Enregistrement Light-réponses évoquées dans les neurones post-synaptiques dans Dark-adaptés, Préparations rétine de la souris tranche en utilisant des techniques de patch clamp
Summary
We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.
Abstract
La rétine est la porte d'entrée du système visuel. Pour comprendre les mécanismes de traitement des signaux visuels, nous étudions les fonctions de réseau de neurones de la rétine. Neurones rétiniens dans le réseau comprennent de nombreux sous-types. Plus de 10 sous-types de cellules bipolaires, les cellules ganglionnaires, les cellules amacrines et ont été identifiés par des études morphologiques. Sous-types multiples de neurones rétiniens sont pensés pour coder des caractéristiques distinctes de signalisation visuelle, telles que le mouvement et la couleur, et de former de multiples voies neurales. Cependant, les rôles fonctionnels de chaque neurone en traitement du signal visuel sont pas pleinement compris. La méthode patch clamp est utile de répondre à cette question fondamentale. Ici, un protocole d'enregistrer les réponses synaptiques lumière évoqués dans les neurones de la rétine de souris en utilisant des enregistrements de patch clamp dans des conditions sombres adapté est fourni. Les yeux de souris sont adaptés à l'obscurité O / N, et les préparations de tranche de la rétine sont disséqués dans une pièce sombre en utilisant un éclairage infrarouge et les téléspectateurs. La lumière infrarouge ne est pasactiver photorécepteurs de la souris et préserve ainsi leur réactivité de lumière. Patch clamp est utilisé pour enregistrer les réponses de lumière évoquée dans les neurones de la rétine. Un colorant fluorescent est injecté pendant les enregistrements pour caractériser les sous-types morphologiques neuronales. Cette procédure permet de déterminer les fonctions physiologiques de chaque neurone dans la rétine de souris.
Introduction
The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms.
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6.
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.
Protocol
Representative Results
Discussion
Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings.
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| mice (28-60 days old, male) | Jackson laboratory | C57BL/6J strain | |
| Ames' medium powder | Sigma | A1420 | excellent |
| Stereo microscope | Nikon | SMZ745 | excellent |
| dissecting tool_forceps | Dumont | #4, #5, #55 | excellent |
| dissecting tool_scissors | Roboz | RS-5605 | excellent |
| dissecting tool_surgery knife | Surgistar | 7514 | excellent |
| razor blade (for chopper) | EMS | 71970 | excellent |
| chopper | handmade | ||
| infrared viewer | Night Owl Optics | NOBG1 | It shows bright view. Focusing small objects is an issue. |
| infrared pocket scopes | B.E. Meyers | OWL Gen 3 NV pocketscope | excellent view |
| puller | Sutter | P-1000 | excellent. Make consistent size pipettes. |
| dark box | Pelican | dark box | excellent |
| patch clamp system | Scientifica | slice scope 2000 | Excellent setup. Most key components are included in one package. Micromanipulators are excellent. |
| amplifier | Molecular Devices | multiclamp 700B | Excellent and easy control. |
| acquiring software | Molecular Devices | pClamp software | Excellent and easy control. |
| light source (LED) | Cool LED | pE-2 4 channel system | Excellent |
| CCD camera | Q-imaging | Retiga 2000 | Excellent |
| Faraday cage | handmade |
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