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.

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)&#160;inject the virus; 3) implant the headplate; 4)&#160;after 3 weeks, implant the plug; 5)&#160;after a ~3 day recovery and habituation on the treadmill, perform two-photon/wide-field imaging.
1. Preparation of a suction cup (Figure 1A)
<ol>
	<li>Deposit a drop of phosphate-buffered saline (PBS, 1x) in an acrylic dish and touch it with a 21 G flat needle to fill the tip by capillarity.</li>
	<li>Cover the tip of the needle with a translucent silicone adhesive and set for about ~10 min.</li>
	<li>Cut the silicone adhesive with scissors to a disk with a ~2 mm diameter.</li>
</ol>
2. Preparation of plugs (Figure 1B)
<ol>
	<li>Prepare a 0.75 mm thick plastic shim stock and cut out a 1&#160;cm x 1&#160;cm&#160;square in the center. Prepare two acrylic blocks. Clean the shim stock and acrylic blocks with alcohol and wipe them with lens paper.</li>
	<li>Put the shim stock on one acrylic block, deposit the silicone adhesive into the center to fill ~90% of the opening (avoid bubbles), add another acrylic block and ~1 kg force, and wait 20 min.</li>
	<li>Under a surgical microscope, cut the silicone adhesive with a scalpel to 1 mm high by 1.5 mm wide triangular prisms above a piece of paper with triangles printed on it.</li>
	<li>Remove any dust from the triangular prisms with plastic sticky tape and put it on a glass coverslip of 5 mm diameter and 0.15 mm thickness.</li>
	<li>Put the coverslip with the triangular prisms under a corona treater, turn on the corona treater, and bring it closer to the coverslip until lightening appears. Hold it at that position for a few minutes until they are attached to each other. 
	NOTE: This step might be different for other corona treaters.</li>
	<li>Put the plugs in a Petri dish and place it in an incubator at 70 &#176;C overnight.</li>
</ol>
3. Preparation of animals and injection of viral construct
<ol>
	<li>Use C57BL/6J (wild-type) and Emx1-Cre:Pals1flox/wt (partial-cortex)20 mice of both sexes at 6 weeks of age (9 weeks at the time of imaging).</li>
	<li>Sterilize all materials and instruments used for the surgical procedure.</li>
	<li>Anesthetize the mouse with 5% isoflurane and then maintain the isoflurane at 1%-2% with a flow rate of 1 L/min during the surgery. Confirm the level of anesthesia by the lack of toe pinch reflex.</li>
	<li>Head-fix the mouse on a stereotaxic frame with ear bars. Throughout the surgery, put ophthalmic ointment on the mouse eyes to prevent drying, and maintain its body temperature at 37 &#176;C with a thermostatic heating pad.</li>
	<li>Shave the fur and sterilize the skin surface with iodophor and 75% ethanol. Inject lidocaine (5 mg/kg, subcutaneous injection [SQ]), tilidine (2 mg/kg, SQ), and meloxicam (2 mg/kg, SQ). 
	NOTE: The surgical procedure for sterilization, analgesia, and local anesthesia may differ across institutions.</li>
	<li>Remove the skin over the SC with surgical scissors to expose the skull. Clean the skull with a cotton swab.</li>
	<li>Adjust the stereotaxic frame until the skull surface is flat. Drill a hole 0.5 mm lateral and 0.42 mm anterior to the lambda until the dura is exposed.</li>
	<li>Inject adeno-associated virus (AAV) expressing GCaMP6m (rAAV2/9-hsyn-GCaMP6m; 1 x 1013 GC/mL dissolved in 1x PBS) at depths of 1 mm and 1.6 mm from the lambda with a beveled glass micropipette (tip is beveled to 25&#176; with a diameter of 40-50 &#181;m).</li>
	<li>At each depth, inject 100 nL at a speed of 50 nL/min and wait 5 min after each injection. After the injection, slowly withdraw the injector.</li>
</ol>
4. Implantation of the headplate
<ol>
	<li>Clean the muscles and tissues over the skull and scratch the skull with a blade. If bleeding occurs, stop it with gel foam and clean the blood. Attach the headplate (Figure 1C) to the skull with a self-curing dental adhesive resin cement. Specifically, mix a 3/4 spoon of polymer, three drops of monomer, and one drop of catalyst in a ceramic bowl on ice. Apply the mixture on the headplate and the skull. Attach them together and wait 5 min for the adhesive to set.</li>
	<li>Inject antibiotics (ceftiofur sodium, 5 mg/kg, SQ) to prevent infections. Remove the mouse from the stereotaxic frame and place it on a heating pad for recovery. Return it to the home cage after recovering from anesthesia. For pain management, provide ibuprofen-containing drinking water&#160;(0.5 mL/50 mL) to the mouse for 3 days after the surgery.</li>
</ol>
5. Implantation of the plug (Figure 2)
<ol>
	<li>Implant the plug 3 weeks after the viral injection. Anesthetize and fix the mouse on a stereotaxic frame, as described in steps 3.2-3.5. Prepare gel foam soaked in saline to stop potential bleeding.</li>
	<li>Drill a 3 mm x 2 mm oval with a microdrill centered at 0.5 mm posterior to the lambda. Thin the boundary of the oval until it cracks. Thin the skull in front of the oval recording window to make the coverslip close to the SC.</li>
