 |
||||
|
||||
|
||||
|
||||
Previous Article | Next Article
Volume 16, Number 18, Issue of September 15, 1996 pp. 5661-5671
Copyright ©1996 Society for NeuroscienceDeterminants of the Time Course of Facilitation at the Granule Cell to Purkinje Cell Synapse
Pradeep P. Atluri and Wade G. Regehr Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
Short-term facilitation is a widely observed form of synaptic enhancement that is not well understood. Although presynaptic calcium has long been implicated in this process, its role is unclear, particularly at synapses in the mammalian brain. We tested the role of presynaptic residual free calcium ([Ca]res) in facilitation of synapses between granule cells and Purkinje cells in rat cerebellar slices. Paired-pulse facilitation of synaptic currents resulted in an approximately 2.5-fold enhancement that decayed with a time constant of ~200 msec, as assessed by voltage-clamp recordings. Measurements of [Ca]res using fluorescent calcium-sensitive indicators revealed that [Ca]res decayed more rapidly than did facilitation. Manipulation of [Ca]res dynamics by introducing EGTA into presynaptic terminals sped the decays of [Ca]res and facilitation in a dose-dependent manner. When [Ca]res was reduced to a brief impulse lasting several milliseconds, facilitation was still present, although reduced in amplitude and duration. Facilitation decayed with an intrinsic time constant of ~40 msec. These results suggest that facilitation at this synapse is produced by a calcium-driven process with a high affinity and a slow effective off-rate. A combination of [Ca]res dynamics and the properties of a calcium-driven reaction determine the time course and amplitude of facilitation.
Key words: synaptic transmission; parallel fiber; presynaptic terminal; short-term enhancement; calcium; magnesium green; cerebellum
INTRODUCTION
At many synapses, when two action potentials depolarize a presynaptic bouton in rapid succession, the second action potential releases more neurotransmitter than the first (Magleby, 1987; Zucker, 1989
We are particularly interested in elucidating the mechanism of facilitation of synapses in the mammalian CNS. Residual free calcium ([Ca]res) that persists in the presynaptic terminal after termination of release has long been implicated in facilitation at synapses with large presynaptic terminals, such as the neuromuscular junction (NMJ) and the squid giant synapse (Katz and Miledi, 1968
The rapid spatial equilibration of calcium in these small boutons confers an important technical advantage for studying the role of calcium in facilitation: the ability to measure [Ca]res dynamics on a tens of milliseconds time scale using calcium-sensitive fluorescent indicators. This has not been possible at the NMJ and squid synapses, because direct measurement of the time course of [Ca]res cannot be made until calcium has spatially equilibrated by buffered diffusion. A conservative estimate of the equilibration time for calcium in a presynaptic bouton of radius r can be obtained using the equation for the characteristic diffusion time of
ch = r2/6D, and using a rather small effective diffusion coefficient for calcium, D, of 1 × 10
7 cm2/sec. We estimate the equilibration time of calcium to be ~3 msec for a 0.8-µm-diameter bouton, such as at the granule cell
Purkinje cell synapse, and 200 msec for a 7-µm-diameter terminal, such as at the crayfish NMJ.
Using modeling and indirect measures of [Ca]res dynamics, several hypotheses concerning the role of calcium in facilitation have emerged from the study of the NMJ and squid synapses (Charlton et al., 1982
According to the simplest form of the residual calcium hypothesis, in facilitation calcium acts at the same low-affinity calcium binding sites involved in triggering release. In this model, the very high (>100 µM) submembrane calcium levels that trigger release dissipate by diffusion into the terminal, and calcium unbinds rapidly from the low-affinity trigger sites to terminate release of neurotransmitter (Zucker, 1989
Another possibility is that [Ca]res acts at a site with a higher calcium affinity than those involved in triggering release (Stanley, 1986
Less is known about the role of calcium in setting the time course of facilitation at synapses with small presynaptic boutons. In frog tectal synapses, the magnitude but not the time course has been shown to be influenced by changes in [Ca]res (Feller et al., 1996
Here we examine the issue of what sets the time course of paired-pulse facilitation at the synapse between cerebellar granule cells and Purkinje cells. By using fast low-affinity calcium indicators, we are able to track changes in [Ca]res on a time scale appropriate for the study of facilitation (Regehr and Atluri, 1995
MATERIALS AND METHODS
Transverse slices (300 µm thick) were cut from the cerebellar vermis of 11- to 14-d-old rats, as described previously. Experiments were conducted at 24 ± 0.5°C, except for the experiments associated with Figure 3B, which were performed at 34 ± 0.5°C. The external solution (2 ml/min flow rate) consisted of (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 25 glucose, and 0.02 bicuculline, bubbled with 95% O2 and 5% CO2.
