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Representative Results






Calcium Imaging in Mouse Superior Colliculus

Published: April 21st, 2023



1Chinese Institute for Brain Research
* These authors contributed equally

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,

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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.......

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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 th.......

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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.......

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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.


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Name Company Catalog Number Comments
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
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
ImageJ NIH Image
Labview National Instruments
MATLAB Mathworks

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