	<li>Take off bone flaps using fine forceps. Make a cut on the dura posterior to the transverse sinus and remove the piece of dura. If necessary, add artificial cerebrospinal fluid (ACSF) to the brain with a 3 mL syringe to prevent drying. Dry the skull and recording window with gel foam and cotton swabs.</li>
	<li>Deposit a drop of silicone adhesive into the recording window. Use the suction cup to hold the plug with negative pressure. Use a motorized micromanipulator to move the suction cup and lower the plug into the silicone adhesive until the coverslip touches the skull. Then, move the plug forward ~1 mm to push away the transverse sinus and expose the SC. This step should be done in 1-2 min before the silicone adhesive becomes sticky.</li>
	<li>Clean the headplate and apply butyl cyanoacrylate and resin cement around the boundary of the plug to fix it to the headplate. 
	NOTE: Provide postoperative care, as described in step 4.2.</li>
</ol>
6. Two-photon imaging (Figure 3)
<ol>
	<li>Head-fix the animal on a rotating treadmill with its headplate. Place the treadmill under a two-photon microscope and adjust the height to an appropriate position. Put a black aluminum cone between the objective and the headplate to prevent light contamination from the monitor for visual stimulation. Habituate the mouse for ~15 min.</li>
	<li>Perform two-photon imaging on the microscope with a 16x magnification, 0.8 numerical aperture (NA), and 3 mm working distance (WD) objective. Use a Ti:sapphire laser with mode-locking technique controlled by two galvo scanners to excite GCaMP6m at 920 nm. Adjust the laser power at the sample plane between 20-80 mW.</li>
	<li>Scan a 600 &#181;m x 600 &#181;m field of view at 4.8 Hz with a spatial resolution of 2.4 &#181; m/pixel, and image neural activity across the depth up to 350 &#181; m. 
	NOTE: The sampling rate and spatial resolution are for galvo scanners, and should be adjusted for resonant scanners.</li>
	<li>Collect emitted fluorescence with a dichroic mirror and detect it using a photomultiplier tube (PMT)&#160;after passing it through a bandpass filter.</li>
	<li>Record the mouse&#39;s locomotion on the treadmill with a rotary encoder. Use a camera with a 50 mm lens to record its pupil size and position. Synchronize these recordings and image acquisitions to visual stimulation by recording triggers sent by the stimulation computer.</li>
</ol>
7. Analysis of calcium responses measured by two-photon imaging
<ol>
	<li>Correct the brain motion during imaging using SIMA21 or NoRMCorre22.</li>
	<li>Manually draw a region of interest (ROI) using the Cell Magic Wand tool (ImageJ) and fit it with an ellipse in MATLAB. Extract fluorescence traces of each ROI after removing neuropil contamination: 
	<img alt="Equation 1" src="/files/ftp_upload/65181/65181eq01.jpg" style="margin: auto;" /> 
	Ftrue&#160;and Fraw are the corrected and raw fluorescence amplitudes of the ROI, respectively, Fneuropil is the fluorescence amplitude of the surrounding neuropil, and r is the out-of-focus neuropil contamination factor (0.7 for our setup). The r is estimated by measuring the signals on a blood vessel for which Ftrue = 0, as described previously23,24.</li>
	<li>Remove slow baseline fluctuations by subtracting the eighth percentile value from a 15 s window centered on each frame25.</li>
	<li>Define visual responses of a neuron as the relative change of fluorescence amplitude in its ROI during the stimulus period: 
	<img alt="Equation 2" src="/files/ftp_upload/65181/65181eq02.jpg" style="margin: auto;" /> 
	Fpeak&#160;is the peak amplitude of the fluorescence trace during the visual stimulation, and Fbaseline is the mean amplitude during a 0.5 s period prior to stimulation.</li>
</ol>
8. Wide-field imaging and data analysis (Figure 4)
<ol>
	<li>Build a wide-field macroscope with a tandem lens4 (Figure 4A). The tandem-lens macroscope is two camera lenses (50 mm and 105 mm) connected via an adapter, and the magnification is ~2x.</li>
	<li>Prepare partial-cortex mutant mice as described in steps 3.2-3.5. Inject 100 nL of AAV expressing GCaMP6m at a depth of 200 &#181;m at the center of each side of the SC.</li>
	<li>After ~3 weeks, implant a 5 mm diameter coverslip over the SC. Perform an oval craniotomy of 4 mm x 3 mm centered at the SC and thin the bone around it. Then, press the coverslip directly onto the SC and fix it with resin cement.</li>
	<li>Excite GCaMP6m with a blue light emitting diode (LED) with an excitation filter (469 nm &#177; 35 nm). Acquire images using a complementary metal oxide semiconductor (CMOS) camera after passing an emission filter (525 nm &#177; 39 nm) at 10 Hz. The camera covers an area of 5.36 mm x 2.85 mm at a spatial resolution of 2.63 &#181;m/pixel.</li>
	<li>Convolve each acquired image with a normalized box filter and downsample it to 1/4 of the original size. For each pixel in the image, define the response, as described in step 7.4. 
	NOTE: Provide postoperative care, as described in step 4.2.</li>
</ol>

