Name: Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse
Uploaded: 2023-04-21T15:00:00
Description: Watch this Scientific Journal Video about Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse at JoVE.com

preparation of a suction cup and plugs for calcium imaging in mouse

上丘的双光子和宽场钙成像

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.

作者聚焦：揭示上丘的神经编码和视觉处理机制 

小鼠上丘的钙成像

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.

Watch this Scientific Journal Video about Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse at JoVE.com

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

用于小鼠钙成像的吸盘和吸盘的制备

通过将一滴 PBS 沉积在亚克力盘中来开始制作吸盘的过程。使用 21 号扁平针通过毛细作用用 PBS 填充针尖。用半透明的硅胶粘合剂覆盖针尖，并放置约 10 分钟。用剪刀将硅胶粘合剂剪成直径为两毫米的圆盘。要准备塞子，请取一个 0.75 毫米厚的塑料垫片，并在中心切出一个 1 厘米的正方形。用酒精清洁垫片和两个亚克力块，然后用镜头纸擦拭。将垫片放在一个丙烯酸块上，将硅胶粘合剂沉积到中心以填充 90% 的开口。再添加一个亚克力块。施加大约一公斤的力，然后等待 20 分钟。在手术显微镜下，用手术刀将硅胶粘合剂切成一个 1.5 毫米的三棱柱。用粘性塑料胶带清除三棱镜上的所有灰尘，然后将它们放在玻璃盖玻片上。将带有三棱镜的盖玻片放在电晕处理机下并打开它。将电晕处理机靠近盖玻片，直到出现闪电， 并将其保持在该位置几分钟，直到安装棱镜和盖玻片.将塞子放入培养皿中后，在 70 摄氏度下孵育过夜。

Research

JoVE Journal

Neuroscience

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

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 ...