Name: Pleins feux sur l’auteur : Dévoilement du codage neuronal et des mécanismes de traitement visuel dans le colliculus supérieur
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

Ces travaux sont soutenus par la Fondation nationale des sciences naturelles de Chine (32271060). Y.-t.L. a conçu la recherche, réalisé l’expérience, analysé les données et rédigé le manuscrit. Z.L. et R.W. ont réalisé l’expérience.

Toutes les procédures expérimentales ont été réalisées conformément aux directives sur le bien-être animal et approuvées par l’IACUC de l’Institut chinois de recherche sur le cerveau de Beijing.
REMARQUE : L’échéancier de ce protocole est le suivant : 1) faire la ventouse ; 2) injecter le virus ; 3) implanter la plaque de tête ; 4) après 3 semaines, implantez le bouchon ; 5) Après une récupération et une accoutumance de ~3 jours sur le tapis roulant, effectuez une i...

Imagerie calcique à deux photons et à grand champ dans le colliculus supérieur

<ol>
	<li>May, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+superior+colliculus:+laminar+structure+and+connections.">The mammalian superior colliculus: laminar structure and connections.</a> Progress in Brain Research. 151, 321-378 (2006).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Ohki, K., Chung, S., Ch'ng, Y. H., Kara, P., Reid, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+with+cellular+resolution+reveals+precise+micro-architecture+in+visual+cortex.">Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex.</a> Nature. 433 (7026), 597-603 (2005).</li><li>Ratzlaff, E. H., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tandem-lens+epifluorescence+macroscope:+Hundred-fold+brightness+advantage+for+wide-field+imaging.">A tandem-lens epifluorescence macroscope: Hundred-fold brightness advantage for wide-field imaging.</a> Journal of Neuroscience Methods. 36 (2-3), 127-137 (1991).</li><li>de Vries, S. E. J., 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=A+large-scale+standardized+physiological+survey+reveals+functional+organization+of+the+mouse+visual+cortex.">A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.</a> Nature Neuroscience. 23 (1), 138-151 (2020).</li><li>Mrsic-Flogel, T. D., 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=Altered+map+of+visual+space+in+the+superior+colliculus+of+mice+lacking+early+retinal+waves.">Altered map of visual space in the superior colliculus of mice lacking early retinal waves.</a> The Journal of Neuroscience. 25 (29), 6921-6928 (2005).</li><li>Cang, J., Wang, L., Stryker, M. P., Feldheim, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+ephrin-as+and+structured+activity+in+the+development+of+functional+maps+in+the+superior+colliculus.">Roles of ephrin-as and structured activity in the development of functional maps in the superior colliculus.</a> The Journal of Neuroscience. 28 (43), 11015-11023 (2008).</li><li>Feinberg, E. H., 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=Orientation+columns+in+the+mouse+superior+colliculus.">Orientation columns in the mouse superior colliculus.</a> Nature. 519 (7542), 229-232 (2015).</li><li>Ahmadlou, M., Heimel, J. 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. L., DePiero, V. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+tuned+response+properties+of+diverse+inhibitory+neuron+subtypes+in+mouse+visual+cortex.">Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex.</a> Neuron. 67 (5), 858-871 (2010).</li><li>G&#246;bel, W., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calcium+imaging+of+neural+network+function.">In vivo calcium imaging of neural network function.</a> Physiology. 22 (6), 358-365 (2007).</li><li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake,+mobile+mice.">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56 (1), 43-57 (2007).</li><li>Evans, D. 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=A+synaptic+threshold+mechanism+for+computing+escape+decisions.">A synaptic threshold mechanism for computing escape decisions.</a> Nature. 558 (7711), 590-594 (2018).</li></ol>

Étapes critiques du protocole L’étape la plus critique est la craniotomie aux étapes 5.2 et 5.3. Tout d’abord, l’os à 0,5 mm en arrière de la lambda est épais et contient des vaisseaux sanguins, ce qui peut provoquer des saignements pendant le processus de forage. Une mousse de gel adéquate doit être préparée pour arrêter le saignement. Deuxièmement, il y a un bon risque d’angiorrhexie lors de l’ablation de l’os juste au-dessus du sinus transverse. Pour le dépannage, une autre...

Le colliculus supérieur (SC) est un centre visuel important chez tous les vertébrés. Chez les mammifères, il reçoit des entrées directes de la rétine et du cortex visuel1. Alors que l’enregistrement optique a été largement appliqué au cortex 2,3,4,5, son application dans le SC est entravée par de mauvais accès optiques 6,7,8,9,10,11,12,13,14,15,16,17 ,18,19. L’objectif de ce protocole est de fournir des détails sur deux méthodes complémentaires d’enregistrement optique de l’activité neuronale dans le CS.
Le SC est situé sous le cortex et le sinus transverse, ce qui limite l’accès optique aux neurones colliculaires. Une approche pour surmonter cette limitation est d’aspirer le cortex superposé et d’exposer le SC antéro-latéral 7,9,10,13,14,19. Cependant, comme le SC reçoit des entrées corticales, une telle opération peut affecter la façon dont les neurones du SC répondent aux stimuli visuels. Pour pallier cette limitation, nous détaillons ici un protocole alternatif permettant d’imager la couche superficielle du SC postérieur-médial avec un bouchon en silicone, tout en laissant le cortex intact 8,11. Plus précisément, pour obtenir une résolution unicellulaire, nous avons appliqué la microscopie à deux photons pour imager les réponses calciques dans le SC postérieur-médial de souris de type sauvage. De plus, pour obtenir une large couverture, nous avons appliqué la microscopie à grand champ pour imager l’ensemble du SC d’une souris mutante dont le cortex postérieur ne s’est pas développé20.
Les deux méthodes décrites dans ce protocole sont complémentaires l’une de l’autre. L’imagerie calcique à deux photons sans ablation du cortex est appropriée pour enregistrer l’activité neuronale à une résolution de cellule unique avec des entrées corticales intactes. L’imagerie calcique à grand champ est appropriée pour enregistrer l’activité neuronale dans l’ensemble du SC tout en sacrifiant la résolution spatiale.

