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

上丘における2光子および広視野カルシウムイメージング

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.

著者スポットライト:上腺における神経コーディングと視覚処理のメカニズムの解明 

マウス上結腸におけるカルシウムイメージング

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  

マウスにおけるカルシウムイメージングのための吸盤とプラグの作製

アクリル皿にPBSを一滴垂らして、吸盤を作るプロセスを開始します。21ゲージの平らな針を使用して、毛細管現象により針の先端にPBSを充填します。針先を半透明のシリコン接着剤で覆い、約10分間セットします。シリコーン接着剤をハサミで直径2ミリの円盤に切ります。プラグを準備するには、厚さ0.75ミリのプラスチックシムストックを用意し、中央を1センチメートルの正方形に切り取ります。シムストックとアクリルブロック2個をアルコールで洗浄し、レンズペーパーで拭きます。シムストックを1つのアクリルブロックの上に置き、中央にシリコン接着剤を堆積させて開口部の90%を埋めます。アクリルブロックをもう1つ追加します。約1キログラムの力を加え、20分間待ちます。手術用顕微鏡下で、メスでシリコーン接着剤を1.5ミリメートルの三角柱に切断します。粘着性のあるプラスチックテープで三角柱のほこりを取り除き、ガラスカバースリップの上に置きます。三角柱の入ったカバースリップをコロナ処理器の下に置き、電源を入れます。コロナ処理装置を稲妻が現れるまでカバースリップに近づけ、プリズムとカバースリップが取り付けられるまで数分間その位置に保持します。プラグをペトリ皿に入れた後、摂氏70度で一晩インキュベートします。

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 ...

神経科学

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

カルシウムイメージングのためのマウス上結腸へのウイルスコンストラクト(AAV)注射

Implantation of the Headplate in Mouse for Calcium Imaging 

カルシウムイメージングのためのマウスへのヘッドプレートの移植 

Implanting the Plug in the Mouse for Calcium Imaging

カルシウムイメージングのためのマウスへのプラグの埋め込み