Vesicle Pool Size at the Salamander Cone Ribbon Synapse

Abstract

Cone light responses are transmitted to postsynaptic neurons by changes in the rate of synaptic vesicle release. Vesicle pool size at the cone synapse constrains the amount of release and can thus shape contrast detection. We measured the number of vesicles in the rapidly releasable and reserve pools at cone ribbon synapses by performing simultaneous whole cell recording from cones and horizontal or off bipolar cells in the salamander retinal slice preparation. We found that properties of spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs) are representative of mEPSCs evoked by depolarizing presynaptic stimulation. Strong, brief depolarization of the cone stimulated release of the entire rapidly releasable pool (RRP) of vesicles. Comparing charge transfer of the EPSC with mEPSC charge transfer, we determined that the fast component of the EPSC reflects release of approximately 40 vesicles. Comparing EPSCs with simultaneous presynaptic capacitance measurements, we found that horizontal cell EPSCs constitute 14% of the total number of vesicles released from a cone terminal. Using a fluorescent ribeye-binding peptide, we counted approximately 13 ribbons per cone. Together, these results suggest each cone contacts a single horizontal cell at approximately 2 ribbons. The size of discrete components in the EPSC amplitude histogram also suggested approximately 2 ribbon contacts per cell pair. We therefore conclude there are approximately 20 vesicles per ribbon in the RRP, similar to the number of vesicles contacting the plasma membrane at the ribbon base. EPSCs evoked by lengthy depolarization suggest a reserve pool of approximately 90 vesicles per ribbon, similar to the number of additional docking sites further up the ribbon.

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors

“…S2), far shorter than the time constant for replenishment. (e) Fusion of vesicles in the IRP is much faster ( 6 ms; Thoreson et al, 2004;Rabl et al, 2005;Bartoletti et al, 2010) than replenishment, and this fast component of release was not affected by CaM inhibition. These considerations leave only two steps as likely to be Ca 2+ dependent and rate limiting for vesicle replenishment: (1) the time required for priming of newly attached vesicles by the ribbon and (2) the rate of vesicle attachment to the ribbon.…”

“…train of pulses empties the remainder of the releasable pool of vesicles Bartoletti et al, 2010). Release soon reaches a steady-state where it is limited by the rate of replenishment Schneggenburger et al, 2002;Babai et al, 2010).…”

“…4 A). When we converted the slope of the cumulative charge transfer to a vesicle release rate (16 fC/vesicle) and scaled it to the amplitude of the postsynaptic current (47 pA/ribbon; Cadetti et al, 2008;Bartoletti et al, 2010Bartoletti et al, , 2011, this corresponded to a change of sustained release rate from 151 ± 24 vesicles (v)/s/ribbon at 30 mV to 334 ± 55 v/s/ribbon Figure 4. Ca 2+ -dependent acceleration of replenishment measured using trains of depolarizing pulses.…”

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

“…In each, depolarizing the cone to -10 mV from a holding potential of -70 mV evoked a voltage-gated calcium current in the cone and fast inward EPSC in the horizontal or bipolar cell. This strong stimulus is sufficient to empty the readily-releasable pool of ~20 vesicles from each synaptic ribbon, resulting in an EPSC of ~47 pA/ribbon 10 . The horizontal cell EPSC in Figure 5A was 232 pA, suggesting that it received 5 ribbon contacts from the presynaptic cone.…”

“…Retinal neurons can also be loaded with fluorescent dyes sensitive to Ca 2+ , Cl -, or Na + introduced through the patch pipette or by bath-application 15,[18][19][20] . A fluorescent peptide that binds to the synaptic ribbon 21 can be introduced through the patch pipette and used for imaging the ribbon 10 or, when conjugated to fluorescein, for acutely and selectively damaging the ribbon 22 . We have also used retinal slices in combination with quantum dots to monitor the movements of individual calcium channels at rod and cone synaptic terminals 23 .…”