Staking out the TIRF
Fluorescence technique helps colocalize sites in synaptic transmission
Vesicle fusion and transmitter release are set in motion at most chemical synapses by a punctuate increase in intracellular calcium concentration. The transmitter is released within a fraction of a millisecond after calcium entry and, for this reason, researchers originally suggested that transmitter release occurs very close to the sites of high intracellular calcium concentration profiles known as calcium concentration microdomains.
Using total internal reflection fluorescence microscopy, researchers have spatially correlated calcium concentration microdomains and transmitter release sites in synaptic transmission. Shown here are images of samples from the squid optic lobe (A) and the rat cerebellum (B), with plated synaptosomes (left), fluorescence images of the same synaptosomes (center) and fluorescence images after depolarization (right), now showing the presence of calcium concentrations. Reprinted with permission of PNAS.
Imaging studies have provided direct evidence of such microdomains. However, because of the limitations of the imaging techniques used, investigators have not been able to correlate microdomain location directly to transmitter release sites.
“It’s one of the really basic questions in synaptic transmission,” said Rodolfo R. Llinàs, a researcher with New York University School of Medicine. “How closely organized, in space, are the sites for calcium entry and those for transmitter release at the synaptic junction?” Llinàs and colleagues Mutsuyuki Sugimori and Yafell Serulle used total internal reflection fluorescence (TIRF) microscopy, at both the university and at Marine Biological Laboratory in Woods Hole, Mass., to determine whether the calcium concentration domains and transmitter release sites coregister at the synapse. They reported their findings in the Jan. 30 issue of PNAS.
Total internal reflection fluorescence microscopy enables visualization of cellular events occurring at, or at least near, the plasma membrane by using evanescent waves to excite fluorophores in an area of the sample adjacent to the glass-water interface. In recent years, researchers have used the technique to image calcium entry sites and exocytosis in a variety of cell types.
In the present study, Llinàs and colleagues employed high-speed sequential imaging of vesicle fusion and calcium entry in the same synaptosome to test the assumption that calcium concentration microdomains and transmitter release sites are strictly colocalized.
Although the advantages of total internal reflection fluorescence microscopy are well-known, the researchers are not aware of any previous attempts to perform these types of synaptosomal measurements with the technique. “The experimental hardware worked beautifully,” Llinàs said. “Any problems we encountered during the study were biological in nature.”
The system consisted of an Olympus microscope outfitted with a 60×, 1.45-NA oil-immersion objective and an argon-ion laser. A fast CCD camera made by SciMedia of Irvine, Calif., with a spatial resolution of 100 × 100 pixels monitored the optical signals. Using it, the researchers probed the relationship between calcium concentration microdomains and transmitter release sites in synaptic terminals, which they had isolated from the squid optic lobe and rat cerebellar mossy fibers and had loaded with fluorescent dyes.
Isolation of the synaptic terminals provided the first potential obstacle. “It wasn’t known if one synaptosomal transmission in vitro would yield the information we wanted; that is, whether the presynaptic ‘active zone structure’ would remain intact when the postsynaptic side of the junction is gone,” Llinàs said. They found that the integrity of the structure remains, indicating a robustness that had not been known previously.
Thus, the scientists imaged calcium concentration microdomains and transmitter release sites. They visualized surface areas of 15 × 15 μm, with each pixel collecting light from an area roughly 0.15 × 0.15 μm, and sampled images every 1 ms for trials of roughly 4 s. They analyzed the images with BrainVision software from SciMedia and with ImageJ software from the National Institutes of Health.
The study yielded important insights into vesicle fusion and transmitter release. First, it validated the assumption that calcium concentration domains and transmitter release sites are strictly colocalized. Furthermore, kinetic analysis of vesicle exocytosis demonstrated two distinct types of exocytosis: full-fusion and “kiss and run,” a transient release mechanism in which only a portion of the synaptic vesicle contents is discharged.
The researchers also found that the vesicle’s speed, as it moves intracellularly toward the release site at the active zone, is calcium concentration-dependent. But most surprising, Llinàs said, was the observation that vesicles apparently are tethered to the membrane for a distance larger than the evanescence gap.
These findings are significant for a variety of reasons. “Synaptic transmission is one of the universal mechanisms in brain function,” Llinàs explained. “There’s much to be learned about normal brain function by understanding transmission — for instance, issues concerning nervous system development.” Such insight also could benefit our understanding of physiopathology. And, finally, it could contribute to studies of the effects of drugs, with an eye toward either treatment or understanding how a particular system works.
In fact, Llinàs and his colleagues are now preparing a study of the pharmacology of synaptic release using total internal reflection fluorescence.
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