Name: Forfatter i søkelyset: Avduking av nevral koding og mekanismer for visuell prosessering i den overlegne kolliculus
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

Dette arbeidet støttes av National Natural Science Foundation of China (32271060). Y.-t.L. designet forskningen, utførte eksperimentet, analyserte dataene og skrev manuskriptet. Z.L. og R.W. utførte eksperimentet.

Alle eksperimentelle prosedyrer ble utført i samsvar med dyrevelferdsretningslinjene og godkjent av IACUC ved Chinese Institute for Brain Research, Beijing.
MERK: Tidslinjen for denne protokollen er som følger: 1) lag sugekoppen; 2) injisere viruset; 3) implantere hodeplaten; 4) etter 3 uker, implanter pluggen; 5) Etter en ~ 3 dagers utvinning og habituation på tredemølle, utføre to-foton / bredfeltsavbildning.
1. Tilberedning av en sugekopp (<st...

To-foton og vidfelts kalsiumavbildning i den overlegne kolleksjonen

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+concentric+orientations+in+the+mouse+superior+colliculus.">Preference for concentric orientations in the mouse superior colliculus.</a> Nature Communications. 6, 6773 (2015).</li><li>de Malmazet, D., K&#252;hn, N. K., Farrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinotopic+separation+of+nasal+and+temporal+motion+selectivity+in+the+mouse+superior+colliculus.">Retinotopic separation of nasal and temporal motion selectivity in the mouse superior colliculus.</a> Current Biology. 28 (18), 2961-2969 (2018).</li><li>Li, Y. T., Turan, Z., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+motion+direction+in+the+mouse+superior+colliculus.">Functional architecture of motion direction in the mouse superior colliculus.</a> Current Biology. 30 (17), 3304-3315 (2020).</li><li>Gribizis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+cortex+gains+independence+from+peripheral+drive+before+eye+opening.">Visual cortex gains independence from peripheral drive before eye opening.</a> Neuron. 104 (4), 711-723 (2019).</li><li>Inayat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+in+the+most+superficial+lamina+of+the+mouse+superior+colliculus+are+highly+selective+for+stimulus+direction.">Neurons in the most superficial lamina of the mouse superior colliculus are highly selective for stimulus direction.</a> The Journal of Neuroscience. 35 (20), 7992-8003 (2015).</li><li>Barchini, J., Shi, X., Chen, H., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+encoding+of+motion+contrast+in+the+mouse+superior+colliculus.">Bidirectional encoding of motion contrast in the mouse superior colliculus.</a> eLife. 7, 35261 (2018).</li><li>Savier, E. L., Chen, H., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+locomotion+on+visual+responses+in+the+mouse+superior+colliculus.">Effects of locomotion on visual responses in the mouse superior colliculus.</a> The Journal of Neuroscience. 39 (47), 9360-9368 (2019).</li><li>Schr&#246;der, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arousal+modulates+retinal+output.">Arousal modulates retinal output.</a> Neuron. 107 (3), 487-495 (2020).</li><li>Ge, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+waves+prime+visual+motion+detection+by+simulating+future+optic+flow.">Retinal waves prime visual motion detection by simulating future optic flow.</a> Science. 373 (6553), (2021).</li><li>Chen, H., Savier, E. 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J., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+evidence+for+stereotypical+direction+columns+in+the+mouse+superior+colliculus.">Lack of evidence for stereotypical direction columns in the mouse superior colliculus.</a> The Journal of Neuroscience. 41 (3), 461-473 (2021).</li><li>Kasai, M., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+light+isoflurane+anesthesia+on+organization+of+direction+and+orientation+selectivity+in+the+superficial+layer+of+the+mouse+superior+colliculus.">Effects of light isoflurane anesthesia on organization of direction and orientation selectivity in the superficial layer of the mouse superior colliculus.</a> The Journal of Neuroscience. 42 (4), 619-630 (2022).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+apical+complex+couples+cell+fate+and+cell+survival+to+cerebral+cortical+development.">The apical complex couples cell fate and cell survival to cerebral cortical development.</a> Neuron. 66 (1), 69-84 (2010).</li><li>Kaifosh, P., Zaremba, J. D., Danielson, N. B., Losonczy, A. S. I. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Python+software+for+analysis+of+dynamic+fluorescence+imaging+data.">Python software for analysis of dynamic fluorescence imaging data.</a> Frontiers in Neuroinformatics. 8, 80 (2014).</li><li>Pnevmatikakis, E. A., Giovannucci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NoRMCorre:+An+online+algorithm+for+piecewise+rigid+motion+correction+of+calcium+imaging+data.">NoRMCorre: An online algorithm for piecewise rigid motion correction of calcium imaging data.</a> Journal of Neuroscience Methods. 291, 83-94 (2017).</li><li>Kerlin, A. M., Andermann, M. L., Berezovskii, V. K., Reid, R. 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Kritiske trinn i protokollen Det mest kritiske trinnet er kraniotomi i trinn 5.2 og 5.3. For det første er benet på 0,5 mm bakenfor lambda tykt og har blodkar inni, noe som kan forårsake blødning under boreprosessen. Tilstrekkelig gelskum bør tilberedes for å stoppe blødningen. For det andre er det en god sjanse for angiorrhexis når du fjerner beinet like over den tverrgående sinus. For feilsøking er en alternativ tilnærming å tynne beinet inne i ovalen og fjerne det bit for bit. Et annet ...

