Cellular NeuroscienceResearch PaperVesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons
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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2011, MicronCitation Excerpt :Detailed protocols to stain nerve terminals with FM1-43 and perform photo-oxidation are given by Schikorski (2010b) and Opazo and Rizzoli (2010). Photo-oxidation of FM1-43 or related styryl dyes has been used to study the size and distribution of various synaptic vesicle pools (readily releasable, recycling, and reserve pools) and the routes of endocytosis by varying the duration and frequency of stimulation, timing of dye application, or affinity of the dye for membranes (Teng and Wilkinson, 2000; Richards et al., 2000, 2003; Rizzoli and Betz, 2004; LoGiudice et al., 2009; Denker et al., 2009; Akbergenova and Bykhovskaia, 2009a,b). Employing a correlative approach to the study of synaptic vesicle release and recycling with FM1-43 and photo-oxidation provides detailed information about synaptic strength and performance.
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