April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Fast fusion kinetics of primed vesicles at a mammalian cone photoreceptor synapse
Author Affiliations & Notes
  • Chad Grabner
    Ophthalmology, Northwestern University, Chicago, IL
  • Steven H DeVries
    Ophthalmology, Northwestern University, Chicago, IL
  • Footnotes
    Commercial Relationships Chad Grabner, None; Steven DeVries, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 4526. doi:
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      Chad Grabner, Steven H DeVries; Fast fusion kinetics of primed vesicles at a mammalian cone photoreceptor synapse. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4526.

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

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Purpose: Cone photoreceptor ribbon synapses signal the end of a light step by releasing a burst of transmitter into the synaptic cleft. The timing and synchronicity of release determine the peak cleft glutamate concentration and hence influence the amplitude of the postsynaptic response; however, postsynaptic responses are subject to non-linearities. Membrane capacitance measurements provide a direct readout of vesicle fusion, and such measurements have not been obtained at the mammalian cone synapse.

Methods: Recordings were obtained from ground squirrel (Ictidomys tridecemlineatus) retina. Membrane capacitance (Cm) was measured in whole-cell voltage clamp using the ‘sine+dc’ routine and a HEKA EPC-10 amplifier. Cells were held at -70 mV, excepted during stimulation. Patch electrodes contained a CsCl based solution with 10 mM EGTA. Slices were bathed in a bicarbonate buffered saline (5% CO2) supplemented with TBOA (200 µM) and CsCl (5 mM).

Results: Release kinetics were examined by varying stimulus duration over a range of 1-30 ms and stepped to -10 mV. The change in Cm at 1 and 30 ms differed by only 2-fold (11.4 ± 1.7 vs. 22.6 ± 1.8 fF; n = 5 cells), and the plot of Cm over pulse duration was well-fit by a double exponential with time constants of 0.9 ms (16 fF ~290 vesicles) and 10 ms (7 fF ~127 vesicles). From a separate set of cells the relationship between Cm and membrane voltage levels was explored with a family of steps between -50 and 30 mV, given for 1 or 30 ms. Release reached a maximum at -10 mV for both short and long steps, the release function was bell-shaped, which suggests the profile of Ca2+ entry greatly influences the apparent vesicle pool size. HEPES (15 mM), which blocks inhibitory proton feedback onto cone voltage-dependent Ca2+ channels, was able to shift release to the first kinetic phase.

Conclusions: The combined size of the two fast pools equals or slightly exceeds the size of the vesicle pool that is estimated to be membrane docked at a cone’s ~20 ribbons. The bulk of transmitter is released at a very high rate upon depolarization, but there also seems to be a slower component that may be a consequence of the retarding effect of proton feedback.

Keywords: 688 retina • 648 photoreceptors • 644 pH regulation/protons  

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