A highly Ca2+-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse

Abstract

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.

Figure 1. Depolarization Triggers Exocytosis in an Isolated Rod

(A) Capacitance increase evoked by a voltage step (−65 to −5 mV, 5 s) given at the arrow. No significant change in membrane or series conductance associated with the voltage step was observed (B and C).

Figure 2. Pulse Duration Experiments Reveal Two Kinetic Components of Exocytosis in Rods

(A) Increasing the duration of a step depolarization (−65 to −5 mV) from 0.5 s (open circles) to 2 s (closed circles) increased the amplitude of the capacitance jump. Arrow indicates beginning of the voltage step. (B) Depolarization-evoked capacitance increase plotted as a function of step duration. The capacitance/duration relation was fit with a dual exponential (τ₁ = 5.6 ms, amplitude₁ = 92.4 fF; τ₂ = 298 ms, amplitude₂ = 210.4 fF). Sample sizes: 12 ms, n = 50; 25 ms, n = 42; 50 ms, n = 43; 100 ms, n = 66; 200 ms, n = 46; 500 ms, n = 45; 2 s, n = 41. Inset magnifies the initial fast rise in capacitance.

Figure 3. The Capacitance Jump Accurately Reports Neurotransmitter Release

(A) Increasing the duration of a depolarizing step (−70 to −10 mV) applied to a rod from 10 ms (top trace) to 1.0 s (lower trace) increased the amplitude of the PSC evoked in a postsynaptic OFF bipolar cell. (B) PSC charge transfer, obtained from paired pre- and postsynaptic recordings, was linearly related to the capacitance increase evoked by depolarizing steps of the same duration (25, 50, 100, 200, and 500 ms; r² = 0.92). Charge transfer was normalized to the charge transfer evoked by a 500 ms step. PSC data in (A) and (B) were obtained in the presence of cyclothiazide (0.1 mM) and were from 2 horizontal cells and 4 OFF bipolar cells. Capacitance data are taken from the experiments shown in Figure 2.

Figure 4. The Voltage Dependence of Depolarization-Evoked Capacitance Increases Matches the Voltage Dependence of I_Ca

The amplitude of the capacitance jump evoked by a 100 ms step from a holding potential of −65 to a test potential is plotted against the test potential (left axis, circles, n = 11). The magnitude of exocytosis shows a sigmoidal relationship to voltage similar to I_Ca (right axis, data trace). I_Ca was recorded using ruptured patch recording techniques from isolated rods and evoked with a ramp depolarization (0.5 mV/ms, −90 to +60 mV). 1.8 mM Ca²⁺ was the charge carrier. K⁺ channels were blocked with Cs⁺ in the pipette and TEA in both the pipette and bathing solutions. Niflumic acid (0.1 mM) was added to inhibit Ca²⁺-activated Cl⁻ channels. The same pipette solution was used in measuring both I_Ca and capacitance. I_Ca from 7 cells were normalized to 100 pA and then averaged to yield the current/voltage relationship shown.

Figure 5. Exocytosis Was Triggered by the Elevation of Ca²⁺ to Submicromolar Levels by Flash Photolysis of Caged Ca²⁺

(A) Flash photolysis of Ca²⁺-loaded DM-nitrophen produced a rapid and spatially homogenous elevation of [Ca²⁺]_i in the rod synaptic terminal to 780 nM. [Ca²⁺]_i was measured with the low-affinity Ca²⁺ indicator dye Fura-FF. (B–D) High-resolution traces show that the increase in [Ca²⁺]_i triggered a slowly developing increase in membrane capacitance (B) and an uncorrelated rapid increase in membrane conductance (C). No change in access resistance was observed (D). Note the difference in time scales between (A) and (B)–(D). For all panels, the UV flash is given at time = 0.

Figure 6. The Relationship between the Rate of Membrane Addition and [Ca²⁺]_i Is Approximately Linear within a Restricted Range of Physiological [Ca²⁺]_i

Exponential rate constants, derived from a single exponential fit to the rising phase of each capacitance response evoked by the flash photolysis of caged Ca²⁺, are plotted against the intraterminal [Ca²⁺] measured immediately after the flash (n = 45). Data points in the Ca²⁺ range from 0.5 to 3 μM (Rieke and Schwartz, 1996) are shown in black (n = 36); data points outside this range are shown in gray. Rate constants in the physiological Ca²⁺ range show a nearly linear relationship with Ca²⁺ as illustrated by the linear regression (solid line, slope = 6.70 ± 2.10, Y intercept = −2.72 ± 2.90, r² = 0.23). Across the entire range, the data could be simulated (dashed line) using a mathematical model of exocytosis that assumes three independent Ca²⁺ binding reactions followed by a final, Ca²⁺-independent fusion step (see Experimental Procedures; also Heinemann et al., 1994; Voets, 2000).

Figure 7. Vesicles that Exocytose in Response to Flash Photolysis of Caged Ca²⁺ also Fuse in Response to Ca²⁺ Influx through Voltage-Gated Channels

Bar graph compares the mean amplitude of the capacitance response evoked by a maximal intensity flash in control cells (170.7 ± 21.5 fF, n = 45) with the mean capacitance response evoked by the identical flash in cells in which the releasable pool was predepleted by a 2 s depolarization from −65 to −5 mV (42.2 ± 38.0 fF, n = 7). Prior depletion of the releasable pool significantly reduced the response to a flash relative to control (p = 0.03).

Figure 8. For Ultrastructural Analysis of Vesicle Pools that May Participate in Release, the Size of Ribbons, Number of Vesicles Tethered to the Presynaptic Ribbon, and the Diameter of Vesicles Were Determined by Examining Serially and Randomly Sectioned Rod Terminals

Ribbon-style active zones in isolated cells (A) are similar to those in the intact retina (B). Ribbons (arrows) are surrounded by vesicles and attached at a perpendicular angle to the membrane. In the retina, membrane-attached ribbons (arrows) are present opposite postsynaptic processes (B). At the far right of (B), a tangentially sectioned ribbon shows the hexagonal packing of tethered vesicles (arrowheads). Same magnification in (A) and (B). Bar = 200 nm.