Name: Auteur in de schijnwerpers: onthulling van neurale codering en mechanismen van visuele verwerking in de superieure colliculus
Uploaded: 2023-04-21T15:00:00
Description: Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

Dit werk wordt ondersteund door de National Natural Science Foundation of China (32271060). Y.-t.L. ontwierp het onderzoek, voerde het experiment uit, analyseerde de gegevens en schreef het manuscript. Z.L. en R.W. voerden het experiment uit.

Alle experimentele procedures werden uitgevoerd in overeenstemming met de richtlijnen voor dierenwelzijn en goedgekeurd door de IACUC van het Chinese Institute for Brain Research, Beijing.
NOTITIE: De tijdlijn voor dit protocol is als volgt: 1) maak de zuignap; 2) injecteer het virus; 3) implanteer de kopplaat; 4) implanteer na 3 weken de plug; 5) Voer na een herstel en gewenning van ~3 dagen op de loopband twee-foton/breedveldbeeldvorming uit.
1. Voorbereidin...

Beeldvorming van twee fotonen en breedveldcalcium in de superieure colliculus

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Kritieke stappen in het protocol De meest kritische stap is de craniotomie in stap 5.2 en 5.3. Ten eerste is het bot op 0,5 mm achter de lambda dik en bevat het bloedvaten aan de binnenkant, die tijdens het boorproces bloedingen kunnen veroorzaken. Er moet voldoende gelschuim worden voorbereid om het bloeden te stoppen. Ten tweede is er een goede kans op angiorrhexis bij het verwijderen van het bot net boven de transversale sinus. Voor het oplossen van problemen is een alternatieve aanpak om het bot i...

De superieure colliculus (SC) is een belangrijk visueel centrum bij alle gewervelde dieren. Bij zoogdieren ontvangt het directe input van het netvlies en de visuele cortex1. Hoewel optische opname op grote schaal is toegepast op de cortex 2,3,4,5, wordt de toepassing ervan in de SC belemmerd door slechte optische toegangen 6,7,8,9,10,11,12,13,14,15,16,17 18,19. Het doel van dit protocol is om details te geven over twee complementaire methoden voor optische registratie van de neurale activiteit in de SC.
De SC bevindt zich onder de cortex en de transversale sinus, wat de optische toegang tot de colliculaire neuronen beperkt. Een benadering om deze beperking te overwinnen is om de overlappende cortex op te zuigen en de anterieure-laterale SC 7,9,10,13,14,19 bloot te leggen. Omdat de SC echter corticale input ontvangt, kan een dergelijke operatie van invloed zijn op hoe de SC-neuronen reageren op visuele stimuli. Om deze beperking te overwinnen, beschrijven we hier een alternatief protocol om de oppervlakkige laag van de posterieure-mediale SC af te beelden met een siliciumplug, terwijl de cortex intact blijft 8,11. Om een eencellige resolutie te bereiken, pasten we twee-fotonmicroscopie toe om calciumresponsen in de posterieure-mediale SC van wildtype muizen in beeld te brengen. Om een brede dekking te bereiken, pasten we bovendien breedveldmicroscopie toe om de volledige SC van een gemuteerde muis waarvan de achterste cortex zich niet heeft ontwikkeld20 in beeld te brengen.
De twee methoden die in dit protocol worden beschreven, zijn complementair aan elkaar. De calciumbeeldvorming met twee fotonen zonder de cortex te ablateren is geschikt voor het registreren van neurale activiteit met een resolutie van één cel met intacte corticale inputs. De breedveldcalciumbeeldvorming is geschikt voor het registreren van neurale activiteit in de gehele SC terwijl de ruimtelijke resolutie wordt opgeofferd.

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	<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

