A Well-Defined Readily Releasable Pool with Fixed Capacity for Storing Vesicles at Calyx of Held

Abstract

The readily releasable pool (RRP) of vesicles is a core concept in studies of presynaptic function. However, operating principles lack consensus definition and the utility for quantitative analysis has been questioned. Here we confirm that RRPs at calyces of Held from 14 to 21 day old mice have a fixed capacity for storing vesicles that is not modulated by Ca2+. Discrepancies with previous studies are explained by a dynamic flow-through pool, established during heavy use, containing vesicles that are released with low probability despite being immediately releasable. Quantitative analysis ruled out a posteriori explanations for the vesicles with low release probability, such as Ca2+-channel inactivation, and established unexpected boundary conditions for remaining alternatives. Vesicles in the flow-through pool could be incompletely primed, in which case the full sequence of priming steps downstream of recruitment to the RRP would have an average unitary rate of at least 9/s during heavy use. Alternatively, vesicles with low and high release probability could be recruited to distinct types of release sites; in this case the timing of recruitment would be similar at the two types, and the downstream transition from recruited to fully primed would be much faster. In either case, further analysis showed that activity accelerates the upstream step where vesicles are initially recruited to the RRP. Overall, our results show that the RRP can be well defined in the mathematical sense, and support the concept that the defining mechanism is a stable group of autonomous release sites.

Fig 1. Stimulus artifact elimination.

A. Sequence of examples of isolated EPSCs without KYN, in 1mM KYN, in 25μM CNQX, and the difference trace obtained by digitally subtracting the recording in CNQX from the recording in 1mM KYN. B. EPSCs during 300Hz stimulation; artifacts were removed by blanking a window of 1ms for traces in 1mM KYN and 0.5ms for the difference traces. C. Integral of sequential 3.33ms segments vs time of stimulation for traces in 1mM KYN and of the difference after subtracting matching traces in CNQX (n = 5 preparations). D. Ratio of corresponding integrals for traces in 1mM KYN and the difference traces vs time of stimulation.

Fig 2. Larger initial responses are balanced by faster induction of depression in high Ca²⁺ when stimulation is 300Hz.

A. Example traces recorded in 2mM Ca²⁺, 4mM Ca²⁺, and again in 2mM Ca²⁺; each trace is the average of 3 consecutive trials; KYN was 2mM; stimulus artifacts are blanked. The mechanism causing the rightward shifts in the time courses as individual EPSCs become smaller have been investigated elsewhere [27]. B. Integrated 3.33ms segments corresponding to the interval between action potentials vs time (n = 5). C. Cumulative plot of integrated responses showing that the total amount of release was the same; the symbols corresponding to 2mM Ca²⁺ (black circles) are the average of the blue and green symbols from Panel (B). D. Summary data showing relative sizes of 1^st responses (left), and integrals of all 45 responses (right; p < 0.001, Kolmogorov-Smirnov).

Fig 3. Frequency jump experiments.

A. Average traces from n = 16 preparations (3 trials per preparation) where the stimulation frequency was abruptly increased to 300Hz after either 500ms (blue) or 750ms (magenta) of 100Hz stimulation. Black trace is 200ms of 300Hz stimulation from the same preparations. Stimulus artifacts were blanked. Ca²⁺ was 2mM throughout; KYN was 1mM. 4 preparations included in the full analysis in the Results were excluded from these display traces because stimulation during 300Hz stimulation was 150ms instead of 200ms. B. Overlaid integrals from 3.33ms segments. The double lines during 100Hz stimulation are because $\raisebox{1ex}{$2$}\!\left/ \!\raisebox{-1ex}{$3$}\right.$ of segments contained only asynchronous responses whereas every 3^rd contained a synchronous response, which was larger. C. Overlaid integrals of 10ms segments revealing a robust increase in the release rate caused by increasing stimulation to 300Hz.D. Indices of increase in response caused by frequency jumps (see Results). Solid bars are for trials where stimulation frequency was increased to 300Hz, open bars are matched baseline values where stimulation was instead maintained at 100Hz. E. Average of traces from n = 3 preparations from frequency jump experiments where extracellular Ca²⁺ was 4mM. KYN was 2mM, explaining why the initial response sizes were not larger than in Panel (A). F. Integrals of segments for experiments in 4mM Ca²⁺analogous to Panel (C).G. Response increase after frequency jumps initiated after 500 or 750ms of 100Hz stimulation in 2mM Ca²⁺ (n = 7; segments were 3.33ms). The baseline response size during matched 100Hz stimulation was calculated by averaging three consecutive 3.33ms segments to smooth out the variation between sequential segments seen in Panel (B), and was subtracted beforehand. Colored lines are estimates of vesicle recruitment to sites newly vacated by the frequency jump: blue assumes Eq 1 with ${\widehat{\alpha }}_{fixed}=4.33/s$; green is the overestimate where bulk refilling is maximal from the start; brown is the underestimate where recruitment does not begin until all release sites have been vacated (see Results).

