implanting the plug in the mouse for calcium imaging

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

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

Watch this Scientific Journal Video about Implanting the Plug in the Mouse for Calcium Imaging at JoVE.com

Implanting the Plug in the Mouse for Calcium Imaging  

Implantar el enchufe en el ratón para obtener imágenes de calcio

Tres semanas después de la infección viral, anestesiar y fijar el ratón con una placa de cabeza en un marco estereotáxico. Remoja una espuma de gel en solución salina para detener un posible sangrado. Utiliza un microtaladro para crear un óvalo de tres por dos milímetros, centrado a 0,5 milímetros detrás de la lambda.Adelgace el límite del óvalo hasta que se agriete, luego adelgace el cráneo antes de la ventana de registro ovalada para cerrar el cubreobjetos al SC. Con unas pinzas finas, retire los colgajos óseos. Haga un corte en la duramadre, posterior al seno transverso y retire el trozo de duramadre. Seque el cráneo y la ventana de grabación con espuma de gel e hisopos de algodón.A continuación, deposite una gota de adhesivo de silicona en la ventana de grabación. Use la ventosa para sostener el tapón con presión negativa y use un micromanipulador motorizado para mover la ventosa y bajar el tapón en el adhesivo de silicona hasta que el cubreobjetos toque el cráneo. Una vez que se limpia la placa de la cabeza, aplique cianoacrilato de butilo y cemento de resina alrededor del límite del tapón para fijarlo a la placa de la cabeza y proceda con la obtención de imágenes.

implanting-the-plug-in-the-mouse-for-calcium-imaging

Research

JoVE Journal

Neuroscience

Implanting the Plug in the Mouse for Calcium Imaging

140 vistas. Tres semanas después de la infección viral, anestesiar y fijar el ratón con una placa de cabeza en un marco estereotáxico. Remoja una espuma de gel en solución salina para detener un posible sangrado. Utiliza un microtaladro para crear un óvalo de tres por dos milímetros, centrado a 0,5 milímetros detrás de la lambda.Adelgace el límite del óvalo hasta que se agriete, luego adelgace el cráneo antes de la ventana de registro ovalada para cerrar el cubreobjetos al SC. Con unas...

Video: Implanting the Plug in the Mouse for Calcium Imaging  

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.

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

Investigación

Neurociencias

Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse

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

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

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

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons

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

Wide-Field Calcium Imaging of Mouse Superior Colliculus Neurons

Obtención de imágenes de calcio de campo amplio de neuronas del colículo superior de ratón