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

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The authors have nothing to disclose.

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...

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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-mm lens</td>
			<td>Computar</td>
			<td>M5018-MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mm coverslip</td>
			<td>Warner instruments</td>
			<td>CS-5R</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter</td>
			<td>Chroma Technology</td>
			<td>HQ575/250 m-2p</td>
			<td></td>
		</tr>
		<tr>
			<td>butyl cyanoacrylate</td>
			<td>Vetbond, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera for monitoring pupil</td>
			<td>FLIR</td>
			<td>BFS-U3-04S2M-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>camera for widefield imaging</td>
			<td>Basler</td>
			<td>acA2000-165&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic</td>
			<td>Chroma Technology</td>
			<td>T600/200dcrb&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bead sterilizer</td>
			<td>RWD</td>
			<td>RS1502</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrill</td>
			<td>RWD</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>QUAD</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td></td>
		</tr>
		<tr>
			<td>rotory encoder</td>
			<td>USdigital</td>
			<td>MA3-A10-125-N</td>
			<td></td>
		</tr>
		<tr>
			<td>self-curing dental adhesive resin cement&#160;</td>
			<td>SuperBond C&#38;B, Sun Medical Co, Ltd. Moriyama, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>thermostatic heating pad&#160;</td>
			<td>RWD</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>translucent silicone adhesive&#160;</td>
			<td>Kwik-Sil, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>treadmill</td>
			<td>Xinglin Biology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Virus Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV2/9-hsyn-Gcamp6m</td>
			<td>Vector Core at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild type</td>
			<td>Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emx1-Cre</td>
			<td>The Jackson Laboratory&#160;</td>
			<td>5628</td>
			<td></td>
		</tr>
		<tr>
			<td>Pals1flox/wt</td>
			<td>Christopher A. Walsh Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH Image</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium imaging in mouse superior colliculus

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&#160;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.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

Calcium Imaging in Mouse Superior Colliculus

My research focuses on neural coding and mechanisms for visual processing. We are trying to answer how various information is encoded in the brain, especially in the superior colliculus under the underlying neuro-mechanisms. To understand visual processing, scientists combine various approaches, including neuronal morphology, antegrade and retrograde tracing, IL sequencing, neuro modeling, machine learning, is understood well.The challenge is to remove the skull over the SSA, cutting and left in the bone in one larger piece, is likely to fail because the bone is attached to the dura. By combining two photo on a wide field calcium image, our recent work revealed the function architecture of motion direction in the mouse SC at both single cell resolution and a global scale. We found that neurons with similar preference form patches up to 500 micrometer under global to the upward on the nasal motion.With our protocol, we are addressing two research gaps. First, researchers can perform long-term cast imaging in mouse SC at a single cell resolution without breaking the cortex. Secondly, researchers can record the neuroactivity across the entire SC using widefield microscoping.Our protocol provides a measure to image the posterior medial SC at single cell resolution with a intact cortex in mice. Also, we use a biocompatible plug to expose the SC, which reduces infection for chronic imaging. Our results provide a way to study the neural coding of visual information across a large visual field.Combined with optogenetics, one can study half inputs from different brain regions modulating the neuroactivity in SC.