De superieure colliculus (SC), een evolutionair geconserveerde middenhersenstructuur bij alle gewervelde dieren, is het meest geavanceerde visuele centrum vóór het ontstaan van de hersenschors. Het ontvangt directe input van ~30 soorten retinale ganglioncellen (RGC's), die elk coderen voor een specifiek visueel kenmerk. Het blijft ongrijpbaar of de SC gewoon retinale kenmerken erft of dat er aanvullende en mogelijk de novo verwerking plaatsvindt in de SC. Om de neurale codering van visuele informatie in de SC te onthullen, bieden we hier een gedetailleerd protocol om visuele reacties optisch vast te leggen met twee complementaire methoden bij wakkere muizen. De ene methode maakt gebruik van twee-fotonmicroscopie om calciumactiviteit in beeld te brengen met een resolutie van één cel zonder de overlappende cortex te ablateren, terwijl de andere breedveldmicroscopie gebruikt om de hele SC van een gemuteerde muis af te beelden waarvan de cortex grotendeels onontwikkeld is. Dit protocol beschrijft deze twee methoden, waaronder diervoorbereiding, virale injectie, implantatie van de kopplaat, implantatie van pluggen, data-acquisitie en data-analyse. De representatieve resultaten tonen aan dat de calciumbeeldvorming met twee fotonen visueel opgewekte neuronale reacties onthult met een resolutie van één cel, en de calciumbeeldvorming met een breed veld onthult neurale activiteit over de hele SC. Door deze twee methoden te combineren, kan men de neurale codering in de SC op verschillende schalen onthullen, en een dergelijke combinatie kan ook worden toegepast op andere hersengebieden.

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.

Auteur in de schijnwerpers: onthulling van neurale codering en mechanismen van visuele verwerking in de superieure colliculus 

Calciumbeeldvorming bij superieure colliculus bij muizen

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.

Figuren 1A,B laten zien hoe respectievelijk de zuignap en de pluggen moeten worden gemaakt. Figuur 2 laat zien hoe u de plug met succes kunt implanteren. Na het implanteren van de plug wordt de posterieure-mediale SC blootgelegd, zoals weergegeven in figuur 2D. Figuur 3 toont calciumresponsen van SC-neuronen van een voorbeeld van een wild-type muis die is afgebeeld met behulp van twee-f...

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

Dit protocol beschrijft de procedure voor het in beeld brengen van calciumresponsen in de superieure colliculus (SC) van wakkere muizen, inclusief het in beeld brengen van de activiteit van één neuron met twee fotonen met behulp van twee-fotonmicroscopie terwijl de cortex intact blijft bij wild-type muizen, en het in beeld brengen van de gehele SC met breedveldmicroscopie bij mutante muizen met gedeeltelijke cortex.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Mijn onderzoek richt zich op neurale codering en mechanismen voor visuele verwerking. We proberen een antwoord te vinden op de vraag hoe verschillende informatie in de hersenen wordt gecodeerd, met name in de superieure colliculus onder de onderliggende neuromechanismen. Om visuele verwerking te begrijpen, combineren wetenschappers verschillende benaderingen, waaronder neuronale morfologie, antegrade en retrograde tracing, IL-sequencing, neuromodellering, machine learning, wordt goed begrepen.De uitdaging is om de schedel over de SSA te verwijderen, snijden en in het bot in één groter stuk laten, zal waarschijnlijk mislukken omdat het bot aan de dura is bevestigd. Door twee foto's te combineren op een calciumbeeld met een breed veld, onthulde ons recente werk de functiearchitectuur van bewegingsrichting in de SC van de muis bij zowel een enkele celresolutie als een globale schaal. We ontdekten dat neuronen met een vergelijkbare voorkeur patches vormen tot 500 micrometer onder globaal naar boven op de nasale beweging.Met ons protocol pakken we twee lacunes in het onderzoek aan. Ten eerste kunnen onderzoekers langdurige castbeeldvorming uitvoeren in SC van muizen met een enkele celresolutie zonder de cortex te breken. Ten tweede kunnen onderzoekers de neuroactiviteit over de hele SC registreren met behulp van widefield microscoping.Ons protocol biedt een maat om de posterieure mediale SC in beeld te brengen met een resolutie van één cel met een intacte cortex bij muizen. Ook gebruiken we een biocompatibele plug om de SC bloot te leggen, wat infectie vermindert voor chronische beeldvorming. Onze resultaten bieden een manier om de neurale codering van visuele informatie over een groot gezichtsveld te bestuderen.In combinatie met optogenetica kan men de helft van de input van verschillende hersengebieden bestuderen die de neuroactiviteit in SC moduleren.

calcium-imaging-in-mouse-superior-colliculus

Research

JoVE Journal

Neuroscience

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.

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.

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.

Voorbereiding van een zuignap en pluggen voor calciumbeeldvorming bij muizen

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.

Viral Construct (AAV)-injectie in superieure colliculus van muizen voor calciumbeeldvorming

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.

Implantatie van de kopplaat bij muizen voor calciumbeeldvorming 

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.

De plug in de muis implanteren voor calciumbeeldvorming

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.

Twee-foton calciumbeeldvorming van superieure colliculusneuronen van muizen

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.