Journal Club—A New Application for pHrodo™ Dextrans

Chen M, Van Hook MJ, Zenisek D, Thoreson WB (2013) J Neurosci 33(5):2071–2086.

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.