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High-Resolution Optical Imaging of Zebrafish Larval Ribbon Synapse Protein RIBEYE, RIM2, and CaV 1.4 by Stimulation Emission Depletion Microscopy

Published online by Cambridge University Press:  26 July 2012

Caixia Lv ,
Travis J. Gould ,
Joerg Bewersdorf  and
David Zenisek
Caixia Lv
Affiliation:
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA Kavli Institute of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA
Travis J. Gould
Affiliation:
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA Kavli Institute of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA
Joerg Bewersdorf
Affiliation:
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA Kavli Institute of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA
David Zenisek*
Affiliation:
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA Department of Ophthalmology and Visual Sciences, Yale University School of Medicine, New Haven, CT 06520, USA Kavli Institute of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA Center for Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06520, USA
*
Corresponding author. E-mail: [email protected]
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Abstract

The synaptic ribbon is a unique presynaptic structure with an intricate morphology in photoreceptors. Because of the resolution limit in conventional fluorescence microscopy, investigating ribbon protein locations has been challenging, especially in the early development stages of model animals. Here, we used stimulated emission depletion microscopy, a super-resolution imaging technique, to look at retina sections in 4 days post-fertilization (dpf) zebrafish. We observed that in photoreceptor cells, RIBEYE and RIM2 are expressed along the synaptic ribbon, with RIM2 consistently located inside of the horseshoe-shaped synaptic ribbon structure with RIBEYE located on the outside. The L-type calcium channel subunit, CACNA1F, exhibited small spot-like staining beneath the RIM2 and RIBEYE structures. Using morpholino antisense oligonucleotides to knock down RIBEYE expression, we observed fewer and shorter ribbons in the photoreceptor outer plexiform layers of 4 dpf fish retina as well as a reduction in RIM2 expression. The clustering of CACNA1F in these blind fish was no longer observed, but instead showed a diffuse expression in the photoreceptor terminal.

Keywords

STEDRIBEYEzebrafishretinasynapsesynaptic ribboncalciumvesicle
Type
Special Section: Seventh Omaha Imaging Symposium
Information
Microscopy and Microanalysis , Volume 18 , Issue 4 , August 2012 , pp. 745 - 752
Copyright
Copyright © Microscopy Society of America 2012

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