Name: Autor destacado: Desvelando la codificación neuronal y los mecanismos de procesamiento visual en el colículo superior
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

Este trabajo cuenta con el apoyo de la Fundación Nacional de Ciencias Naturales de China (32271060). Y.-t.L. diseñó la investigación, realizó el experimento, analizó los datos y escribió el manuscrito. Z.L. y R.W. realizaron el experimento.

Todos los procedimientos experimentales se realizaron de acuerdo con las directrices de bienestar animal y fueron aprobados por la IACUC en el Instituto Chino de Investigación del Cerebro, Beijing.
NOTA: El cronograma para este protocolo es el siguiente: 1) hacer la ventosa; 2) inyectar el virus; 3) implantar la placa de la cabeza; 4) después de 3 semanas, implantar el tapón; 5) Después de una recuperación de ~ 3 días y habituación en la cinta de correr, realice imágenes de dos fotones...

Imágenes de calcio de dos fotones y de campo amplio en el colículo superior

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Pasos críticos en el protocolo El paso más crítico es la craneotomía en los pasos 5.2 y 5.3. En primer lugar, el hueso a 0,5 mm posterior a la lambda es grueso y tiene vasos sanguíneos en su interior, lo que puede causar sangrado durante el proceso de perforación. Se debe preparar una espuma de gel adecuada para detener el sangrado. En segundo lugar, existe una buena probabilidad de angiorrexis cuando se extirpa el hueso justo por encima del seno transverso. Para la resolución de problemas, un ...

El colículo superior (SC) es un centro visual importante en todos los vertebrados. En los mamíferos, recibe información directa de la retina y la corteza visual1. Si bien el registro óptico se ha aplicado ampliamente a la corteza 2,3,4,5, su aplicación en el SC se ve obstaculizada por accesos ópticos deficientes 6,7,8,9,10,11,12,13,14,15,16,17 ,18,19. El objetivo de este protocolo es proporcionar detalles sobre dos métodos complementarios para el registro óptico de la actividad neuronal en el SC.
El SC se encuentra debajo de la corteza y el seno transverso, lo que limita el acceso óptico a las neuronas coliares. Un enfoque para superar esta limitación es aspirar la corteza suprayacente y exponer el SCanterolateral 7,9,10,13,14,19. Sin embargo, debido a que el SC recibe entradas corticales, tal operación podría afectar la forma en que las neuronas SC responden a los estímulos visuales. Para superar esta limitación, detallamos aquí un protocolo alternativo para obtener imágenes de la capa superficial del SC posterior-medial con un tapón de silicio, dejando intacta la corteza 8,11. Específicamente, para lograr la resolución de una sola célula, aplicamos microscopía de dos fotones para obtener imágenes de las respuestas de calcio en el SC posterior-medial de ratones de tipo salvaje. Además, para lograr una amplia cobertura, aplicamos microscopía de campo amplio para obtener imágenes de todo el SC de un ratón mutante cuya corteza posterior no se ha desarrollado20.
Los dos métodos descritos en este protocolo son complementarios entre sí. Las imágenes de calcio de dos fotones sin ablar la corteza son apropiadas para registrar la actividad neuronal a una resolución de una sola célula con entradas corticales intactas. Las imágenes de calcio de campo amplio son apropiadas para registrar la actividad neuronal en todo el SC al tiempo que sacrifican la resolución espacial.

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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

