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


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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    • "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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    ABSTRACT: At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.
    The Journal of General Physiology 10/2014; 144(5). DOI:10.1085/jgp.201411229 · 4.79 Impact Factor
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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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    ABSTRACT: An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.
    PLoS ONE 06/2014; 9(6):e100757. DOI:10.1371/journal.pone.0100757 · 3.23 Impact Factor
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    • "Extrasynaptic exocytosis of vesicles or prefusion of vesicles before reaching the active zone are alternative scenarios consistent with this idea. Supporting this view, long depolarization pulses (>200 ms) applied to rods evoked the exocytosis of vesicles located far from synaptic ribbons (Chen et al., 2013). These ectopic exocytic events were triggered by CICR, suggesting the potential contribution of ER to maintained synaptic release of vesicles (Chen et al., 2014). "
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    ABSTRACT: Intracellular calcium stores control many neuronal functions such as excitability, gene expression, synaptic plasticity, and synaptic release. Although the existence of calcium stores along with calcium-induced calcium release (CICR) has been demonstrated in conventional and ribbon synapses, functional significance and the cellular mechanisms underlying this role remains unclear. This review summarizes recent experimental evidence identifying contribution of CICR to synaptic transmission and synaptic plasticity in the CNS, retina and inner ear. In addition, the potential role of CICR in the recruitment of vesicles to releasable pools in hair-cell ribbon synapses will be specifically discussed.
    Frontiers in Cellular Neuroscience 06/2014; 8:162. DOI:10.3389/fncel.2014.00162 · 4.29 Impact Factor
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