Evidence that exocytosis is driven by calcium entry through multiple calcium channels in goldfish retinal bipolar cells

Abstract

Ribbon-containing neurons represent a subset of neural cells that undergo graded membrane depolarizations rather than Na(+)-channel evoked action potentials. Bipolar cells of the retina are one type of ribbon-containing neuron and extensive research has demonstrated kinetically distinct pools of vesicles that are released and replenished in a calcium-dependent manner. In this study, we look at the properties of the fastest pool of releasable vesicles in these cells, often referred to as the immediately releasable pool (IRP), to investigate the relationships between vesicle release and calcium channels in these terminals. Using whole cell capacitance measurements, we monitored exocytosis in response to different magnitude and duration depolarizations, with emphasis on physiologically relevant step depolarizations. We find that release rate of the IRP increases superlinearly with membrane potential and that the IRP is sensitive to elevated EGTA concentrations in a membrane-potential-dependent manner across the physiological range of membrane potentials. Our results are best explained by a model in which multiple Ca(2+) channels act in concert to drive exocytosis of a single synaptic vesicle. Pooling calcium entering through many calcium channels may be important for reducing stochastic noise in neurotransmitter release associated with the opening of individual calcium channels.

FIG. 1.

Exocytosis as a function of depolarization step length. A, left, raw current signal from a bipolar cell terminal loaded with 0.5 mM ethylene glycol tetraacetic acid (EGTA) and depolarized from −60 to 0 mV for 250 ms. A, right: raw capacitance signal from the same terminal as at left. B, left, raw current signal from a bipolar cell terminal loaded with 10 mM EGTA and depolarized from −60 to 0 mV for 250 ms. B, right: the raw capacitance signal from the same terminal as at left. C, left: composite data from all terminals. Number of terminals loaded with 0.5 mM EGTA (black squares) by stimulus duration: 5 ms (n = 8), 10 ms (n = 6), 15 ms (n = 19), 30 ms (n = 7), 60 ms (n = 7), and 120 ms (n = 6). Number of terminals loaded with 10 mM EGTA (red circles) by stimulus duration: 5 ms (n = 8), 10 ms (n = 8), 15 ms (n = 10), 30 ms (n = 10), 60 ms (n = 8), and 120 ms (n = 6). C, right: composite data from terminals loaded with 0.5 mM EGTA and depolarized for indicated times. Red curve shows a single-exponential fit to these data using OriginLab 7.5. The curve had R² = 0.96 and τ = 2.9 ms.

FIG. 2.

Exocytosis as a function of short-duration step depolarizations and total internal reflection fluorescent microscopy (TIRFM) imaging of synaptic ribbon density. A, left: composite data of exocytosis from terminals loaded with 0.5 mM EGTA (filled circles) or 10 mM EGTA (open triangles) and depolarized to 0 mV for the indicated time (same data as in Fig. 1). A, right: experiment with 2 step depolarizations of the same duration given 50 ms apart. This experiment is designed to determine the length of time required for a single pulse to deplete the immediately releasable pool (IRP). Ordinate represents the capacitance jump due to the 2nd depolarization as a percentage of the capacitance jump due to the 1st depolarization; 0.5 mM EGTA is represented by filled circles, whereas 10 mM EGTA terminals are represented by open triangles. B, left: brightfield microscopy image of the synaptic terminal of a whole bipolar neuron. B, middle: TIRFM image of the same cell as on left. B, right: TIRFM image of the synaptic terminal of a different whole bipolar neuron. The scale bars for all images indicate 1.70 μm in both axes. C: histogram of number of synaptic ribbons compared with the full-width at half-maximum (FWHM) determined from fitting fluorescent intensity with a Gaussian function (Coggins et al. 2007).

FIG. 3.

Exocytosis due to step depolarizations to physiological membrane potentials using 0.5 mM EGTA. A, left: pulse protocol (P1) used to probe the amount of exocytosis elicited by 250-ms step depolarizations to various membrane potentials (−XX mV) from a holding potential of −60 mV. A, right: pulse protocol (P2) designed to probe IRP size and terminal capacity to exocytose the remainder of the IRP—using a 15-ms pulse to 0 mV—if a portion had been predepleted by a 235-ms step depolarization to a physiological membrane potential. B, left: composite data for all terminals. Bars, from leftmost to rightmost, represent terminals depolarized for −35, −30, −28, and [minus[25 mV. B, right: comparison of capacitance jump for terminals depolarized to −28 mV using P1 (leftmost bar), depolarized to −28 mV using P2 (middle bar), and depolarized to 0 mV for 15 ms (rightmost bar). The P2 and 15 ms to 0 mV protocols are significantly different (P < 0.05) from the P1 protocol. C: average charge for terminals loaded with 0.5 mM EGTA and step depolarized to −35, −30, −28, and −25 mV (from left to right, respectively). The number of terminals stimulated at each condition is: −35 mV (n = 4), −30 mV (n = 4), −28 mV (n = 6), and −25 mV (n = 11).

