Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Summary

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Abstract

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass¹. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release^2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43®) by co-puffing the dye and a ringer solution containing a high K⁺ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE⁴, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

Protocol

Part 1: Dissection and Bipolar Cell Isolation

1. Prepare solutions listed in Table 2; The pH of ringers’ (external) solutions should be adjusted to 7.4 with NaOH and the pH of the internal solution should be adjusted to 7.2 with CsOH. Protect internal solution from light with aluminum foil and keep it at 4 °C until use;
2. Dark-adapt a goldfish for at least 30 minutes prior to dissection;
3. While the animal dark-adapts, prepare 5 mL of the hyaluronidase (type V hyaluronidase,1100 units/mL in low Ca²⁺ ringer’s; Sigma, St. Louis, MO) and 10 mL of the L-cysteine (0.5 mg/mL in low Ca²⁺ ringer’s) solutions and weigh the papain....

Discussion

The advantages of objective-type TIRF microscopy are that 1) it provides excellent optical sectioning by restricting excitation light to a narrow region within the focal plane of the objective, thereby minimizing out-of-focus light; 2) since light drops exponentially with distance, movement in a vertical direction can be monitored as a change in fluorescence intensity; 3) efficient light collection through the high numerical aperture objective^1,5.

The main drawback of the technique.......