Journal Club—A New Application for pHrodo™ Dextrans
Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles
The release of synaptic vesicles by rod photoreceptors is mediated by Ca2+ flux through L-type channels, which is triggered by membrane potential changes upon illumination. In rod photoreceptors, this release appears to be associated with synaptic ribbons—electron-dense structures that function in the delivery, priming, and clustering of vesicles near the L-type Ca2+ channels—as well as with non-ribbon sites. Using total internal reflection fluorescence microscopy (TIRFM), Chen and coworkers characterized the kinetics of vesicle release from ribbon and non-ribbon sites in salamander rods by visualizing individual synaptic vesicles loaded with either pHrodo™ Red 10,000 MW dextran or the well-established FM 1-43 membrane probe.
As described in the recent Journal of Neuroscience article, the vesicle-loading protocol included short incubation times (3 minutes for pHrodo™ dextran, 1 minute for FM 1-43 dye) in order to label only a fraction of the ~100,000 vesicles in the rod terminal. With a small number of vesicles labeled, the researchers were able to visualize individual vesicles as they approached, paused, and then fused with the plasma membrane. Vesicle release was also studied with longer loading times that labeled a larger population of vesicles (Figure 1).
The 10,000 MW dextran conjugated to the pHrodo™ Red pH indicator (a pH-sensitive rhodamine derivative) is a new endocytosis marker that exhibits bright red fluorescence once it is incorporated into acidic organelles such as endosomes and synaptic vesicles but is only weakly fluorescent in extracellular medium (pH 7.8). The authors validated the use of pHrodo™ Red dextran for visualizing synaptic vesicles by showing that, when used simultaneously, the pHrodo™ Red dextran and FM 1-43 dye fluorescently labeled the same set of organelles. They also report that vesicles labeled with pHrodo™ Red dextran were slightly less fluorescent than those labeled with FM 1-43 dye but provided a better signal-to-noise ratio, possibly due to residual FM 1-43 membrane staining and more cellular autofluorescence at FM 1-43 dye wavelengths.
|Figure 1. Vesicle release sites in rod photoreceptor terminals revealed with pHrodo™ Red 10,000 MW dextran. Synaptic vesicles of salamander rods were loaded with pHrodo™ Red 10,000 MW dextran (500 μg/mL) by incubation at room temperature for 30 min in darkness. Intact rod photoreceptors are depolarized in darkness, promoting synaptic vesicle exocytosis and endocytosis. After loading, rods were placed in a low-Ca2+ solution, exposed to light to inhibit further synaptic release, and then enzymatically isolated and plated on high NA glass coverslips. pHrodo™ Red dextran–loaded vesicles were illuminated with a 561 nm solid-state laser (Melles Griot) and visualized by total internal reflection fluorescence microscopy (TIRFM) using a 1.65 NA objective (Olympus). The footprint of the rod terminal attached to the cover glass is outlined in white. Release was stimulated by a 1 sec application of 50 mM KCl. Upon depolarization, vesicles advanced toward the plasma membrane, causing a fluorescence increase. After a brief pause at the membrane surface, vesicles then fused, causing a decrease in fluorescence (not shown). Here vesicle release sites were mapped by measuring the depolarization-evoked increase in near-membrane fluorescence. Release occurred mostly near the terminal center (middle arrow) but was also observed in other parts of the terminal (left and right arrows). Scale bar: 1 μm. Figure provided by Minghui Chen and Wallace B. Thoreson, University of Nebraska Medical Center, Omaha, Nebraska, and reproduced with permission.|