This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.
The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.
The superior colliculus (SC) is an important visual center in all vertebrates. In mammals, it receives direct inputs from the retina and the visual cortex1. While optical recording has been widely applied to the cortex2,3,4,5, its application in the SC is hindered by poor optical accesss6,7,8,9,10,11,12,13,14,15,16,17,18,19. The goal of this protocol is to provide details about two complementary methods for optical recording of the neural activity in the SC.
The SC is located beneath the cortex and transverse sinus, which limits optical access to the collicular neurons. One approach to overcome this limitation is to aspirate the overlaying cortex and expose the anterior-lateral SC7,9,10,13,14,19. However, because the SC receives cortical inputs, such an operation might affect how the SC neurons respond to visual stimuli. To overcome this limitation, we detail here an alternative protocol to image the superficial layer of the posterior-medial SC with a silicon plug, while leaving the cortex intact8,11. Specifically, to achieve single-cell resolution, we applied two-photon microscopy to image calcium responses in the posterior-medial SC of wild-type mice. In addition, to achieve broad coverage, we applied wide-field microscopy to image the entire SC of a mutant mouse whose posterior cortex has not developed20.
The two methods described in this protocol are complementary to each other. The two-photon calcium imaging without ablating the cortex is appropriate for recording neural activity at single-cell resolution with intact cortical inputs. The wide-field calcium imaging is appropriate for recording neural activity in the entire SC while sacrificing spatial resolution.
All experimental procedures were performed in accordance with the animal welfare guidelines and approved by the IACUC at the Chinese Institute for Brain Research, Beijing.
NOTE: The timeline for this protocol is as follows: 1) make the suction cup; 2) inject the virus; 3) implant the headplate; 4) after 3 weeks, implant the plug; 5) after a ~3 day recovery and habituation on the treadmill, perform two-photon/wide-field imaging.
1. Preparation of a suction cup (Figure 1A)
2. Preparation of plugs (Figure 1B)
3. Preparation of animals and injection of viral construct
4. Implantation of the headplate
5. Implantation of the plug (Figure 2)
6. Two-photon imaging (Figure 3)
7. Analysis of calcium responses measured by two-photon imaging
8. Wide-field imaging and data analysis (Figure 4)
Figures 1A,B show how to make the suction cup and the plugs, respectively. Figure 2 shows how to implant the plug successfully. After implanting the plug, the posterior-medial SC is exposed, as shown in Figure 2D. Figure 3 shows calcium responses of SC neurons from an example wild-type mouse imaged using two-photon microscopy. The triangular prism, which is easily captured under the microscope, can be used to locate the imaging site (Figure 3B). Visual responses are shown at single-cell resolution (Figures 3C ,Dand Video 1). Figure 4 shows calcium responses of SC neurons from an example partial-cortex mutant mouse imaged using wide-field microscopy. The acquired image was first downsampled to one-quarter of the original size (Figure 4B). Visually evoked calcium responses are shown at both sides of the SC (Figure 4C and Video 2). Figure 5 shows the expression of GCaMP6m in the SC at the injection site and the imaging site; there are less neurons at the injection site.
Figure 1: Preparation. (A) Three steps for making the suction cup (steps 1.1-1.3); PBS inside the needle is not shown. (B) Three steps for making the plugs (steps 2.1-2.3). (C) Two types of headplate with an 8 mm diameter for wild-type mice. Two types of headplate with a 7 mm diameter for partial-cortex mutant mice. Please click here to view a larger version of this figure.
Figure 2: Implantation of the plug (steps 5.2-5.5). (A) The bone is thinned. The surroundings are resin cement. (B) The bone and dura are removed to expose the inferior colliculus and cerebellum. (C) The plug is lowered, and it pushed the transverse sinus forward to expose the posterior-medial SC. The green dashed circle marks the coverslip. The blue dashed triangle marks the plug. The red dashed circle marks the suction cup. (D) The plug is fixed with butyl cyanoacrylate and resin cement. The image is overexposed to show the posterior-medial SC clearly. Please click here to view a larger version of this figure.
Figure 3: Two-photon calcium imaging. (A) Schematic of the mouse brain anatomy after implanting the plug. The dark dashed line marks the outline of the SC. The yellow triangle indicates the triangular prism. The green color indicates the GCaMP expression. (B) A picture of the silicon triangular prism under the microscope before scanning. (C) An image of the fluorescence of GCamMP from SC neurons, measured by two-photon microscopy from an example wild-type mouse. (D) A standard deviation projection of the fluorescence across images (see Video 1). Please click here to view a larger version of this figure.
Figure 4: Wide-field calcium imaging. (A) A photo of the wide-field imaging setup. (B) An example raw image and the downsampled image. The dashed yellow line marks the outline of the SC. (C) A single frame and a standard deviation projection of the calcium responses of SC neurons, measured by wide-field microscopy from an example partial-cortex mouse (see Video 2). Please click here to view a larger version of this figure.
Figure 5: Coronal sections of a mouse brain at different distances from the injection site. The bottom row shows the high magnification of the boxes marked at the top row. The yellow dashed rectangle marks the injection site, which contains less neurons than the region on its right. Please click here to view a larger version of this figure.