Fig 4. Higher p_v when 300Hz stimulation was initiated after rest vs after 100Hz stimulation.

The main point is that the mean value for p_v for vesicles within the flow-through pool during 100Hz stimulation is less than the mean for vesicles in the RRP at the start of stimulation (n = 20 preparations; data points were quantified from segments of 3.33ms throughout). A. Cumulative plot of time-integrated segments (3.33ms) when 300Hz stimulation was initiated after rest. Brown and green lines match steady state responses attributed to release of newly recruited vesicles. The brown line is back-extrapolated assuming new vesicle recruitment began only after 100ms of stimulation, making the intercept at Time = 0 an overestimate of RRP capacity. The y-axis on the left was calibrated so that the first response was 1.0, making the reciprocal of the intercept a lower bound estimate for the mean value of p_v for all vesicles within the RRP when fully replenished. The green line is back-extrapolated assuming that the bulk recruitment rate was constant from Time = 0; in this case, the reciprocal of the left y-axis intercept is an upper bound for p_v. The y-axis on the right is calibrated in absolute units (pC) to facilitate comparisons with Panel (B). B. Cumulative plot of segments when 300Hz stimulation was initiated following 500ms of 100Hz stimulation. The matching response rate from interleaved trials when stimulation was 100Hz was subtracted beforehand. The y-axis on the left was calibrated so that the first response during 300Hz stimulation was 1.0, making the reciprocal of the Time = 0 intercept (green line) an upper bound for p_v of vesicles within the RRP immediately before increasing the stimulation frequency to 300Hz; the first response during 300Hz stimulation is the last response during 100Hz stimulation. The calibration of the left y-axis is not directly comparable to the left y-axis in Panel (A), but the right y-axes are directly comparable. C. Scaled response sizes during 300Hz stimulation after rest and after frequency jumps; steady state values were subtracted before scaling.

Fig 5. Synchronous release of reluctant vesicles.

A. Overlaid segments from the entire 200ms after the frequency jump and controls where stimulation was 100Hz over a matching time window. $\raisebox{1ex}{$2$}\!\left/ \!\raisebox{-1ex}{$3$}\right.$ of segments from control trials (left) only contained asynchronous responses, which are the nearly horizontal traces in the plot. In contrast, all segments after the frequency jump (right) contained an EPSC synchronized to an action potential because stimulation was 300Hz. Arrows indicate regions that were removed because they contained residual components of the stimulus artifact when stimulation was 300Hz. B. Quantification of relative sizes of synchronous and asynchronous components of the mean response over the first 50ms at 300Hz during the frequency jump trials, and the 100Hz controls at matching times. Note that most of the transmitter continued to be released synchronously after the frequency jump (n = 7). C. Summed segments over the first 50ms after the frequency jump and the 100Hz controls at matching times. The solid red horizontal line is the baseline calculated over the 100ms before stimulation was initiated and the offsets demarcated by green boxes represent the asynchronous component. D. Quantification of relative sizes of synchronous and asynchronous components of the summed response showing that much more transmitter was released synchronously after the frequency jumps compared to controls where stimulation frequency was not increased, but was instead maintained at 100Hz.

Fig 6. Development of general model described by Eq 1.

A. RRP conceptualized as a collection of autonomous release sites (green squares). B. General model for vesicle recruitment and priming at individual release sites described by Eq 1. Release sites in the F-state (Full) contain a vesicle that is included in the RRP, whereas release sites in E-states (Empty) do not. Recruitment to the RRP at release site i is represented by the transition from E_i to F_i. Subsequent priming that increases the probability of release is represented by increasing the value for β_i over time. The general model can describe the behavior of both sequential and parallel models of vesicle priming as explained in the Results. C. A more traditional sequential model, also consistent with Eq 1, where vesicle priming progresses through discrete states on the way to full maturity.

Fig 7. Analysis of vesicle recruitment to the RRP for models covered by Eq 1.