El colículo superior (SC), una estructura evolutivamente conservada del mesencéfalo en todos los vertebrados, es el centro visual más sofisticado antes de la aparición de la corteza cerebral. Recibe entradas directas de ~ 30 tipos de células ganglionares de la retina (RGC), cada una de las cuales codifica una característica visual específica. Sigue siendo difícil de entender si el SC simplemente hereda las características de la retina o si se produce un procesamiento adicional y potencialmente de novo en el SC. Para revelar la codificación neuronal de la información visual en el SC, proporcionamos aquí un protocolo detallado para registrar ópticamente las respuestas visuales con dos métodos complementarios en ratones despiertos. Un método utiliza la microscopía de dos fotones para obtener imágenes de la actividad del calcio con una resolución de una sola célula sin ablacionar la corteza superpuesta, mientras que el otro utiliza la microscopía de campo amplio para obtener imágenes de todo el SC de un ratón mutante cuya corteza está en gran parte subdesarrollada. Este protocolo detalla estos dos métodos, incluida la preparación de animales, la inyección viral, la implantación de la placa de cabeza, la implantación del tapón, la adquisición de datos y el análisis de datos. Los resultados representativos muestran que las imágenes de calcio de dos fotones revelan respuestas neuronales evocadas visualmente a una resolución de una sola célula, y las imágenes de calcio de campo amplio revelan actividad neuronal en todo el SC. Al combinar estos dos métodos, se puede revelar la codificación neuronal en el SC a diferentes escalas, y dicha combinación también se puede aplicar a otras regiones del cerebro.

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.

Autor destacado: Desvelando la codificación neuronal y los mecanismos de procesamiento visual en el colículo superior 

Imágenes de calcio en el colículo superior de ratón

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.

Las figuras 1A, B muestran cómo hacer la ventosa y los tapones, respectivamente. La figura 2 muestra cómo implantar el tapón con éxito. Después de implantar el tapón, el SC posterior-medial queda expuesto, como se muestra en la Figura 2D. La Figura 3 muestra las respuestas de calcio de las neuronas SC de un ejemplo de ratón de tipo salvaje fotografiado mediante microscopía de do...

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

Este protocolo detalla el procedimiento para obtener imágenes de las respuestas de calcio en el colículo superior (SC) de ratones despiertos, incluida la obtención de imágenes de la actividad de una sola neurona con microscopía de dos fotones mientras se deja la corteza intacta en ratones de tipo salvaje, y la obtención de imágenes de todo el SC con microscopía de campo amplio en ratones mutantes de corteza parcial.

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

Mi investigación se centra en la codificación neuronal y los mecanismos de procesamiento visual. Estamos tratando de responder cómo se codifica diversa información en el cerebro, especialmente en el colículo superior bajo los mecanismos neurológicos subyacentes. Para comprender el procesamiento visual, los científicos combinan varios enfoques, incluida la morfología neuronal, el rastreo anterógrado y retrógrado, la secuenciación de IL, el modelado neurológico y el aprendizaje automático.El reto consiste en extraer el cráneo sobre el SSA, cortándolo y dejándolo en el hueso en una pieza más grande, es probable que falle porque el hueso está unido a la duramadre. Al combinar dos fotos en una imagen de calcio de campo amplio, nuestro trabajo reciente reveló la arquitectura de la función de la dirección del movimiento en el ratón SC tanto a una resolución de una sola célula como a escala global. Encontramos que las neuronas con preferencia similar forman parches de hasta 500 micrómetros por debajo del global al ascendente en el movimiento nasal.Con nuestro protocolo, estamos abordando dos brechas de investigación. En primer lugar, los investigadores pueden realizar imágenes de moldeo a largo plazo en SC de ratón con una resolución de una sola célula sin romper la corteza. En segundo lugar, los investigadores pueden registrar la neuroactividad en todo el SC utilizando microscopio de campo amplio.Nuestro protocolo proporciona una medida para obtener imágenes de la SC medial posterior con una resolución de una sola célula con una corteza intacta en ratones. Además, utilizamos un tapón biocompatible para exponer el SC, lo que reduce la infección para la obtención de imágenes crónicas. Nuestros resultados proporcionan una forma de estudiar la codificación neuronal de la información visual a través de un gran campo visual.En combinación con la optogenética, se pueden estudiar las medias entradas de diferentes regiones cerebrales que modulan la neuroactividad en el 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.

Investigación

Neurociencias

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.

Preparación de una ventosa y tapones para la obtención de imágenes de calcio en ratones

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.

Inyección de construcción viral (AAV) en el colículo superior de ratón para obtener imágenes de calcio

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.

Implantación de la placa de la cabeza en ratón para la obtención de imágenes de calcio 

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.

Implantar el enchufe en el ratón para obtener imágenes de calcio

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.

Obtención de imágenes de calcio de dos fotones de neuronas del colículo superior de ratón

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.