Abstract
Despite the growing popularity of blood oxygen level–dependent (BOLD) functional MRI (fMRI), understanding of its underlying principles is still limited. This protocol describes a technique for simultaneous measurement of neural activity using fluorescent calcium indicators together with the corresponding hemodynamic BOLD fMRI response in the mouse brain. Our early work using small-molecule fluorophores in rats gave encouraging results but was limited to acute measurements using synthetic dyes. Our latest procedure combines fMRI with optical detection of cell-type-specific virally delivered GCaMP6, a genetically encoded calcium indicator (GECI). GCaMP6 fluorescence, which increases upon calcium binding, is collected by a chronically implanted optical fiber, allowing longitudinal studies in mice. The chronic implant, placed horizontally on the skull, has an angulated tip that reflects light into the brain and is connected via fiber optics to a remote optical setup. The technique allows access to the neocortex and does not require adaptations of commercial MRI hardware. The hybrid approach permits fiber-optic calcium recordings with simultaneous artifact-free BOLD fMRI with full brain coverage and 1-s temporal resolution using standard gradient-echo echo-planar imaging (GE-EPI) sequences. The method provides robust, cell-type-specific readouts to link neural activity to BOLD signals, as emonstrated for task-free ('resting-state') conditions and in response to hind-paw stimulation. These results highlight the power of fiber photometry combined with fMRI, which we aim to further advance in this protocol. The approach can be easily adapted to study other molecular processes using suitable fluorescent indicators.
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Acknowledgements
This work was supported by an R'Equip grant of the Swiss National Science Foundation (SNSF grant 316030_405019 to F.H. and B.W., grants 310030_141202 and 310030B_160310 to M.R.), a Joint Collaborative Project with F. Hoffmann La-Roche (Y.S. and F.H.) and an ERC Advanced Grant to F.H. (grant 670757; BRAINCOMPATH).
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M.R., F.H., F.S., and Y.S. developed the concept and designed the experiments. F.S. performed the surgeries and experiments with the help of Y.S. and A.S., and prepared the manuscript. F.H. edited the manuscript with contributions from M.R., B.W., A.S., and J.S. Y.S. designed the optical setup and developed the software tools with contributions from F.S. J.S. developed and validated the astrocyte-specific constructs.
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Integrated supplementary information
Supplementary Figure 1 Evaluation of the GCaMP6s expression under neuronal or astrocytic promoter.
(a) Photomicrograph of a coronal brain section at the site of primary sensory cortex of a mouse transduced with the neuron specific AAV‐hSyn1‐GCaMP6s. Shown is the GFP channel indicating the area of GCaMP expression. (b) Confocal microscopic section (70 μm thickness) showing a zoomed-in view of a cortical S1 region 2 months after AAV‐hSyn1‐GCaMP6s transduction. The majority of neurons (morphologically discernible as pyramidal neurons with their apical dendrites pointing upwards) show cytosolic GCaMP6s expression and a dark spot (nucleus). Only a small number of neurons appear to show nuclear filling which may indicate toxicity.
(c-e) Show the results of mice transduced with astrocyte‐specific AAV‐GFAP‐GCaMP6s. All scale bars are 20 μm.
(c) Two‐photon microscopic images of GCaMP6s expression (left), the astrocyte‐specific dye sulforhodamine‐101 (20mg/kg, imaged 100 min after i.v. injection, middle), and the overlay of both channels (right). (d) Immunohistochemistry of brain slices at the virus injection site, further stained with anti‐GFP antibody (left), anti-GFAP antibody (middle), and the overlay of both channels (right). (e) Immunohistochemistry to test for microglial activation. As in (d) anti-GFP antibody was applied (left), but anti-Iba1 was used to label microglia (middle). The overlay revealed no overlap between the two channels (right). For further details and additional control experiments of the astrocyte-specific GCaMP6 construct, see Stobart et al.1
1. Stobart, J. L. et al. Long-term in vivo calcium imaging of astrocytes reveals distinct cellular compartment responses to sensory stimulation. Cereb. Cortex 1–15 (2016). doi:10.1093/gbe/evw245
Supplementary Figure 2 Examples of coronal EPI slices for animals with fiber-optic implants, showing varying degrees of susceptibility artifacts.
(a) Representative EPI image in the absence of any artifacts. (b) Small susceptibility artifact caused by air bubbles enclosed in the Kwik-Sil at the fiber implantation site. (c) Strong susceptibility artifact caused by inadequate dental material. Dental materials typically contain paramagnetic particles to render them radiopaque.
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Supplementary Text and Figures
Supplementary Figures 1 and 2, and Supplementary Methods 1–3. (PDF 2611 kb)
Subcutaneous injection.
Demonstrating the injection of the local analgesic subcutaneously under the scalp. (MP4 24579 kb)
Scalp removal.
Demonstrating the procedure and the amount of the scalp that should be removed. (MP4 16192 kb)
Craniotomy.
Demonstrating the use of the microdrill to prepare the craniotomy for the fiber-optic implant. (MP4 16638 kb)
Dental composite wall.
Demonstrating application of the dental composite and the use of the dental curing lamp to create a barrier at the outer circumference of the skull. (MP4 9228 kb)
Connecting the implant.
Demonstrating how the fiber-optic patch cable is connected with a fiber-optic implant via a ceramic split sleeve. (MP4 27985 kb)
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Schlegel, F., Sych, Y., Schroeter, A. et al. Fiber-optic implant for simultaneous fluorescence-based calcium recordings and BOLD fMRI in mice. Nat Protoc 13, 840–855 (2018). https://doi.org/10.1038/nprot.2018.003
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DOI: https://doi.org/10.1038/nprot.2018.003
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