Properties of release from rods revealed by visualizing individual synaptic vesicles

Abstract

 

Purpose

 

Vesicle release from rods is triggered by Ca²⁺ entry through L-type channels near synaptic ribbons but release may also occur at non-ribbon sites. We visualized fusion of individual synaptic vesicles to characterize sites and kinetics of vesicle release from rods.

 

Methods

 

A small number (1~3%) of vesicles were loaded by briefly incubating salamander retinas with FM1-43 or a dextran-conjugated, pH-sensitive rhodamine, pHrodo in darkness. Synaptic vesicles of isolated rods were imaged using total internal reflection fluorescence microscopy (fig.). Release was stimulated by depolarizing steps applied to voltage-clamped rods or by puffing high KCl or ryanodine.

 

Results

 

Labeled organelles matched the diffraction-limited size of 40-nm microspheres and disappeared rapidly upon stimulation. Disappearance of fluorescent organelles was blocked by inhibiting Ca²⁺ influx. Kinetics of release measured optically matches that measured from capacitance change. We saw few membrane-associated vesicles unless Ca²⁺ entry was inhibited. During stimulation, newly appearing vesicles approached the membrane at ~800 nm/s where they paused for ~60 ms before fusion. With fusion, vesicles advanced ~18 nm closer to the membrane. Release occurred mostly near ribbons, but lengthy depolarization stimulated spread of Ca²⁺ throughout the terminal and caused more release from non-ribbon sites. Activation of Ca²⁺-induced Ca²⁺ release (CICR) by ryanodine (10 μM) stimulated Ca²⁺ increases and vesicle release. Depolarization-evoked Ca²⁺ spread and ryanodine-evoked effects were inhibited by blocking CICR with dantrolene (10 μM, in bath). Consistent with more release from non-ribbon sites during slow release, damaging the ribbon by fluorophore-assisted laser inactivation of RIBEYE only weakly inhibited exocytotic capacitance jumps evoked by 200-ms depolarizing steps. By contrast, damaging the ribbon strongly inhibited capacitance jumps evoked by 25-ms steps suggesting fast release relies more on ribbons.

 

Conclusions

 

We found that ongoing release at depolarized resting potentials depletes membrane-associated vesicles. Sustained release rates are therefore limited by vesicle delivery rates and membrane dwell times of ~60 ms. The overall rate of sustained release is amplified by release from non-ribbon sites involving CICR from intracellular stores. This amplification may improve detection of changes in release caused by changing illumination.

 

 

Keywords: 599 microscopy: light/fluorescence/immunohistochemistry • 728 synapse • 689 retina: distal (photoreceptors, horizontal cells, bipolar cells)