Article

Properties of Ribbon and Non-Ribbon Release from Rod Photoreceptors Revealed by Visualizing Individual Synaptic Vesicles

Departments of Pharmacology and Experimental Neuroscience and Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 01/2013; 33(5):2071-86. DOI: 10.1523/JNEUROSCI.3426-12.2013
Source: PubMed

ABSTRACT

Vesicle release from rod photoreceptors is regulated by Ca(2+) entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1-43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca(2+) influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca(2+) entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light.

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    • "These are illustrated in the examples of rod-and conedriven EPSCs shown in Figure 1. Following fast ribbon-mediated release, the first of the two slow components of roddriven release represents exocytosis at nonribbon release sites that is triggered by CICR (Krizaj et al. 1999; Cadetti et al. 2006; Suryanarayanan and Slaughter 2006; Chen et al. 2013, 2014). A final component of rod-driven EPSCs in salamander retina arises from the spread of depolarizing current into neighboring rods through gap junctions. "
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    • "Recovery is no such mechanism appears to operate in cones as fluorescence recovery after photobleaching measurements indicate that Ca 2+ does not alter vesicle mobility (Rea et al., 2004). In TIRFM experiments in salamander rods, the velocity of vesicle approach to the presynaptic membrane in response to a depolarizing step was the same at nonribbon and ribbon-associated sites, at which [Ca 2+ ] reaches lower and higher levels, respectively (M. Chen et al., 2013), consistent with a lack of an effect of [Ca 2+ ] on vesicle mobility in photoreceptors. Likewise, Ca 2+ also appears to have no effect on vesicle mobility in goldfish BCs (Holt et al., 2004). "
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    • "However, upon internalization, the acidic environment (pH≈5.5) of the vesicles elicits a bright green fluorescent signal from this dextran conjugate. This probe has been used to study autophagy [49] as well as to describe the sites and kinetics of vesicle release [50]. FM1-43 (1 mM) and pHrodo Green (0.5 mg/ml) stock solutions were aliquoted and store at −20°C, protected from light. "
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