Limit on the role of activity in controlling the release-ready supply of synaptic vesicles

Abstract

Typical fast chemical synapses in the brain weaken transiently during normal high-frequency use after expending their presynaptic supply of release-ready vesicles. Although it takes several seconds for the readily releasable pool (RRP) to refill during periods of rest, it has been suggested that the replenishment process may be orders of magnitude faster when synapses are active. Here, we measure this replenishment rate at active Schaffer collateral terminals by determining the maximum rate of release that can still be elicited when the RRP is almost completely exhausted. On average, we find that spent vesicles are replaced at a maximum unitary rate of 0.24/sec during periods of intense activity. Because the replenishment rate is similar during subsequent periods of rest, we conclude that no special mechanism accelerates the mobilization of neurotransmitter in active terminals beyond the previously reported, several-fold, residual calcium-driven modulation that persists for several seconds after bouts of intense synaptic activity. In the course of this analysis, we provide new evidence supporting the hypothesis that a simple enzymatic step limits the rate at which reserve synaptic vesicles become ready to undergo exocytosis.

Fig. 1.

The RRP is emptied by 60 action potentials at 20 Hz. A, Schaffer collaterals were stimulated 60 times at 20 Hz and then 21 more times either at 40 or 20 Hz while postsynaptic responses were recorded by patch clamping CA1 pyramidal neuron somata. Fluctuations caused by the quantal nature of transmitter release, including frequent transmission failures, dominated the recordings during the 40 Hz stimulation, and so traces were averaged across all experiments before being measured (each point represents the average of 18 trials from 4 slices). The synaptic response sizes were binned in groups of three, normalized by the size of the first response, and plotted versus the stimulus number.Diamonds represent responses of synapses stimulated at 20 Hz throughout; circles are from trials where the frequency was doubled. The filled circles represent 20 Hz responses; the open circles are 40 Hz responses. The value of the first bin is greater than 1 because of the short-term enhancement apparent during the first several responses. The top gray dashed line matches the steady-state response size for the last 21 responses when recorded at 20 Hz; the bottom line is drawn at exactly half that. Note that during the 40 Hz stimulation, the synaptic strength quickly settled to half that recorded at 20 Hz, matching the bottom dashed line and indicating that the RRP is left exhausted by the first 60 action potentials. Inset, The averages of the first 10 individual responses at 20 Hz, responses numbered 51–60 at 20 Hz, and the first 10 responses at 40 Hz are all scaled by their peak size and overlaid. Because the individual responses evoked at 40 Hz do not decay away completely in the 25 msec interstimulus interval, the corresponding tail of the average 20 Hz response was first scaled to match the prestimulus artifact baseline and then subtracted from the average 40 Hz response. Note that the shape of the EPSC did not change during the experiment. B, The stimulus intensity was set within the narrow threshold window as described in Results, and antidromic action potentials were recorded in CA3 pyramidal neurons as Schaffer collaterals were stimulated repetitively for 3 sec at 20 Hz and then for 1 sec at 40 Hz. The average probability of action potential firing given a stimulus (47 experiments, 2 neurons) was calculated for each second of stimulation and plotted versus time. Note that the firing probability did not change when the stimulus frequency was doubled (compare bars 3 and4).

Fig. 2.

Readily releasable vesicles are replaced slowly during 20 Hz stimulation. A, Average relative synaptic strength is plotted as the Schaffer collaterals are stimulated at 20 Hz for 4 sec (each response was normalized by the size of the response to the first stimulus; experiments are from 14 slices). Thedashed gray line represents the fraction of the synaptic response that is predicted to result from the exocytosis of vesicles that became available for release during the experiment (as derived in the ). B, Working model of RRP replenishment.Left panel, In the resting synapse the spontaneous rate of exocytosis is low, and the RRP fills completely. The pool has a maximum capacity, and the high-energy barrier that keeps vesicles from fusing spontaneously is the rate-limiting element that controls how quickly synaptic vesicles undergo exocytosis. Right panel, Episodes of high-frequency activity drive exocytosis quickly enough to exhaust the RRP. With the pool empty, the rate at which new vesicles are made available limits the rate of transmitter release; the energy barrier to fusion prevents vesicles from undergoing exocytosis in the interval between action potentials, but as long as the high-frequency spike train continues, the barrier is no longer the rate-limiting element in the exocytic/endocytic cycle. This model accounts only for rate-limiting steps in the exocytic/endocytic cycle during the first several seconds of heavy use. Thereafter an additional element plays a role in controlling the dynamics of neurotransmitter mobilization. Endocytosis does not play a rate-limiting role in this scheme and is not represented here. C, The simultaneous solutions for Equations 1A and 3A () are plotted for the data shown in A. The two independent equations relate the rate constant of pool replenishment to the fraction of the full pool triggered to release by isolated action potentials (fusion efficiency). When the corresponding values for the two parameters are plotted against each other, the resulting lines intersect where the refilling rate constant equals 0.24/sec and the initial fusion efficiency is 0.044. Equation 1A depends only on the steady-state response size after the pool has reached a near-empty steady state and is represented by the gray diagonal line. Equation 3A depends on the changing rate at which transmitter was released over the complete course of the experiment and is represented by the black line. D, The initial fusion efficiency and the pool replenishment rate constant were estimated separately by simultaneously solving Equations 1A and 3A for each of the 14 experiments summarized in A and plotted versus each other. Note that there is no evident correlation between these two parameters.

Fig. 3.

