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

Tvåfoton- och bredfältsavbildning av kalcium i Superior Colliculus

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.

Författare i fokus: Avslöja neural kodning och mekanismer för visuell bearbetning i Superior Colliculus 

Kalciumavbildning hos mus Superior Colliculus

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  

Förberedelse av en sugkopp och pluggar för kalciumavbildning i mus

Börja processen med att skapa en sugkopp genom att deponera en droppe PBS i en akrylskål. Använd en 21 gauge platt nål för att fylla nålspetsen med PBS genom kapilläritet. Täck nålspetsen med ett genomskinligt silikonlim och låt verka i cirka 10 minuter.Klipp silikonlimmet med en sax till en skiva med en diameter på två millimeter. För att förbereda pluggarna, ta ett 0,75 millimeter tjockt mellanläggslager av plast och skär ut en kvadrat på en och en centimeter i mitten. Rengör mellanläggsmaterialet och två akrylblock med alkohol och torka av dem med linspapper.Placera mellanläggsmaterialet på ett akrylblock och lägg silikonlim i mitten för att fylla 90 % av öppningen. Lägg till ytterligare ett akrylblock. Applicera cirka ett kilo kraft och vänta 20 minuter.Skär silikonlimmet med en skalpell i ett gånger 1,5 millimeter triangulärt prisma under ett kirurgiskt mikroskop. Ta bort eventuellt damm från de triangulära prismorna med självhäftande plasttejp och placera dem på ett glasglas. Placera täckglaset med de triangulära prismorna under en koronabehandlare och slå på den.För koronabehandlaren närmare täckglaset tills blixten dyker upp och håll den i det läget i några minuter tills prismorna och täckglaset sitter fast. Efter att ha placerat pluggarna i en petriskål, inkubera vid 70 grader Celsius över natten.

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 ...