Name: Författare i fokus: Avslöja neural kodning och mekanismer för visuell bearbetning i Superior 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

Detta arbete stöds av National Natural Science Foundation of China (32271060). Y.-t.L. utformade forskningen, utförde experimentet, analyserade data och skrev manuskriptet. Z.L. och R.W. utförde experimentet.

Alla experimentella procedurer utfördes i enlighet med djurskyddsriktlinjerna och godkändes av IACUC vid Chinese Institute for Brain Research, Peking.
OBS: Tidslinjen för detta protokoll är som följer: 1) gör sugkoppen; 2) injicera viruset; 3) implantera huvudplattan; 4) efter 3 veckor, implantera pluggen; 5) Efter en ~3 dagars återhämtning och tillvänjning på löpbandet, utför två-foton/vidvinkelavbildning.
1. Beredning av en sugkopp (<st...

Tvåfoton- och bredfältsavbildning av kalcium i Superior Colliculus

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Kritiska steg i protokollet Det mest kritiska steget är kraniotomin i steg 5.2 och 5.3. För det första är benet 0,5 mm bakom lambdan tjockt och har blodkärl inuti, vilket kan orsaka blödning under borrningsprocessen. Tillräckligt med gelskum bör förberedas för att stoppa blödningen. För det andra finns det en god risk för angiorrhexis när man tar bort benet strax ovanför den tvärgående sinusen. För felsökning är ett alternativt tillvägagångssätt att tunna ut benet inuti ovalen o...

Superior colliculus (SC) är ett viktigt syncentrum hos alla ryggradsdjur. Hos däggdjur får den direkta intryck från näthinnan och synbarken1. Medan optisk inspelning har tillämpats i stor utsträckning på cortex 2,3,4,5, hindras dess tillämpning i SC av dålig optisk åtkomst 6,7,8,9,10,11,12,13,14,15,16,17 ,18,19. Målet med detta protokoll är att ge detaljer om två kompletterande metoder för optisk registrering av den neurala aktiviteten i SC.
SC är belägen under cortex och tvärgående sinus, vilket begränsar optisk åtkomst till kollikulära neuroner. Ett tillvägagångssätt för att övervinna denna begränsning är att aspirera den överliggande cortex och exponera den främre-laterala SC 7,9,10,13,14,19. Men eftersom SC tar emot kortikala ingångar kan en sådan operation påverka hur SC-neuronerna svarar på visuella stimuli. För att övervinna denna begränsning beskriver vi här ett alternativt protokoll för att avbilda det ytliga lagret av den posterior-mediala SC med en kiselplugg, samtidigt som cortex lämnas intakt 8,11. Specifikt, för att uppnå encellsupplösning, tillämpade vi tvåfotonmikroskopi för att avbilda kalciumsvar i den posteriora-mediala SC hos vildtypsmöss. Dessutom, för att uppnå bred täckning, tillämpade vi vidvinkelmikroskopi för att avbilda hela SC av en muterad mus vars bakre cortex inte har utvecklat20.
De två metoder som beskrivs i detta protokoll kompletterar varandra. Kalciumavbildningen med två fotoner utan att ablera cortex är lämplig för att registrera neural aktivitet med encellsupplösning med intakta kortikala ingångar. Kalciumavbildningen med brett fält är lämplig för att registrera neural aktivitet i hela SC samtidigt som den rumsliga upplösningen offras.

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			<td>16x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
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			<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>
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</table>

