Elsevier

Neuroscience

Volume 164, Issue 4, 29 December 2009, Pages 1546-1556
Neuroscience

Cellular Neuroscience
Research Paper
Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

https://doi.org/10.1016/j.neuroscience.2009.09.023Get rights and content

Abstract

In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval occurred at discrete sites that were spatially consistent over multiple stimuli, indicative of endocytotic “hot spots.” Retrieval sites were spatially correlated with fluorescently labeled synaptic ribbons. Electron microscopy (EM) analysis of bipolar cell terminals after photoconversion of internalized FM dye revealed that almost all of the dye was contained within vesicles ∼30 nm in diameter. Clathrin-coated vesicles were observed budding from the plasma membrane and within the cytosol, and application of dynasore, a dynamin inhibitor, arrested membrane retrieval just after the budding stage. We conclude that synaptic vesicles in the fine branches of mouse bipolar axon terminals are retrieved locally near active zones, at least in part via a clathrin-mediated pathway.

Section snippets

Bipolar cell isolation

We used adult male mice of strain C57BL/6J (Taconic, Germantown, NY, USA). Animal use was approved by the Institutional Animal Care and Use Committee of SUNY Stony Brook and was in accord with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. The number of animals and their suffering were minimized. A mouse was killed by CO2 inhalation, and both eyes were removed and hemisected. Neural retinas were detached and cut into six pieces. To isolate bipolar

“Hot spots” of membrane retrieval

To detect sites of membrane retrieval, we depolarized single bipolar cells by focally applying the styryl dye FM4-64 in high K+ for several seconds. Approximately 12 s after the onset of stimulation, the cells were washed with a dye-free external solution containing Advasep-7 to eliminate plasma membrane fluorescence. Trapped FM4-64 was confined to the synaptic terminals and the adjacent axon (Fig. 1A), and was located discretely, suggesting that endocytosis occurred at distinct sites along the

Direct recycling vs bulk endocytosis

We have found that mouse bipolar cell terminals retrieve the membrane added during neurotransmitter release by direct recovery of small vesicles. Furthermore, recently recycled vesicles were found to be associated with synaptic ribbons, even after brief stimulation with a single voltage-clamp depolarization, which indicates that at least some newly retrieved vesicles are reused quickly in mouse bipolar cells. This scheme contrasts with goldfish bipolar cells and frog saccular hair cells, where

Acknowledgments

Supported by NIH grants EY003821 (G.M.) and EY008124 (P.S.). We thank Jian Li and Wendy Akmentin for electron microscopy.

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