Video 1: Raw and motion-corrected videos of the calcium responses of SC neurons to the visual stimulus in Video 3 by two-photon microscopy. The images were acquired at 4.8 Hz and displayed at 20 Hz. Please click here to download this Video.
Video 2: Raw and motion-corrected videos of the calcium responses of SC neurons to the visual stimulus in Video 3 by wide-field microscopy. The images were acquired at 10 Hz and displayed at 20 Hz. Please click here to download this Video.
Video 3: A black full-screen bar (5° wide) drifting in 12 different directions (50°/s). Please click here to download this Video.
Critical steps in the protocol
The most critical step is the craniotomy in steps 5.2 and 5.3. First, the bone at 0.5 mm posterior to the lambda is thick and has blood vessels inside, which can cause bleeding during the drilling process. Adequate gel foam should be prepared to stop the bleeding. Second, there is a good chance of angiorrhexis when removing the bone just above the transverse sinus. For troubleshooting, one alternative approach is to thin the bone inside the oval and remove it piece by piece. Another trick is to soak the bone prior to lifting to make the detachment of the dura from the bone easier and prevent bleeding.
Another critical step is the plug implantation in step 5.4, for which the whole process should last 1-2 min. For troubleshooting, one can first move the plug with the micromanipulator to an appropriate position in the ACSF and set the final position of the plug. Then, add the silicon adhesive and move the plug at an appropriate speed to the final position.
Significance
This protocol has two merits. First, it provides details for imaging the posterior-medial SC at single-cell resolution with an intact cortex in wild-type mice, while in previous reports the overlaying cortex was aspirated to expose the anterior-lateral SC. Second, it details how to image the entire SC of partial-cortex mutant mice using the wide-field calcium imaging technique. These two approaches can be applied to image other brain regions in behaving animals at different scales.
Two alternative methods have been reported to image the posterior-medial SC without aspirating the overlaying cortex15,16. Schröder et al. had a circular 4 mm craniotomy and appled a stainless-steel tube with a washer and a glass coverslip at each end to expose the SC16. The larger craniotomy and more rigid material, compared to what we used, are more likely to cause bleeding during the implantation. In addition, because stainless steel is not biocompatible, it is more likely to cause infection for chronic imaging. Similarly, the glass coverslip used in Savier et al. was also more rigid and not biocompatible, and more likely cause bleeding and infections15. Another difference from these two methods is that our viral injection site is outside the imaging window, and thus the imaging quality is less affected by the potential damage at the injection site (Figure 5).
Limitations of the protocol
This protocol provides details for imaging calcium responses in the superficial layer of the SC. Imaging deeper layers, for example using a prism26, is not covered. The two-photon calcium imaging approach is used for recording the neural activity in the posterior-medial SC and does not cover the anterior-lateral SC7,13. Entire SC calcium imaging with wide-field microscopy was applied to mutant partial-cortex mice. For imaging the entire SC of wide-type mice, one needs to aspirate the overlaying cortex. An alternative way to image the entire SC, without viral injection, is the intrinsic imaging technique; however, it has a lower signal-to-noise ratio6,8.
The authors have nothing to disclose.
This work is supported by the National Natural Science Foundation of China (32271060). Y.-t.L. designed the research, performed the experiment, analyzed the data, and wrote the manuscript. Z.L. and R.W. performed the experiment.
16x objective | Nikon | ||
50-mm lens | Computar | M5018-MP2 | |
5-mm coverslip | Warner instruments | CS-5R | |
bandpass filter | Chroma Technology | HQ575/250 m-2p | |
butyl cyanoacrylate | Vetbond, World Precision Instruments | ||
camera for monitoring pupil | FLIR | BFS-U3-04S2M-CS | |
camera for widefield imaging | Basler | acA2000-165µm | |
corona treater | Electro-Technic Products | BD-20AC | |
dichroic | Chroma Technology | T600/200dcrb | |
galvanometers | Cambridge Technology | ||
glass bead sterilizer | RWD | RS1502 | |
microdrill | RWD | 78001 | |
micromanipulator | Sutter Instruments | QUAD | |
photomultiplier tube | Hamamatsu | R3896 | |
rotory encoder | USdigital | MA3-A10-125-N | |
self-curing dental adhesive resin cement | SuperBond C&B, Sun Medical Co, Ltd. Moriyama, Japan | ||
thermostatic heating pad | RWD | 69020 | |
Ti:Sapphire laser | Spectra-Physics | Mai Tai HP DeepSee | |
translucent silicone adhesive | Kwik-Sil, World Precision Instruments | ||
treadmill | Xinglin Biology | ||
Virus Strains | |||
rAAV2/9-hsyn-Gcamp6m | Vector Core at Chinese Institute for Brain Research, Beijing | ||
Animals | |||
C57BL/6J wild type | Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing | ||
Emx1-Cre | The Jackson Laboratory | 5628 | |
Pals1flox/wt | Christopher A. Walsh Lab | ||
Software | |||
ImageJ | NIH Image | ||
Labview | National Instruments | ||
MATLAB | Mathworks |