Conical Tomography of a Ribbon Synapse: Structural Evidence for Vesicle Fusion

Guido A Zampighi,Lorenzo M Zampighi,Michael Woodruff,Nicholas C Brecha,Cataldo Schietroma,Ernest M Wright,Steven Barnes

doi:10.1371/journal.pone.0016944

Guido A Zampighi, Lorenzo M Zampighi + Show 5 more

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https://doi.org/10.1371/journal.pone.0016944

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Abstract

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3–15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.

Highlights

There is a remarkable diversity in the structure and protein composition of specialized regions of the plasma membrane, called ‘‘active zones,’’ where synaptic vesicles dock, fuse and release their transmitter [1,2,3,4,5]
Our findings support a conventional mechanism for neurotransmitter release from rod photoreceptors where several thousand synaptic vesicles fuse at active zones in ribbon-associated and ribbon-free regions of the rod spherule that are in apposition to horizontal cell endings. Supporting this conclusion is a synchronized series of events that occur with depolarization of the rod spherule in the dark, including a decrease in the rod spherule volume (Table 2), a large increase of hemi-fused vesicles (Fig. 7) and an increase in the number of fully fused vesicles during the first 30 minutes of darkness (Table 2; Figs. 2A–D; 6A–D)
There was an absence of intracellular cisterns or vacuoles near the synaptic ribbon that would have been indicative of compound vesicle fusion [57,58,59,60]

Summary

Introduction

There is a remarkable diversity in the structure and protein composition of specialized regions of the plasma membrane, called ‘‘active zones,’’ where synaptic vesicles dock, fuse and release their transmitter [1,2,3,4,5]. The active zone of photoreceptors, auditory and vestibular hair cells, and electroreceptors differ from conventional synapses. They contain at least one large and distinct electron-dense structure, which is shaped as a plate or sphere, called a synaptic ribbon or body [11,12,13,14,15,16]. Cone synaptic ribbons are distributed at variable distances to nearby horizontal cell endings, and more distant OFF-bipolar cell dendrites [13,28,29]

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