Katalin Rabl

A Highly Ca2+-Sensitive Pool of Vesicles Contributes to Linearity at the Rod Photoreceptor Ribbon Synapse

Studies of the properties of synaptic transmission have been carried out at only a few synapses. We analyzed exocytosis from rod photoreceptors with a combination of physiological and ultrastructural techniques. As at other ribbon synapses, we found that rods exhibited rapid kinetics of release, and the number of vesicles in the releasable pool is comparable to the number of vesicles tethered at ribbon-style active zones. However, unlike other previously studied neurons, we identified a highly Ca(2+)-sensitive pool of releasable vesicles with a relatively shallow relationship between the rate of exocytosis and [Ca(2+)](i) that is nearly linear over a presumed physiological range of intraterminal [Ca(2+)]. The low-order [Ca(2+)] dependence of release promotes a linear relationship between Ca(2+) entry and exocytosis that permits rods to relay information about small changes in illumination with high fidelity at the first synapse in vision.

Role of the synaptic ribbon in transmitting the cone light response

Cone photoreceptors distinguish small changes in light intensity while operating over a wide dynamic range. The cone synapse encodes intensity by modulating tonic neurotransmitter release, but precise encoding is limited by the quantal nature of synaptic vesicle exocytosis. Cones possess synaptic ribbons, structures that are thought to accelerate the delivery of vesicles for tonic release. Here we show that the synaptic ribbon actually constrains vesicle delivery, resulting in a maintained state of synaptic depression in darkness. Electron microscopy of cones from the lizard Anolis segrei revealed that depression is caused by the depletion of vesicles on the ribbon, indicating that resupply, not fusion, is the rate-limiting step that controls release. Responses from postsynaptic retinal neurons from the salamander Ambystoma tigrinum showed that the ribbon behaves like a capacitor, charging with vesicles in light and discharging in a phasic burst at light offset. Phasic release extends the operating range of the cone synapse to more accurately encode changes in light intensity, accentuating features that are salient to photopic vision.

Calcium‐dependent inactivation and depletion of synaptic cleft calcium ions combine to regulate rod calcium currents under physiological conditions

L‐type Ca2+ currents (ICa) in rod photoreceptors exhibit Ca2+‐dependent inactivation. Perforated‐patch whole‐cell recordings were obtained from isolated rods of the tiger salamander using 1.8 mm Ca2+ in the bathing medium to determine the extent of Ca2+‐dependent inactivation of ICa with physiological [Ca2+] and endogenous buffering. ICa was measured with voltage ramps applied before and after 5‐s steps to −40, −30, −20, or −10 mV. Long depolarizing steps in isolated rods produced inactivation of ICa ranging from 15% at −40 mV to > 80% at −10 mV. Because, in addition to Ca2+‐dependent inactivation, depletion of synaptic cleft Ca2+ accompanying activation of ICa can reduce presynaptic ICa at calycal synapses, we investigated whether a similar mechanism worked at the invaginating rod synapse. Rods from retinal slices with intact synapses were compared with isolated rods in which synaptic cleft depletion is absent. ICa was more strongly depressed by depolarization of rods in retinal slices, with ICa reduced by 47% following voltage steps to −40 mV. The depression of currents by depolarization was also greater for rods from retinal slices than isolated rods when Ca2+ was replaced with Ba2+ to reduce Ca2+‐dependent inactivation. The stronger depolarization‐evoked inhibition of ICa in retinal slices compared to isolated rods probably reflects depletion of synaptic cleft Ca2+ arising from sustained Ca2+ influx. Inactivation of ICa exhibited slow onset and recovery. These findings suggest that Ca2+‐dependent inactivation and depletion of synaptic cleft Ca2+ may combine to regulate ICa in response to light‐evoked changes in rod membrane potential.

Calcium-induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization.

We examined the contribution of calcium‐induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole‐cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (−70 to −10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage‐clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 µm) had no significant effect on rod calcium current (Ica) although it slightly diminished rod light‐evoked voltage responses. Bath application of 50 µm ryanodine strongly inhibited light‐evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 µm) also inhibited excitatory post‐synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods.

Paired-Pulse Depression at Photoreceptor Synapses

Synaptic depression produced by repetitive stimulation is likely to be particularly important in shaping responses of second-order retinal neurons at the tonically active photoreceptor synapse. We analyzed the time course and mechanisms of synaptic depression at rod and cone synapses using paired-pulse protocols involving two complementary measurements of exocytosis: (1) paired whole-cell recordings of the postsynaptic current (PSC) in second-order retinal neurons and (2) capacitance measurements of vesicular membrane fusion in rods and cones. PSCs in ON bipolar, OFF bipolar, and horizontal cells evoked by stimulation of either rods or cones recovered from paired-pulse depression (PPD) at rates similar to the recovery of exocytotic capacitance changes in rods and cones. Correlation between presynaptic and postsynaptic measures of recovery from PPD suggests that 80–90% of the depression at these synapses is presynaptic in origin. Consistent with a predominantly presynaptic mechanism, inhibiting desensitization of postsynaptic glutamate receptors had little effect on PPD. The depression of exocytotic capacitance changes exceeded depression of the presynaptic calcium current, suggesting that it is primarily caused by a depletion of synaptic vesicles. In support of this idea, limiting Ca2+ influx by using weaker depolarizing stimuli promoted faster recovery from PPD. Although cones exhibit much faster exocytotic kinetics than rods, exocytotic capacitance changes recovered from PPD at similar rates in both cell types. Thus, depression of release is not likely to contribute to differences in the kinetics of transmission from rods and cones.

Quantitative Analysis of Synaptic Release at the Photoreceptor Synapse

Exocytosis from the rod photoreceptor is stimulated by submicromolar Ca(2+) and exhibits an unusually shallow dependence on presynaptic Ca(2+). To provide a quantitative description of the photoreceptor Ca(2+) sensor for exocytosis, we tested a family of conventional and allosteric computational models describing the final Ca(2+)-binding steps leading to exocytosis. Simulations were fit to two measures of release, evoked by flash-photolysis of caged Ca(2+): exocytotic capacitance changes from individual rods and postsynaptic currents of second-order neurons. The best simulations supported the occupancy of only two Ca(2+) binding sites on the rod Ca(2+) sensor rather than the typical four or five. For most models, the on-rates for Ca(2+) binding and maximal fusion rate were comparable to those of other neurons. However, the off-rates for Ca(2+) unbinding were unexpectedly slow. In addition to contributing to the high-affinity of the photoreceptor Ca(2+) sensor, slow Ca(2+) unbinding may support the fusion of vesicles located at a distance from Ca(2+) channels. In addition, partial sensor occupancy due to slow unbinding may contribute to the linearization of the first synapse in vision.