Fig. 3. Comparison of the decay times of calcium and facilitation for control conditions. The time courses of synaptic facilitation and normalizedF/F changes produced by single parallel fiber stimulation are shown for 24°C (A) and 34°C (B).
fac). Fit parameters {C0, C1,
[View Larger Version of this Image (20K GIF file)]
For EGTA wash-in experiments, a 100 mM stock solution of EGTA-AM in dimethyl sulfoxide (DMSO) was aliquoted and frozen. Immediately before use, aliquots were diluted in external saline to final EGTA concentrations (in µM) of 100, 20, or 1, and DMSO concentrations (%) of 0.1, 0.02, and 0.001. Wash-ins were 15 min long, with flow rates of 2 ml/min. To control for the effects of DMSO on facilitation, control experiments were performed with wash-in of DMSO alone. DMSO had no significant effect on the time course of facilitation in these experiments. Some experiments were also performed in which DMSO was present in the external solution, bathing the slice both before and after application of EGTA.
Detecting presynaptic calcium transients. Parallel fibers, made up of granule cell axons and presynaptic terminals, were labeled by local application of a solution containing the membrane-permeant forms of calcium indicators, as described previously (Regehr and Tank, 1991
The filter set that was used for fura-2 and mag-fura-5 was a 380 HT 15 excitation, a 430DCLP02 dichroic, and a 510WB40 emission filter (Omega Optical, Brattleboro, VT), and for magnesium green, a 450-490 excitation, an FT510 dichroic, and an LP520 emission filter (Zeiss, Thornwood, NY). Fluorescence was detected with a photomultiplier tube (Hamamatsu HC124-06MOD; Hamamatsu, Bridgewater, NJ). Because calcium increases produce a decrease in fluorescence of fura-2 and mag-fura-5 with 380 nm excitation,
Fig. 2. Detection of calcium transients evoked by single stimuli. Time courses of
[View Larger Version of this Image (18K GIF file)]
Correction of fura-2 traces. As a consequence of its high affinity, fura-2 is known to distort the large calcium transients that occur in skeletal muscle (Konishi et al., 1988
where K* and (
(1) where c is a constant.
![[Ca]<SUB>res</SUB>=c <FR><NU>&Dgr;F</NU><DE>F</DE></FR> <FR><NU>K*</NU><DE><FENCE>(&Dgr;F/F)<SUB>max</SUB>−&Dgr;F/F</FENCE></DE></FR>,](/content/vol16/issue18/fulltext/5661/img002.gif)
(2) The similarity of the time course of calcium decay for the corrected fura-2 transients and the low-affinity calcium indicators (see Fig. 2) supports the view that the time course of [Ca]res decay is reported faithfully by mag-fura-5 and magnesium green. Although the procedure described above corrects for the distortion produced by steady-state saturation of the indicator, it does not correct for dye kinetics (Regehr and Atluri, 1995
Measuring synaptic currents. Whole-cell recordings of Purkinje neurons were obtained as described previously (Regehr and Mintz, 1994
glass pipettes containing an internal solution of (in mM): 35 CsF, 100 CsCl, 10 EGTA, 10 HEPES, and 0.1 D600, pH 7.3 with CsOH. The access resistance (<5 M
200 pA) were monitored continuously.