The RRP replenishment time course during periods of rest is predicted by the refreshment rate when synapses are active. The RRP was emptied with 80 action potentials (20 Hz), and subsequent recovery was monitored after an experimentally varied delay with an identical burst of stimulation (diagrammed at top). The sums of the responses to the second stimulus train were normalized as described in Results and plotted against the recovery interval. The curve is the predicted RRP refilling time course calculated with Equation 2 from the replenishment rate measured when the synapses were active (0.30/sec for these synapses; mean ± SEM; 9 cells, at least 10 trials for each point). Inset, Average synaptic strengths of the responses elicited during the first and second stimulus trains for the 2 sec recovery interval are plotted versus time during the trial (filled circles represent the responses that were used to estimate the RRP recovery after 2 sec of rest, i.e., the first 60 responses of each stimulus train).

Fig. 4.

The capacity of the RRP does not depend on the measurement protocol. A, Short-term depression was induced with 80 stimuli (20 Hz) with either 2.5 mm Ca/2.5 mm Mg (squares) in the bath or 4.5 Ca/0.5 Mg (circles) at the same synapses. i, Responses were normalized by the average size of the first response recorded at the lower calcium concentration and plotted versus stimulus number (3 slices, 7 trials for each). ii, The RRP replenishment rate constant and the initial fusion efficiency were calculated by simultaneously solving Equations 1A and 3A, as in Figure2C. The gray lines represent the analysis of responses evoked under the high calcium condition; the black lines are for the lower calcium experiments. The dashed lines represent the solutions for Equation 1A; the solid lines represent Equation 3A. Points of intersection represent the common solutions. Note that, as predicted, the fusion efficiency in high calcium is greater than the fusion efficiency in low calcium by a factor similar to the amount of enhancement in synaptic strength observed after switching into the higher calcium-containing solution.B, Depression was induced with 81 stimuli at 20 and 40 Hz for the same synapses. i, The response sizes were normalized by the average size of the first responses in the stimulus trains and plotted versus stimulus number for both sets of trials (3 slices, 11 trials for each). ii, Simultaneous solution of Equations 1A (dashed lines) and 3A (solid lines). The gray lines represent the analysis of the 40 Hz responses; black lines represent the analysis of 20 Hz responses.

Fig. 5.

The working model predicts the synaptic response settling time course when the stimulation frequency is doubled after the RRP has been emptied. The circles represent the relative synaptic strength during the 40 Hz stimulation (also plotted on a smaller scale as open circles in Fig.1A). The dashed line is the prediction generated by Equation 1 in the and depends on the integral of the small transient increase in the overall rate of exocytosis that occurs after switching frequencies from 20 to 40 Hz.

Fig. 6.

CTZ and KYN do not affect short-term depression. Ai, Forty Hertz stimulation. Schaffer collaterals were stimulated 80 times at 40 Hz in the presence and absence of 100 μm CTZ. Average responses to the stimulus trains with (gray traces) and without (black traces; scaled by 1.12) CTZ are plotted (data are from 4 slices, 18 trials for each). The first six responses are plotted in the top panel. Note that CTZ amplified the first response slightly more than the rest, possibly because of presynaptic side effects of the drug. The entire electrophysiological recordings (the trace gathered in the absence of drug is scaled by 1.12), with the stimulus artifacts blanked (2 msec windows), are plotted in thebottom panel. Aii, Twenty Hertz and frequency switching. Schaffer collaterals were stimulated 60 times at 20 Hz and then 21 more times at 40 Hz as in Figure1A in the presence and absence of 100 μm CTZ. The size of the synaptic response to each stimulus was normalized by the average size of the first control response (no drug). The size of each response recorded in CTZ is plotted against the size of the corresponding control response to the same stimulus number (open circles represent responses at 40 Hz; filled circles are 20 Hz responses). Thedashed gray line is straight, with a slope of 1.12 (3 slices, 10 trials each). Inset, The average current traces representing the responses to the 20 stimuli preceding and after the frequency switch for each condition are overlaid (the response recorded in the absence of drug was scaled by 1.12; the larger of the two sets of current deflections represents responses recorded at 20 Hz; the baseline of the 40 Hz response average was calculated and subtracted as for the inset of Fig.1A). Note that the scaled responses with and without drug are identical and that the average 40 Hz response size is approximately half that recorded at 20 Hz. B, Schaffer collaterals were stimulated at least 80 times at 40 Hz alternately in the presence and absence of KYN (300 μm: 3 slices, 22 trials each; 1000 μm: 3 slices, 10 trials each). The normalized sizes of the control responses are plotted against the corresponding sizes of the responses gathered with KYN in the bath for each stimulus (i, 300 μm KYN;ii, 1000 μm KYN). The dashed lines are straight with slopes of 0.34 (i) and 0.22 (ii).

Fig. 7.

Individual synapses behave like simultaneously activated populations. Schaffer collaterals were stimulated with pairs of 4-sec-long spike trains (20 Hz) separated by 2 sec intervals. For half of the trials, only a minimal number of afferents were stimulated throughout. For the other half, stimuli of normal intensity were interleaved with the weak stimuli.A, Typical, sequential raw data example of a failure, a large response, and a minimal response during interleaved stimulation.B, C, The average sizes (B) and probability (C) of successful transmissions in response to the minimal stimuli were averaged into 200 msec bins and plotted versus the time of the experiment. The squares represent the interleaved minimal responses; the circles summarize experiments in which only minimal stimuli were used throughout.