FIG. 4.

Exocytosis due to step depolarizations to physiological membrane potentials using 10 mM EGTA. A: composite data for all terminals. Bars, from leftmost to rightmost, represent terminals depolarized for −35, −30, −28, and −25 mV. B: comparison of capacitance jump for terminals depolarized to −28 mV using P1 (leftmost bar), depolarized to −28 mV using P2 (middle bar), and depolarized to 0 mV for 15 ms (rightmost bar). The P2 and 15 ms to 0 mV protocols are significantly different (shown by stars; P < 0.05) from the P1 protocol. C: average charge for terminals loaded with 0.5 mM EGTA and step depolarized to −35, −30, −28, and −25 mV (from left to right, respectively). The number of terminals stimulated at each condition is: −35 mV (n = 4), −30 mV (n = 4), −28 mV (n = 8), and −25 mV (n = 11).

FIG. 5.

Comparison of physiological membrane depolarizations using 0.5 or 10 mM EGTA. A, left: charge introduced during depolarizations as a function of the step depolarization protocol. Black bars (leftmost within protocol) represent terminals loaded with 0.5 mM EGTA; white bars (rightmost within protocol) represent terminals loaded with 10 mM EGTA. Comparing terminals within protocols shows that for −28-mV P1 step depolarizations the charge is significantly larger for 10 mM EGTA-loaded terminals. A, right: capacitance jump as a function of the step depolarization protocol. Black bars (leftmost within protocol) represent terminals loaded with 0.5 mM EGTA; white bars (rightmost within protocol) represent terminals loaded with 10 mM EGTA. Comparing terminals within protocols shows that for all protocols except −28-mV P1 step depolarizations, the capacitance jump is significantly larger for 0.5 mM EGTA-loaded terminals. B, left: charge introduced during a step depolarization to −30 mV compared with that introduced during a step depolarization to −30 mV that was immediately preceded by a 5-ms prepulse to +180 mV, to introduce a calcium tail current (−30-mV tail). B, right: capacitance change due to a simple 250-ms step depolarization to −30 mV (black trace) compared with the capacitance change elicited by a 250-ms step depolarization to −30 mV immediately preceded by a 5-ms prepulse to +180 mV (red trace). C, left: current records from representative terminals stimulated with a step depolarization from −60 to −30 mV for 250 ms (black trace) or stimulated with a step depolarization from −60 to −30 mV for 250 ms immediately preceded by a 5-ms depolarization to +180 mV (red trace). These currents gave rise to capacitance changes of −1.4 fF (terminal from black trace) and 4.6 fF (terminal from red trace). C, right: capacitance jump comparing simple 250-ms step depolarizations to step depolarizations with 5-ms prepulse to +180 mV. Black bars are from terminals loaded with 10 mM and depolarized to −30 mV. Red bars are from terminals loaded with 15 mM EGTA and 7.5 mM Ca²⁺, to give a measured internal [Ca²⁺] of about 300 nM (see methods).

FIG. 6.

Rates of exocytosis due to different calcium currents. A: linear plot of exocytic rate compared with calcium current in response to step depolarizations to different membrane potentials for terminals loaded with 0.5 mM EGTA (black squares; data from Figs. 1 and 3) or 10 mM EGTA (open circles; data from Figs. 1 and 4). See text for a description of how the exocytic rate was calculated. B: log–log plot of the data in A. The vertically hatched line represents a least-squares best-fit linear line to data from terminals loaded with 0.5 mM EGTA implemented in OriginLab (see methods). The solid line represents a best-fit linear line to data from terminals loaded with 10 mM EGTA.

FIG. 7.

