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Different roles of ribbon-associated and ribbon-free active zones in retinal bipolar cells

Nature Neuroscience volume 10, pages 1268–1276 (2007)Cite this article

Abstract

Synaptic ribbons with a halo of synaptic vesicles are seen at the active zones of sensory neurons that release transmitter tonically. Thus, ribbons are assumed to be a prerequisite for sustained exocytosis. By applying total internal reflection fluorescence microscopy to goldfish retinal bipolar cell terminals, we visualized Ca2+ entry sites, ribbons, and vesicle fusion events. Here we show that the main Ca2+ entry sites were located at ribbons, and that activation of the Ca2+ current induced immediate and delayed vesicle fusion events at ribbon-associated and ribbon-free 'hot spots', respectively. The activation of protein kinase C (PKC) specifically potentiated vesicle fusion at ribbon-free sites. Electron microscopy showed that PKC activation selectively increased the number of docked vesicles at ribbon-free sites, which faced neuronal processes with the postsynaptic density. Retinal bipolar cells have both ribbon-associated and ribbon-free active zones in their terminals and might send functionally distinct signals through ribbon-associated and ribbon-free synapses to postsynaptic neurons.

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Figure 1: Ca2+ entry sites and ribbons.
Figure 2: Identification of vesicle fusion.
Figure 3: Spatio-temporal distribution of fusion events.
Figure 4: Effects of PMA on ribbon displacement and spatio-temporal distribution of vesicle fusion events.
Figure 5: Effects of PMA on spatio-temporal distribution of fusion events.
Figure 6: Aggregation of vesicle fusion events at 'hot spots'.
Figure 7: Distribution of synaptic vesicles in the Mb1 bipolar cell terminal.
Figure 8: Ribbon-associated and ribbon-free synapses in a terminal of the Mb1 bipolar cell.

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Acknowledgements

We thank T. Asaka for participation in early experiments and S. Tokimura for excellent technical assistance. This work was supported by JSPS Grants-in-Aid for Scientific Research (18300132) and Grant-in-Aid for Scientific Research on Priority Areas from MEXT (12053212, 18019012) to M.T.

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Author notes

  1. Mitsuharu Midorikawa

    Present address: Present address: Almers Lab, Vollum Institute, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97239, USA.,

Authors and Affiliations

  1. Department of Psychology, Graduate School of Humanities and Sociology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Mitsuharu Midorikawa, Ken Berglund & Masao Tachibana

  2. Department of Biology, Hyogo College of Medicine, 1-1 Mukogawa Nishinomiya, 663-8501, Japan

    Yoshihiko Tsukamoto & Masaaki Ishii

  3. Department of Neurobiology, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Ken Berglund

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  3. Ken Berglund
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  4. Masaaki Ishii
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  5. Masao Tachibana
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Contributions

M.M. conducted the main body of the physiological experiments, Y.T. carried out the electron microscopic studies, K.B. contributed the data analysis of the electron microscopy, M.I. carried out the immunohistochemistry, and M.T. wrote the manuscript and supervised the project.

Corresponding author

Correspondence to Masao Tachibana.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Table 1 (PDF 128 kb)

Supplementary Video 1

Synaptic ribbons and Ca2+ entry sites. Visualization by fluorescein-conjugated CtBP-biding peptide and Fluo-5F. A goldfish Mb1 bipolar cell terminal under a TIRF microscope with 488-nm illumination. Before depolarization, five ribbons are observed. Upon depolarization, Ca2+ emerged at ribbons. Picture size is 10.24 × 10.24 μm (40 nm pixel−1). (MOV 123 kb)

Supplementary Video 2

Fusion of FM-labeled vesicles in a goldfish Mb1 bipolar cell terminal under a TIRF microscope. Images were acquired for 3.5 s every 36.4 ms. Picture size is 10.24 × 10.24 μm (40 nm pixel−1). (MOV 257 kb)

Supplementary Video 3

Fusion of 'Resident'. The vesicle intensity was already high before fusion. Picture size is 1.24 × 1.24 μm (40 nm pixel−1). Averaged image from ten fusion events. (MOV 57 kb)

Supplementary Video 4

Fusion of 'Newcomer'. The vesicle intensity was increased abruptly from background before fusion. Picture size is 1.24 × 1.24 μm (40 nm pixel−1). Averaged image from five fusion events. (MOV 71 kb)

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Midorikawa, M., Tsukamoto, Y., Berglund, K. et al. Different roles of ribbon-associated and ribbon-free active zones in retinal bipolar cells. Nat Neurosci 10, 1268–1276 (2007). https://doi.org/10.1038/nn1963

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