A. Integral of segments vs time during the frequency jump experiments; segments were 3.33ms for the 300Hz trials (left panel) and 10ms for the frequency-jump (right panel). Values are directly comparable to each other because they were identically normalized by the integral of the first 3.33ms segment. Colored lines are estimates from a range of models of the response generated by release of vesicles that were recruited to the RRP to replace vesicles expended earlier during stimulation; colors match Panel (B). B. Bar graph of estimates of the fraction of the RRP released by 100Hz (solid) and subsequent 300Hz (checkerboard) stimulation for a variety of models as indicated. C. 100Hz conditioning slows RRP replenishment over subsequent rest intervals. Top: stimulus protocols. Red represents trials that included 5s of 100Hz conditioning stimulation, black (dashed) is without prior conditioning. 1s-long rest intervals were preceded by 150ms of 300Hz stimulation for both types of trials to ensure the RRP was empty at the start of the rest interval. Middle and Bottom: Responses from sample traces are plotted as integrals of 10ms segments vs time. All values were normalized by the integrated trace segment corresponding to the first 3ms of stimulation; the 2X value on the scale bar signifies 2-times the value used for normalization. Inset: Integrals of segments (3.33ms) during 300Hz stimulation after the rest interval for both types of trials. Note reduced response after conditioning (red trace). Scale bar in inset is 100ms by 0.5-times the value used for normalization. D. Estimates of ${\widehat{\alpha }}_t$ from Eq 1 that were used to incorporate activity-dependent acceleration and later fatigue into models of vesicle recruitment. The black line is the estimate used for trials where stimulation was 300Hz throughout and the red is for the frequency jump trials. The exponential constant for acceleration (τ ⋅ ν in Eq 2) was 10 action potentials for 100Hz and 300Hz stimulation alike, and fatigue was modeled as a 10%/s linear decrease. The dashed section of the black line is an extrapolation that was not tested empirically.

Fig 8. Recovery from depression during rest intervals.

A. Incomplete RRP replenishment in 1s. Stimulation was pairs of 150ms-long trains at 300Hz separated by 1s rest intervals as diagrammed at top. Top trace: Averaged raw data across all experiments after blanking stimulation artifacts; trace is colored magenta during the second train to match the panels below. Bottom left: Quantification; responses were divided into 3.33ms segments, integrated, normalized by the value for the first segment, and averaged across trials (black is first train, magenta is second). Bottom right: Overlaid traces from the first 10ms of each train illustrate slightly more paired-pulse facilitation during the second train. B. Integrated segments (10ms) for a series of experiments with a range of inter-train intervals. Data are from a single preparation; multiple traces for each inter-train interval were averaged digitally before segmentation and integration. C. Time course of recovery from depression (n ≥ 14 trials from 7 preparations). Circles (black) are the integral of the entire second train divided by the integral of the first train. Squares (green) are the integral of the first 3.33ms segment of the second train divided by the first segment of the first train. The Y-axis is calibrated so that recovery was 0 when the inter-train interval was 0. The values in parentheses are uncorrected integral of the second train divided by the integral of the first; the value for inter-train intervals of 0 was 0.44, even though the RRP was empty, because of ongoing release of newly recruited neurotransmitter during stimulation. The dashed line (magenta) is Eq 3 in the Results. D. Time course of decay of the small enhancement in the paired-pulse ratio caused by prior stimulation. The dashed line is the single exponential $e^{\frac{-t}{\tau }}$ where τ = 2s.

Fig 9. Correlations between steady state supply of reluctant vesicles and short-term plasticity during submaximal stimulation.

Data are color coded by age: magenta circles are from animals aged 14–15 days; green squares were 16–18 days; and blue x’s were 19–21 days. A. Steady state unreleased fraction of the RRP remaining after 100Hz stimulation vs the paired-pulse ratio when synapses were rested (top panel) and vs the half-decay time for responses during the preceding 100Hz stimulation (bottom panel). The steady state fraction was calculated without theory by dividing the cumulative response after the frequency jump by the cumulative response during trials where stimulation was 300Hz from the start. Half-decay time was estimated as the 50% point in the cumulative response when stimulation was 100Hz from the start. In both cases, cumulative responses were calculated after subtracting the steady state (green lines in Fig 7A); calculating the steady state unreleased fraction instead as the index generated for Fig 3D yielded a similar result. Straight lines are best fits; p < 0.001 for both panels. B. Capacity of the low p_v subdivision of the RRP assuming the parallel model of vesicle recruitment vs the same measures of short-term plasticity used in Panel (A). The procedure factors out variation in new vesicle recruitment and release (see Lemma 7; upper bound for ${\widehat{\alpha }}_{LpD,ss100}$ was used; one experiment was excluded because the recording quality was too low to estimate the steady state response size, which is required for calculating ${\widehat{\alpha }}_{LpD,ss100}$; p < 0.01 for both correlations; R² = 64% for the top panel, and 50% for the lower).