<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

Le colliculus supérieur (SC), une structure du mésencéphale conservée au cours de l’évolution chez tous les vertébrés, est le centre visuel le plus sophistiqué avant l’émergence du cortex cérébral. Il reçoit des entrées directes de ~30 types de cellules ganglionnaires rétiniennes (RGC), chacune codant pour une caractéristique visuelle spécifique. Il reste difficile de savoir si le CS hérite simplement des caractéristiques rétiniennes ou si un traitement supplémentaire et potentiellement de novo se produit dans le CS. Pour révéler le codage neuronal de l’information visuelle dans le SC, nous fournissons ici un protocole détaillé pour enregistrer optiquement les réponses visuelles avec deux méthodes complémentaires chez les souris éveillées. Une méthode utilise la microscopie à deux photons pour imager l’activité du calcium à une seule résolution cellulaire sans ablation du cortex superposé, tandis que l’autre utilise la microscopie à grand champ pour imager l’ensemble du SC d’une souris mutante dont le cortex est en grande partie sous-développé. Ce protocole détaille ces deux méthodes, y compris la préparation des animaux, l’injection virale, l’implantation de la plaque de tête, l’implantation du bouchon, l’acquisition de données et l’analyse des données. Les résultats représentatifs montrent que l’imagerie calcique à deux photons révèle des réponses neuronales évoquées visuellement à une résolution unicellulaire, et que l’imagerie calcique à grand champ révèle une activité neuronale dans l’ensemble du SC. En combinant ces deux méthodes, on peut révéler le codage neuronal dans le CS à différentes échelles, et une telle combinaison peut également être appliquée à d’autres régions du cerveau.

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.

Pleins feux sur l’auteur : Dévoilement du codage neuronal et des mécanismes de traitement visuel dans le colliculus supérieur 

Imagerie calcique dans le colliculus supérieur de souris

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.

Les figures 1A et B montrent comment fabriquer respectivement la ventouse et les bouchons. La figure 2 montre comment implanter le bouchon avec succès. Après l’implantation du bouchon, le CS postérieur-médial est exposé, comme le montre la figure 2D. La figure 3 montre les réponses calciques des neurones SC d’un exemple de souris de type sauvage imagé à l’aide de la micros...

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

Ce protocole détaille la procédure d’imagerie des réponses calciques dans le colliculus supérieur (SC) des souris éveillées, y compris l’imagerie de l’activité d’un seul neurone avec la microscopie à deux photons tout en laissant le cortex intact chez les souris de type sauvage, et l’imagerie de l’ensemble du SC avec la microscopie à grand champ chez les souris mutantes à cortex partiel.

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

Mes recherches portent sur le codage neuronal et les mécanismes du traitement visuel. Nous essayons de répondre à la question de savoir comment diverses informations sont codées dans le cerveau, en particulier dans le colliculus supérieur sous les neuromécanismes sous-jacents. Pour comprendre le traitement visuel, les scientifiques combinent diverses approches, notamment la morphologie neuronale, le traçage antérograde et rétrograde, le séquençage IL, la modélisation neurologique, l’apprentissage automatique, est bien compris.Le défi consiste à retirer le crâne au-dessus de la SSA, coupant et laissé dans l’os en un seul morceau plus grand, est susceptible d’échouer parce que l’os est attaché à la dure-mère. En combinant deux photos sur une image de calcium à grand champ, nos travaux récents ont révélé l’architecture fonctionnelle de la direction du mouvement dans le SC de souris à la fois à la résolution d’une seule cellule et à l’échelle globale. Nous avons constaté que les neurones ayant une préférence similaire forment des patchs jusqu’à 500 micromètres sous le global vers le haut sur le mouvement nasal.Avec notre protocole, nous comblons deux lacunes en matière de recherche. Tout d’abord, les chercheurs peuvent effectuer une imagerie de moulage à long terme dans un SC de souris à une résolution d’une seule cellule sans briser le cortex. Deuxièmement, les chercheurs peuvent enregistrer la neuroactivité sur l’ensemble du SC à l’aide de la microscopation à grand champ.Notre protocole fournit une mesure permettant d’imager le CS médian postérieur à une résolution de cellule unique avec un cortex intact chez la souris. De plus, nous utilisons un bouchon biocompatible pour exposer le SC, ce qui réduit l’infection pour l’imagerie chronique. Nos résultats fournissent un moyen d’étudier le codage neuronal de l’information visuelle à travers un large champ visuel.Combiné à l’optogénétique, on peut étudier les demi-entrées de différentes régions du cerveau modulant la neuroactivité dans le 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.

Recherche

Neurosciences

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.

Préparation d’une ventouse et de bouchons pour l’imagerie calcique chez la souris

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.

Injection de construction virale (AAV) dans un colliculus supérieur de souris pour l’imagerie calcique

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 de la plaque frontale chez la souris pour l’imagerie calcique 

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.

Implantation du bouchon dans la souris pour l’imagerie calcique

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.

Imagerie calcique à deux photons des neurones du colliculus supérieur de souris

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.