Superior colliculus (SC) er et viktig visuelt senter hos alle virveldyr. Hos pattedyr mottar den direkte innganger fra netthinnen og den visuelle cortex1. Mens optisk opptak har blitt mye brukt på cortex 2,3,4,5, hindres anvendelsen i SC av dårlig optisk tilgang 6,7,8,9,10,11,12,13,14,15,16,17 ,18,19. Målet med denne protokollen er å gi detaljer om to komplementære metoder for optisk opptak av nevral aktivitet i SC.
SC ligger under cortex og tverrgående sinus, som begrenser optisk tilgang til de kollikulære nevronene. En tilnærming for å overvinne denne begrensningen er å aspirere den overliggende cortex og eksponere den fremre-laterale SC 7,9,10,13,14,19. Men fordi SC mottar kortikale innganger, kan en slik operasjon påvirke hvordan SC-nevronene reagerer på visuelle stimuli. For å overvinne denne begrensningen, beskriver vi her en alternativ protokoll for å avbilde det overfladiske laget av den bakre-mediale SC med en silisiumplugg, mens du lar cortex være intakt 8,11. Spesielt, for å oppnå enkeltcelleoppløsning, brukte vi to-foton mikroskopi for å avbilde kalsiumresponser i bakre mediale SC av villtype mus. I tillegg, for å oppnå bred dekning, brukte vi bredfeltsmikroskopi for å avbilde hele SC av en mutant mus hvis bakre cortex ikke har utviklet20.
De to metodene beskrevet i denne protokollen er komplementære til hverandre. To-foton kalsiumavbildning uten å ablatere cortex er egnet for opptak av nevral aktivitet ved enkeltcelleoppløsning med intakte kortikale innganger. Kalsiumavbildningen med bredt felt er egnet for registrering av nevral aktivitet i hele SC mens du ofrer romlig oppløsning.

