Wide-field calcium imaging of cortex-wide activity in awake, head-fixed mice

Abstract

Characterizing cortex-wide neural activity is essential for understanding large-scale interactions among distributed cortical regions. Here, we describe a protocol using wide-field calcium imaging to monitor the cortex-wide activity in awake, head-fixed mice. This approach provides sufficient signal-to-noise ratio and spatiotemporal resolution to capture large-scale neural activity in behaving mice on a moment-by-moment basis. The use of genetically encoded calcium indicators allows longitudinal imaging over months and can achieve cell-type specificity. We also describe a pipeline to process the imaging data. For complete details on the use and execution of this protocol, please refer to Makino et al. (2017) and Liu et al. (2021).

Graphical abstract

Figure 1

Surgery setup and key reagents for the skull-intact surgery

Figure 2

The custom-built stainless steel headbar (left) attached to the skull (middle) for head fixation on the stage (right) under the wide-field microscope

Figure 3

Surgical procedures of the skull-intact preparation for wide-field calcium imaging (A) Protect the eyes with copious amounts of Vaseline (indicated by white arrowheads). Clean and antisepticise the surgical region (marked by the white dashed line). Make a small incision (∼2 mm) on the scalp using fine scissors (marked by the white scissor symbol). (B) Starting from the small incision, cut off a circular piece of the scalp to expose the skull covering the dorsal cortex and cerebellum. (C) Gently remove the periosteum on the skull surface using a scalpel. Note that the skull is less glossy after removing the periosteum compared to (B). Next, detach the muscles from the occipital bone (within the black dashed line) with a scalpel and push the muscles away. (D) Insert small pieces of gel foam into the gaps between muscles and the occipital bone to stop bleeding. Wipe the skull with cotton swabs soaked in 3% hydrogen peroxide to remove any remaining soft tissues. (E) Comparison between moisturized and dry skulls. Note that the dry skull is whiter and less transparent. It is critical to dry the skull completely before applying cyanoacrylate glue, as the glue becomes opaque when encountering a wet surface. (F) Remove the gel foam and apply small drops of cyanoacrylate glue to cover the muscles and the occipital bone (marked by the black dashed line). Let the cyanoacrylate glue cure. (G) Add drops of dental acrylic cement with a double-end carver to build a thick layer on top of the glue. Let the dental acrylic cement cure. Apply cyanoacrylate glue with a double-end carver to cover the skin along the cuts (indicated by yellow arrowheads). (H) Implant the headbar. Apply a small amount of cyanoacrylate glue to the interparietal bone (marked by the black dashed ellipse in G) and place the headbar ∼0.5–1 mm posterior to the lambda. Adjust the angle of the headbar by eyeballing until it is parallel to the transverse plane of the skull (the top side of the headbar and the transverse plane of the skull are marked by yellow dashed lines in the bottom panel). Hold the headbar in place until it stays steady. (I) Secure the headbar. Left, add drops of cyanoacrylate glue to build a glue layer between the headbar and cement (indicated by the yellow shading area). Let the glue cure. Middle and right, apply dental acrylic cement around the headbar to build up the attachment between bones and the headbar. Make sure the bones posterior to the lambda and the middle part of the headbar are entirely covered. Let the dental acrylic cement cure. (J) Build the cyanoacrylate glue layer on the skull. Apply a small amount of cyanoacrylate glue and evenly distribute it to form a thin layer covering the entire skull. Wait for the cyanoacrylate glue to cure to form a solid, smooth, and transparent layer. Repeat 3–4 times to thicken the glue layer. The skull should gradually become more transparent within 20 min, and the blood vessels should become clearly visible compared to the dry skull in (E). (K) Protect the eyes from the excitation light of imaging. Cut a chunk (∼5 mm) of black heat shrink tube (∼2 cm diameter) and glue it to the circumference of the skull using cyanoacrylate glue. Then fill the gap between the heat shrink tube and mouse head with black dental acrylic cement from the outside. Scale bars in (D), 1 mm, in (E and J), 0.5 mm.

