dotted white line (left) indicates plane of cross-sectional view in the associated schematic (right). (b) The ipsilateral temporalis muscle is gently retracted (red arrow) using forceps (PROCEDURE Step 30). Images in a and b are from a different mouse than that used in Figs. 3-8.

Supplementary Figure 4 Cranial window and microprism assembly fabrication.

(a-c) Description of three-layered cranial window assembly constructed of round coverglass. (a) Schematic illustrating the three layers of coverglass (no. 1 thickness) used to construct the cranial window shown in b. (b) Image of a three-layered cranial window (view of the surface that will contact the brain). (c) Schematic illustrating steps for constructing a cranial window. (d) Schematic illustrating the components of a microprism assembly. A cannula attached to a vacuum line (red arrow) grips the assembly from above during implantation. (e) Schematic illustrating the steps required to construct a microprism assembly. (f) Schematic illustrating how to attach a microprism to the coverglass at a precise location for implants that are targeted to specific brain areas while avoiding major blood vessels. Left: First, we take a picture parallel to the plane of the chronic cranial window, to maintain the correct aspect ratio. We then draw the template on a printout (black circle, arrow, line, and gray lines). The black line targets a 1 mm line at the proposed incision site and placement of the front imaging face of a 1 mm mircoprism. The black arrow represents the direction that excitation light will travel, following implantation, as it is reflected towards the imaging face of the prism. The yellow 'x' indicates the targeted imaging area. The two line drawings represent the template after the image of the brain is removed (middle), and following reflection 180° about the vertical axis (right); this final image is the template that will be used to fabricate the microprism assembly. (g) An image of the template after it is has been printed on a piece of paper and taped to the bottom of a glass slide (with the image in panel f, middle, facing upwards). The slide is held by a leveled headpost clamp (see Supplementary Figs. 2d,e). (h) Zoomed-in image of the top surface of the glass slide in g after application of a small amount of Kwik-Cast to the surface of the slide. (i) A 5 mm coverslip is then centered using ceramic-tipped forceps, compressing the Kwik-Cast until it is semi-transparent. (j) Image of the coverglass shown in I, five-minutes after the Kwik-Cast has set, so that it now holds the coverglass securely in place. (k) Image showing a small drop of Norland Optical Adhesive being applied to the coverglass using a broken cotton-tipped applicator with a fine point. (l) The 1 mm glass microprism is then placed on the coverglass in k such that the edge of the 1 mm x 1 mm imaging face precisely aligns with the 1 mm black line. (m) Image showing the microprism as it is being cured to the bottom layer of the assembly, using an ELC-410 light curing system. The coverslip assembly is then completed as in panel e (not shown).

Supplementary Figure 5 Fashioned cotton-tipped applicator for implanting the cranial window.

(a) Image of the fashioned cotton-tipped applicator, loaded into the stereotaxic attachment that is attached to the micromanipulator at the time of the cranial window implant. (b) Schematic of the fashioned cotton-tipped applicator (side view) applying force to the cranial window. The diagram shows that as the force increases (straight arrow) downward to implant the cranial window, the fashioned cotton-tipped applicator will being to bow (curved arrow). (c) Image of fashioned end of the cotton-tipped applicator (front view).

Supplementary Figure 6 EEG implant.

(a) Mounting holder used during EEG implant (white arrow; see PROCEDURE Steps 32–36. (b) Image depicting the electrode implant sites drilled using a FG1/4 carbide. Red arrows designate electrode implant sites and the black arrow designates the ground electrode implant site. (c) Image of a single electrode wire (tip bent ~90 degrees) resting on the surface of the skull. (d) Image of an electrode after it has been implanted. (e) Image of an electrode after Vetbond has been applied to hold it in place during the headpost implant (PROCEDURE Steps 37-39). (f-g) Zoomed-out view of the electrode wires prior to (f) and following (g) headpost placement.

Supplementary information

Supplementary Figure 1

No significant glial cell activation under cranial window. (PDF 7294 kb)

Supplementary Figure 2

Description of titanium headpost and surgical rig. (PDF 1882 kb)

Supplementary Figure 3

Marking the center of the craniotomy and retraction of the temporalis muscle. (PDF 480 kb)

Supplementary Figure 4

Cranial window and microprism assembly fabrication. (PDF 512 kb)

Supplementary Figure 5

Fashioned cotton-tipped applicator for implanting the cranial window. (PDF 653 kb)

Supplementary Figure 6

EEG implant. (PDF 952 kb)

Supplementary Methods

Intracerebral injection. (PDF 206 kb)

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Goldey, G., Roumis, D., Glickfeld, L. et al. Removable cranial windows for long-term imaging in awake mice. Nat Protoc 9, 2515–2538 (2014). https://doi.org/10.1038/nprot.2014.165

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