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

calcium imaging in mouse superior colliculus

Superior colliculus (SC), en evolusjonært bevart midthjernestruktur hos alle vertebrater, er det mest sofistikerte visuelle senteret før fremveksten av hjernebarken. Den mottar direkte innganger fra ~ 30 typer retinal ganglionceller (RGC), med hver koding av en bestemt visuell funksjon. Det er fortsatt unnvikende om SC bare arver retinale egenskaper eller om ytterligere og potensielt de novo-behandling skjer i SC. For å avsløre nevral koding av visuell informasjon i SC, gir vi her en detaljert protokoll for optisk registrering av visuelle responser med to komplementære metoder i våkne mus. Den ene metoden bruker to-foton mikroskopi for å avbilde kalsiumaktivitet ved enkeltcelleoppløsning uten å ablatere overliggende cortex, mens den andre bruker bredfeltsmikroskopi for å avbilde hele SC av en mutant mus hvis cortex i stor grad er uutviklet. Denne protokollen beskriver disse to metodene, inkludert dyreforberedelse, virusinjeksjon, implantasjon av hodeplater, pluggimplantasjon, datainnsamling og dataanalyse. De representative resultatene viser at to-foton kalsiumavbildningen avslører visuelt fremkalte nevronresponser ved enkeltcelleoppløsning, og bredfeltkalsiumavbildningen avslører nevral aktivitet over hele SC. Ved å kombinere disse to metodene kan man avsløre nevral koding i SC på forskjellige skalaer, og en slik kombinasjon kan også brukes på andre hjernegrupper.

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.

Forfatter i søkelyset: Avduking av nevral koding og mekanismer for visuell prosessering i den overlegne kolliculus 

Kalsiumavbildning i 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.

Figur 1A,B viser hvordan du lager henholdsvis sugekoppen og pluggene. Figur 2 viser hvordan du implanterer pluggen vellykket. Etter implantering av pluggen eksponeres den bakre-mediale SC, som vist i figur 2D. Figur 3 viser kalsiumresponser av SC-nevroner fra et eksempel på villtype mus avbildet ved hjelp av to-foton mikroskopi. Det trekantede prismet, som lett fanges opp under mikrosk...

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

Denne protokollen beskriver prosedyren for avbildning av kalsiumresponser i overlegen colliculus (SC) av våken mus, inkludert avbildning av enkeltneuronaktivitet med to-foton mikroskopi mens du forlater cortex intakt i villtypemus, og avbildning av hele SC med bredfeltsmikroskopi i partial-cortex mutante mus.

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

Min forskning fokuserer på nevral koding og mekanismer for visuell prosessering. Vi prøver å svare på hvordan forskjellig informasjon er kodet i hjernen, spesielt i superior colliculus under de underliggende nevromekanismene. For å forstå visuell behandling kombinerer forskere ulike tilnærminger, inkludert nevronmorfologi, antegrad og retrograd sporing, IL-sekvensering, nevromodellering, maskinlæring, forstås godt.Utfordringen er å fjerne skallen over SSA, kutte og igjen i beinet i ett større stykke, vil sannsynligvis mislykkes fordi beinet er festet til duraen. Ved å kombinere to bilder på et bredt feltkalsiumbilde, avslørte vårt siste arbeid funksjonsarkitekturen til bevegelsesretning i musens SC på både enkeltcelleoppløsning og global skala. Vi fant at nevroner med lignende preferanse danner flekker opp til 500 mikrometer under global til oppover på nesebevegelsen.Med vår protokoll adresserer vi to forskningshull. For det første kan forskere utføre langsiktig støpt avbildning i musens SC ved en enkelt celleoppløsning uten å bryte cortex. For det andre kan forskere registrere nevroaktiviteten over hele SC ved hjelp av bredfeltmikroskopiering.Vår protokoll gir et mål for å avbilde den bakre mediale SC ved enkeltcelleoppløsning med en intakt cortex hos mus. Vi bruker også en biokompatibel plugg for å eksponere SC, noe som reduserer infeksjon for kronisk bildebehandling. Våre resultater gir en måte å studere nevral koding av visuell informasjon over et stort synsfelt.Kombinert med optogenetikk kan man studere halvinnganger fra forskjellige hjernegrupper som modulerer nevroaktiviteten i 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.

Fremstilling av en sugekopp og plugger for kalsiumavbildning 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) injeksjon i mus Superior Colliculus for kalsiumavbildning

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.

Implantasjon av hodeplaten i mus for kalsiumavbildning 

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.

Implantere pluggen i musen for kalsiumavbildning

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.

To-foton kalsiumavbildning av musens overlegne colliculus-nevroner

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.