June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Endocytosis at rod photoreceptor synapses
Author Affiliations & Notes
  • Karlene Cork
    University of Nebraska Medical Center, Omaha, NE
  • Wallace Thoreson
    University of Nebraska Medical Center, Omaha, NE
  • Footnotes
    Commercial Relationships Karlene Cork, None; Wallace Thoreson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2487. doi:
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      Karlene Cork, Wallace Thoreson; Endocytosis at rod photoreceptor synapses. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2487.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Release from rods is triggered by opening of L-type Ca2+ channels confined beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous studies showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones (Van Hook & Thoreson, in press). We therefore hypothesized that endocytosis in rods may also differ from cones in its dependence upon Ca2+ and/or the amount of prior exocytosis.

Methods: We measured exocytosis and endocytosis from membrane capacitance (Cm) changes evoked by depolarizing steps in voltage-clamped rods from tiger salamander retinal slices. Cm changes were measured using a phase lock amplifier integrated into the Cairn Optopatch amplifier. We manipulated Ca2+ influx and vesicle release by altering step duration and test voltage. To measure the size of Ca2+ channel confinement domains in rods, we labeled Ca2+ channels with quantum dots and used single-particle tracking techniques (Mercer et al., 2011).

Results: As in cones, the time constant for endocytosis in rods was quite fast, averaging ~200ms. Similar time constants were observed when using low frequency sine waves to measure Cm, employing software-based dual sine wave protocols to measure Cm, or blocking Ca2+-activated Cl- channels. Endocytosis kinetics did not change after varying Ca2+ channel activation with voltage steps of -40, -30, -20 and -10 mV or varying step duration between 5, 10, 25, 50, 100, and 200 ms. Replacing 1 mM BAPTA with 5 mM EGTA as the Ca2+ chelator in the pipette solution also did not significantly alter endocytosis time constants. Blocking dynamin with dynasore (80 μM) slowed endocytosis and significantly reduced the total amount of exocytosis. Dynasore also expanded Ca2+ channel confinement domains in rods simultaneously bathed with 20 mM KCl to enhance ongoing release and endocytosis. Although this expansion may increase the distance between Ca2+ channels and release sites, dynasore did not significantly inhibit release efficiency as measured by the ratio between release and Ca2+ influx.

Conclusions: While endocytosis in rods and cones differ in their dependence on dynamin, they exhibit similar rapid kinetics and independence from both Ca2+ and amount of release. Although inhibiting endocytosis expanded Ca2+ channel confinement domains, this did not appear to change release efficiency.

Keywords: 728 synapse • 648 photoreceptors • 689 retina: distal (photoreceptors, horizontal cells, bipolar cells)  
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