calcium imaging in mouse superior colliculus

Superior colliculus (SC), en evolutionärt bevarad mitthjärnstruktur hos alla ryggradsdjur, är det mest sofistikerade syncentret före uppkomsten av hjärnbarken. Den tar emot direkta indata från ~30 typer av retinala ganglieceller (RGC), där var och en kodar för en specifik visuell funktion. Det är fortfarande svårt att avgöra om SC helt enkelt ärver näthinnefunktioner eller om ytterligare och potentiellt ny bearbetning sker i SC. För att avslöja den neurala kodningen av visuell information i SC tillhandahåller vi här ett detaljerat protokoll för att optiskt registrera visuella svar med två kompletterande metoder i vakna möss. Den ena metoden använder tvåfotonmikroskopi för att avbilda kalciumaktivitet med encellsupplösning utan att ablera den överliggande cortex, medan den andra använder vidvinkelmikroskopi för att avbilda hela SC hos en muterad mus vars cortex i stort sett är outvecklad. Detta protokoll beskriver dessa två metoder, inklusive djurförberedelser, virusinjektion, implantation av huvudplattan, pluggimplantation, datainsamling och dataanalys. De representativa resultaten visar att tvåfotonkalciumavbildningen avslöjar visuellt framkallade neuronala svar vid encellsupplösning, och den vidfältskalciumavbildningen avslöjar neural aktivitet över hela SC. Genom att kombinera dessa två metoder kan man avslöja den neurala kodningen i SC på olika skalor, och en sådan kombination kan också tillämpas på andra hjärnregioner.

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.

Författare i fokus: Avslöja neural kodning och mekanismer för visuell bearbetning i Superior Colliculus 

Kalciumavbildning hos mus 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.

Figurerna 1A,B visar hur man tillverkar sugkoppen respektive pluggarna. Figur 2 visar hur man implanterar pluggen framgångsrikt. Efter implantation av pluggen exponeras den posteriora mediala SC, som visas i figur 2D. Figur 3 visar kalciumrespons från SC-neuroner från ett exempel på vildtypsmus avbildad med tvåfotonmikroskopi. Det triangulära prismat, som lätt fångas under mikro...

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

Detta protokoll beskriver proceduren för avbildning av kalciumsvar i den överlägsna colliculus (SC) hos vakna möss, inklusive avbildning av enneuronaktivitet med tvåfotonmikroskopi samtidigt som cortex lämnas intakt i vildtypsmöss, och avbildning av hela SC med vidfältsmikroskopi i partiella cortexmutanta möss.

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

Min forskning fokuserar på neural kodning och mekanismer för visuell bearbetning. Vi försöker svara på hur olika typer av information kodas i hjärnan, särskilt i den övre colliculus under de underliggande neuromekanismerna. För att förstå visuell bearbetning kombinerar forskare olika tillvägagångssätt, inklusive neuronal morfologi, antegrad och retrograd spårning, IL-sekvensering, neuromodellering, maskininlärning, förstås väl.Utmaningen är att ta bort skallen över SSA, skära och lämna kvar i benet i en större bit, kommer sannolikt att misslyckas eftersom benet är fäst vid dura. Genom att kombinera två foton på en kalciumbild med brett fält avslöjade vårt senaste arbete funktionsarkitekturen för rörelseriktning i musens SC vid både encellsupplösning och global skala. Vi fann att neuroner med liknande preferens bildar fläckar upp till 500 mikrometer under global till uppåt på nasal rörelse.Med vårt protokoll tar vi itu med två forskningsluckor. För det första kan forskare utföra långtidsgjuten avbildning i mus SC med en enda cellupplösning utan att bryta cortex. För det andra kan forskare registrera neuroaktiviteten över hela SC med hjälp av vidvinkelmikroskopi.Vårt protokoll ger ett mått för att avbilda den bakre mediala SC vid encellsupplösning med en intakt cortex hos möss. Vi använder också en biokompatibel kontakt för att exponera SC, vilket minskar infektion för kronisk avbildning. Våra resultat ger ett sätt att studera den neurala kodningen av visuell information över ett stort synfält.I kombination med optogenetik kan man studera halva ingångar från olika hjärnregioner som modulerar neuroaktiviteten vid SC.

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.

Förberedelse av en sugkopp och pluggar för kalciumavbildning i mus

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) injektion i mus Superior Colliculus för kalciumavbildning

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.

Implantation av huvudplattan i mus för kalciumavbildning 

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.

Implantera pluggen i musen för kalciumavbildning

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.

Två-fotonkalciumavbildning av mus överlägsna colliculus-neuroner

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.