Simple paired-pulse depression of bipolar neurons. A, left: pulse protocol used in this experiment. Terminals were voltage clamped at −60 mV and given two 20-ms-long depolarizations to 0 mV with variable time between the depolarizations. The number of 0.5 mM EGTA loaded cells for each interpulse interval: 50 ms (n = 6), 100 ms (n = 8), 200 ms (n = 8), 400 ms (n = 6), 800 ms (n = 6), 1 s (n = 7), 2 s (n = 6), 5 s (n = 7), 20 s (n = 8), and 30 s (n = 9). The number of 10 mM EGTA-loaded cells for each interpulse interval: 50 ms (n = 9), 100 ms (n = 7), 200 ms (n = 10), 400 ms (n = 8), 800 ms (n = 7), 1 s (n = 7), 2 s (n = 7), 5 s (n = 6), 20 s (n = 9), and 30 s (n = 9). A, right: average peak current measured for both 0.5 mM and 10 mM EGTA-loaded terminals. The black (leftmost) bars represent current from the 1st depolarization, whereas the white (rightmost) bars represent current from the 2nd depolarization. B, left: raw current signal from a bipolar cell terminal loaded with 0.5 mM EGTA and depolarized twice with 200 ms between stimuli. B, middle: raw capacitance signal from the same terminal as at left. B, right: raw capacitance signal from a different terminal that had 2 s between stimuli. C, left: raw current signal from a bipolar cell terminal loaded with 10 mM EGTA and depolarized twice with 200 ms between stimuli. C, middle: raw capacitance signal from the same terminal as at left. C, right: raw capacitance signal from a different terminal that had 2 s between stimuli. D, left: composite data from all terminals. Filled circles represent terminals loaded with 0.5 mM EGTA, whereas open triangles are terminals loaded with 10 mM EGTA. Right: expanded abscissa from data at left to show short interpulse intervals more clearly.

FIG. 8.

Simple paired-pulse depression of bipolar neurons stimulated to physiological membrane potentials. A, left: raw capacitance of bipolar neuron terminal voltage clamped at −60 mV, loaded with 0.5 mM EGTA and stimulated with two 250-ms depolarizations to −30 mV with a 50-ms interval between depolarizations. A, middle: different bipolar terminal stimulated the same as at left, only to a membrane potential of −28 mV. A, right: different bipolar terminal stimulated the same as at left, only to a membrane potential of −25 mV. B, left: raw capacitance of bipolar neuron terminal voltage clamped at −60 mV, loaded with 10 mM EGTA, and stimulated with two 250-ms depolarizations to −30 mV with a 50-ms interval between depolarizations. B, middle: different bipolar terminal stimulated the same as at left, only to a membrane potential of −28 mV. B, right: different bipolar terminal stimulated the same as at left, only to a membrane potential of −25 mV. C, left: composite capacitance jump data for all terminals loaded with 0.5 mM EGTA. The black bar (leftmost within depolarization protocol) is the capacitance jump from the 1st 250-ms depolarization, whereas the white bar (rightmost within protocol) represents the capacitance jump from the 2nd 250-ms depolarization started 50 ms after the cessation of the 1st stimulation. The 1st and 2nd depolarizations were not significantly different (P < 0.05) for any protocols. C, right: composite capacitance jump data for all terminals loaded with 10 mM EGTA. The black bar (leftmost within depolarization protocol) is the capacitance jump from the 1st 250-ms depolarization, whereas the white bar (rightmost within protocol) represents the capacitance jump from the 2nd 250-ms depolarization started 50 ms after the cessation of the 1st stimulation. The 1st and 2nd depolarizations were not significantly different (P < 0.05) for any protocols.

FIG. 9.

Exocytic rate compared with calcium channel open probability calculated using a multiple-channel model. A: linear plot of vesicle release rate compared with channel open probability (P_open) as calculated by the model, assuming release sites are equidistant from 2 (blue triangles), 5 (green triangles), 10 (red circles), or 20 (black squares) calcium channels, assuming 0.5 mM EGTA. The model calculates the calcium concentration as a function of distance from each number of channels. The traces in A are chosen to have a separation (to the nearest nm) between the channels and release sites that gave a release rate (0.37 s⁻¹) closest to our capacitance measurements at −30 mV at lowest open probability (0.014). B: log–log plot of data in A. Red lines indicate best-fit lines through the data. The slopes of the lines were the following: 1.38 for 2 channels, 1.83 for 5 channels, 2.15 for 10 channels, and 2.38 for 20 channels. C: same as in A, except model calculates release rates in the presence of 10 mM EGTA. The same channel-release site separation distances are used as in A. D: log–log plot of data in C. Lines are linear best-fit curves through the data. The slopes were the following: 1.39 for 2 channels, 1.87 for 5 channels, 2.21 for 10 channels, and 2.54 for 20 channels. Symbols in B through D are the same as in A.