Figure 4

Wide-field calcium imaging in the transgenic mouse with a broad expression of GCaMP6s in cortical excitatory neurons (A) Left, imaging setup. Middle, an example field of view showing the fluorescence signal from most of the dorsal cortex. Note that the lateral blood vessels around the edges are out of focus and appear blurry due to the curvature of the cortex. Right, cortical regions (based on the mouse brain atlas from the Allen Institute (Wang et al., 2020)) simultaneously recorded by wide-field calcium imaging. Orange dashed box represents the area within the FOV. M2, Secondary motor cortex; M1, primary motor cortex; S1, primary somatosensory cortex; PPC, posterior parietal cortex; S2, secondary somatosensory cortex; Aud., auditory cortex; RSC, retrosplenial cortex; Vis., visual cortex. (B) Example image frames of cortex-wide activity (Δf/f) showing different patterns and activity traces of individual pixels in a behaving mouse. Not that the activation in small cortical areas can be resolved by wide-field calcium imaging. Gray dashed lines indicate the time of image frames in activity traces of example pixels. Adapted from Ren and Komiyama (2021). Scale bars in (A), 5 mm, in (B), for image frames, 2 mm, for activity traces, veritical, 0.1 Δf/f, horizontal, 10 s.

Figure 5

Two example cases using wide-field calcium imaging to investigate cortex-wide dynamics (A) Imaging cortex-wide activity with wide-field calcium imaging during a motor learning task. In this task, water-restricted mice are trained to press a lever during an auditory cue to receive a water reward. (B) Cortex-wide activity aligned to the lever-pressing movement onset averaged across movements in a single session from an example animal. Note that the lever-pressing movement evokes distributed activation of most of the cortex. (C) Learning changes the activity flow throughout the cortex, inducing a secondary activity stream flowing from the secondary motor cortex to the rest of the cortex. Black arrows indicate the directions of the activity flow. Adapted from Makino et al. (2017). (D) Simultaneous recordings of cortex-wide activity using wide-field calcium imaging and hippocampal activity using a newly developed transparent and flexible electrode array (Neuro-FITM). Yellow dashed lines mark the edge of the electrode array for visualization. Left, wide-field surgical preparation with the electrode array implanted. Right, example field of view under the wide-field microscope. (E) Diverse cortical activity patterns during hippocampal SWRs. Adapted from Liu et al. (2021). (F) Different cortex-wide activity patterns are associated with distinct hippocampal neural population activity during SWRs. Scale bars in (D), 5 mm, in (E), vertical, 200 μV, horizontal, 50 ms.

Figure 6

Obtain Δf/f time series for each pixel in wide-field images Raw fluorescence signal, estimated baseline (f), and Δf/f traces of two example pixels with different brightness are shown here. Note that the estimated baseline (f) captures the difference in fluorescence intensity between two pixels and the slow drifts in raw fluorescence traces (potentially due to the slow decay time of GCaMP6s). After normalization, the Δf/f time series shows a more stable baseline level while fast transients are retained.

Figure 7

Procedures of using PCA-and-ICA analysis to remove hemodynamic artifacts from wide-field signals (A) Transform the 3D Δf/f image stack (row pixel × column pixel × frame) to a 2D matrix (pixel × frame) for PCA where pixels are treated as variables and frames are treated as observations. (B) Extract spatial components by PCA-and-ICA analysis. Most spatial ICs correspond to known cortical regions or hemodynamic artifacts. ICs corresponding to artifacts are visually identified and excluded. (C) Reconstruct Δf/f images by multiplying the remaining ICs corresponding to cortical regions with their time series, then adding the mean value from the original Δf/f image stack for each pixel. The resulting reconstructed images retain the activity of cortical regions while effectively reducing artifacts. White arrowheads indicate the elimination of hemodynamic signals in reconstructed images compared to the raw images.