Abstract
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