Lucia Cadetti

A comparison of release kinetics and glutamate receptor properties in shaping rod–cone differences in EPSC kinetics in the salamander retina

Synaptic transmission from cones is faster than transmission from rods. Using paired simultaneous recordings from photoreceptors and second‐order neurones in the salamander retina, we studied the contributions of rod–cone differences in glutamate receptor properties and synaptic release rates to shaping postsynaptic responses. Depolarizing steps evoked sustained calcium currents in rods and cones that in turn produced transient excitatory postsynaptic currents (EPSCs) in horizontal and OFF bipolar cells. Cone‐driven EPSCs rose and decayed faster than rod‐driven EPSCs, even when comparing inputs from a rod and cone onto the same postsynaptic neurone. Thus, rod–cone differences in EPSCs reflect properties of individual rod and cone synapses. Experiments with selective AMPA and KA agonists and antagonists showed that rods and cones both contact pharmacologically similar AMPA receptors. Spontaneous miniature EPSCs (mEPSCs) exhibited unimodal distributions of amplitude and half‐amplitude time width and there were no rod–cone differences in mEPSC properties. To examine how release kinetics shape the EPSC, we convolved mEPSC waveforms with empirically determined release rate functions for rods and cones. The predicted EPSC waveform closely matched the actual EPSC evoked by cones, supporting a quantal release model at the photoreceptor synapse. Convolution with the rod release function also produced a good match in rod‐driven cells, although the actual EPSC was often somewhat slower than the predicted EPSC, a discrepancy partly explained by rod–rod coupling. Rod–cone differences in the rates of exocytosis are thus a major factor in producing faster cone‐driven responses in second‐order retinal neurones.

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.

Quantal mEPSCs and residual glutamate: how horizontal cell responses are shaped at the photoreceptor ribbon synapse

At the photoreceptor ribbon synapse, glutamate released from vesicles at different positions along the ribbon reaches the same postsynaptic receptors. Thus, vesicles may not exert entirely independent effects. We examined whether responses of salamander retinal horizontal cells evoked by light or direct depolarization during paired recordings could be predicted by summation of individual miniature excitatory postsynaptic currents (mEPSCs). For EPSCs evoked by depolarization of rods or cones, linear convolution of mEPSCs with photoreceptor release functions predicted EPSC waveforms and changes caused by inhibiting glutamate receptor desensitization. A low‐affinity glutamate antagonist, kynurenic acid (KynA), preferentially reduced later components of rod‐driven EPSCs, suggesting lower levels of glutamate are present during the later sustained component of the EPSC. A glutamate‐scavenging enzyme, glutamic‐pyruvic transaminase, did not inhibit mEPSCs or the initial component of rod‐driven EPSCs, but reduced later components of the EPSC. Inhibiting glutamate uptake with a low concentration of dl‐threo‐β‐benzoyloxyaspartate (TBOA) also did not alter mEPSCs or the initial component of rod‐driven EPSCs, but enhanced later components of the EPSC. Low concentrations of TBOA and KynA did not affect the kinetics of fast cone‐driven EPSCs. Under both rod‐ and cone‐dominated conditions, light‐evoked currents (LECs) were enhanced considerably by TBOA. LECs were more strongly inhibited than EPSCs by KynA, suggesting the presence of lower glutamate levels. Collectively, these results indicate that the initial EPSC component can be largely predicted from a linear sum of individual mEPSCs, but with sustained release, residual amounts of glutamate from multiple vesicles pool together, influencing LECs and later components of EPSCs.