Figures 1A,B show&#160;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...

Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

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 ...

calcium-imaging-in-mouse-superior-colliculus

Research

JoVE Journal

Neuroscience

Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse

Begin the process of creating a suction cup by depositing a drop of PBS in an acrylic dish. Use a 21 gauge flat needle to fill the tip of the needle with PBS by capillarity. Cover the tip of the needle with a translucent silicone adhesive, and set it for approximately 10 minutes.Cut the silicone adhesive with scissors to a disc with a two millimeter diameter. To prepare the plugs, take a 0.75 millimeter thick plastic shim stock and cut out a one by one centimeter square in the center. Clean the shim stock and two acrylic blocks with alcohol, and wipe them with lens paper.Place the shim stock on one acrylic block, and deposit silicone adhesive into the center to fill 90%of the opening. Add another acrylic block. Apply approximately one kilogram of force, and wait 20 minutes.Under a surgical microscope, cut the silicone adhesive with a scalpel into one by 1.5 millimeter triangular prisms. Remove any dust from the triangular prisms with sticky plastic tape, and place them on a glass cover slip. Place the cover slip with the triangular prisms under a corona treater and turn it on.Bring the corona treater closer to the cover slip until lightning appears, and hold it in that position for a few minutes until the prisms and cover slip are attached. After placing the plugs in a Petri dish, incubate at 70 degrees Celsius overnight.

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

Implantation of the Headplate in Mouse for Calcium Imaging 

After injecting the viral construct into the mouse brain, clean the muscles and tissues over the skull, and scratch the skull with a blade. Prepare the dental adhesive resin cement by mixing 3/4 spoonful of polymer, three drops of monomer, and one drop of catalyst in a ceramic bowl on ice. Apply the mixture to the head plate and the skull.Attach them and wait for the adhesive to set for five minutes. Finally, inject antibiotics to prevent infections. After removing the mouse from the stereotaxic frame, place it on a heating pad for recovery, and return it to the home cage after recovering from anesthesia.

Implanting the Plug in the Mouse for Calcium Imaging

Three weeks after the viral infection, anesthetize and fix the mouse with a head plate on a stereotaxic frame. Soak a gel foam in saline to stop potential bleeding. Use a micro-drill to create a three by two millimeter oval, centered at 0.5 millimeter posterior to the lambda.Thin the boundary of the oval until it cracks, then thin the skull before the oval recording window to close the cover slip to the SC.Using fine forceps, take off the bone flaps. Make a cut down the dura, posterior to the transverse sinus and remove the piece of dura. Dry the skull and the recording window with gel foam and cotton swabs.Then deposit a drop of silicone adhesive into the recording window. Use the suction cup to hold the plug with negative pressure, and use a motorized micro-manipulator to move the suction cup and lower the plug into the silicone adhesive until the cover slip touches the skull. Once the head plate is cleaned, apply butyl cyanoacrylate and resin cement around the boundary of the plug to fix it to the head plate and proceed with imaging.

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons

After implanting the head plate and plug, secure the animal on a rotating treadmill with its head plate. Place the treadmill beneath the two-photon microscope and adjust the height to an appropriate position. Place a black aluminum cone between the objective and the head plate to prevent light contamination from the monitor for visual stimulation.To perform two-photon imaging, adjust the laser power at the sample plane between 20 and 80 milliwatts. Scan a 600-by-600 micrometer field of view at 4.8 hertz with a spatial resolution of 2.4 micrometers per pixel and image neural activity up to 350 micrometers in depth. Using a rotary encoder, record the mouse's locomotion on the treadmill.Use a camera with a 50 millimeter lens to record its pupil size and position. Lastly, synchronize these recordings, and image acquisitions to visual stimulation by recording triggers sent by the stimulation computer. The calcium responses of SC neurons from a wild type mouse and the standard deviation projection of the fluorescence across images are presented.

Wide-Field Calcium Imaging of Mouse Superior Colliculus Neurons

To begin, inject 100 nanoliters of AAV expressing GCaMP6m at a depth of 200 micrometers at the center of each side of the SC to the mutant mouse whose posterior cortex has failed to develop. After approximately three weeks, implant a five millimeter diameter cover slip over the SC.After fixing the cover slip, acquire images using a CMOA camera after passing an emission filter at 10 hertz. The calcium responses of SC neurons from a partial cortex mutant mouse were imaged using wide-field microscopy.The acquired image was down sampled to one quarter of the original size, and visually evoked calcium responses are shown.

1.8K Views.  Chinese Institute for Brain Research.  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. 

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Summary