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

Superior Colliculus의 Two-photon 및 Wide-field Calcium 이미징

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.

저자 스포트라이트: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

Mouse Superior Colliculus의 칼슘 이미징

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  

마우스의 칼슘 이미징을 위한 흡입 컵 및 플러그 준비

아크릴 접시에 PBS 한 방울을 넣어 흡입 컵을 만드는 과정을 시작합니다. 21게이지 납작한 바늘을 사용하여 모세관으로 바늘 끝을 PBS로 채웁니다. 바늘 끝을 반투명 실리콘 접착제로 덮고 약 10분 동안 고정합니다.실리콘 접착제를 가위로 직경 2mm의 디스크로 자릅니다. 플러그를 준비하려면 0.75mm 두께의 플라스틱 심 스톡을 사용하여 중앙에 1 x 1 센티미터 정사각형을 잘라냅니다. 심 스톡과 아크릴 블록 2개를 알코올로 닦고 렌즈 페이퍼로 닦습니다.하나의 아크릴 블록에 심 스톡을 놓고 실리콘 접착제를 중앙에 증착하여 개구부의 90%를 채웁니다. 다른 아크릴 블록을 추가하십시오. 약 1kg의 힘을 가하고 20분 동안 기다립니다.수술용 현미경으로 실리콘 접착제를 메스로 1.5mm 삼각형 프리즘으로 자릅니다. 끈적끈적한 플라스틱 테이프로 삼각형 프리즘의 먼지를 제거하고 유리 커버 슬립에 놓습니다. 삼각형 프리즘이 있는 커버 슬립을 코로나 처리기 아래에 놓고 전원을 켭니다.번개가 칠 때까지 코로나 처리기를 커버 슬립에 더 가까이 가져가고 프리즘과 커버 슬립이 부착될 때까지 몇 분 동안 그 위치에 유지합니다. 플러그를 페트리 접시에 넣은 후 섭씨 70도에서 밤새 배양합니다.

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 ...