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.
הרשתית היא השער למערכת הראייה. כדי להבין את מנגנוני עיבוד אותות חזותיים, אנו חוקרים פונקציות רשת עצביות ברשתית. נוירונים ברשתית ברשת מהווים של תת סוגים רבים. יותר מ -10 תת-סוגים של תאים דו קוטביים, תאי הגנגליון ותאי amacrine זוהו על ידי מחקרים מורפולוגיים. תת מרובה של תאי עצב ברשתית הם חשבו לקודד תכונות מובהקות של איתות חזותית, כגון תנועה וצבע, ויוצרים מספר רב של מסלולים עצביים. עם זאת, התפקידים הפונקציונליים של כל נוירון בעיבוד אותות חזותי אינם מובנים במלואם. שיטת מהדק התיקון היא שימושית כדי לטפל בשאלה יסודית זו. כאן, פרוטוקול להקליט תגובות הסינפטי אור עורר בנוירונים ברשתית עכבר באמצעות הקלטות מהדק תיקון בתנאים כהים מותאם מסופק. עיני העכבר הן כהות מותאם O / N, והכנות פרוסת רשתית הם גזורים בחדר חשוך באמצעות תאורה וצופים אינפרא אדום. אור אינפרא אדום לאלהפעיל קולטני אור בעכבר ובכך שומר על היענות האור שלהם. מהדק תיקון משמש להקלטת תגובות אור עורר בנוירונים ברשתית. צבע ניאון מוזרק במהלך הקלטות לאפיין תת מורפולוגיות עצביות. הליך זה מאפשר לנו לקבוע את הפונקציות הפיזיולוגיות של כל נוירון ברשתית העכבר.
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.
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 …
The authors have nothing to disclose.
This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.
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 |