Parallel fibers were stimulated extracellularly with a bipolar electrode placed in the molecular layer several hundred micrometers from the recording electrode. The resulting excitatory postsynaptic current (EPSC) decayed with a time constant of 5-7 msec at 24°C and 3-5 msec at 34°C. Low stimulus intensities were used to keep synaptic currents small and the resulting voltage errors arising from uncompensated series resistance below 4 mV.
Facilitation was computed for each trial according to the equation 100 × (A2
30 msec), the test EPSC was superimposed on the conditioning EPSC. For these trials, the waveform of the conditioning EPSC was subtracted from the superimposed test EPSC before determination of the maximum amplitude of the test EPSC.
Data acquisition and analysis. Outputs of the photomultiplier tube and Axopatch 200A were filtered at 1 kHz with a model 900C9L8L 8-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized with a 16-bit D/A converter (Instrutech, Great Neck, NY), Pulse Control software (Herrington and Bookman, 1995
RESULTS
Facilitation and presynaptic calcium for control conditions
The synapse between cerebellar granule cells and Purkinje cells exhibits robust paired-pulse facilitation, as shown in Figure 1. Synaptic currents evoked by extracellular stimulation of parallel fibers were measured with whole-cell recordings from the soma of cerebellar Purkinje cells (Perkel et al., 1990
Fig. 1. Paired-pulse facilitation at the granule cell to Purkinje cell synapse. Percentage facilitation as a function of interstimulus interval. Points are averages of 19 trials ± SEM. Inset, Synaptic currents evoked by extracellular stimulation with stimulus pulses separated by
[View Larger Version of this Image (15K GIF file)]
To investigate the role of [Ca]res in facilitation, we sought to obtain a measure of [Ca]res in parallel fiber presynaptic terminals on the time scale of facilitation, namely in the 1 sec interval after stimulation. Parallel fiber tracts were labeled with calcium indicators as described in Materials and Methods. We have shown previously that indicators with low calcium affinities, such as mag-fura-5 (Claflin et al., 1994
On the basis of anatomical considerations, such
We also considered the possibility of differential distribution of calcium channels and extrusion mechanisms on the axon relative to the presynaptic terminals. If calcium channels and extrusion mechanisms were both located preferentially on axonal segments, then axonal signals could contribute a rapid component to the fluorescence transient, which would make the fluorescence changes we detect difficult to interpret. This is unlikely, because calcium channels and extrusion mechanisms are generally distributed preferentially in presynaptic terminals relative to axons (Cohen et al., 1991
1A calcium channel subunit (Westenbroek et al., 1995
To compare the time course of calcium decay to the time course of facilitation, it seemed most natural to compare fits to exponential decays. Fits of the fluorescence transients to single exponential decays provide a crude measure of the decay time of calcium, with a control time constant of ~90 msec over the range 10 msec to 1 sec; however, the decay of calcium was not very well approximated by a single exponential, and the decay could not be fit reliably by the sum of two distinct exponentials. Half-decay times (t1/2), the time for [Ca]res to decay to 50% of peak elevation, are useful measures of the decay time of calcium; however, they do not fully describe calcium dynamics and cannot be directly related to time constants of exponential decay. Therefore, to avoid assumptions about the form of the decays, we directly compared the time course of facilitation and residual calcium simply by plotting them on the same graph, as in Figure 3.
After single stimuli, [Ca]res decayed more rapidly than did facilitation. Figure 3A shows that at 24°C the decay of facilitation, fit by a single exponential with
These results have important implications for the role of calcium in facilitation. They are inconsistent with the simplest form of the residual calcium hypothesis for paired-pulse facilitation, in which the calcium sensor that triggers release also mediates facilitation. That model predicts that facilitation decays at the same rate as or faster than [Ca]res. Instead, we see that calcium decays more rapidly than facilitation.
Altering presynaptic calcium transients with EGTA
To pursue further the role of [Ca]res in paired-pulse facilitation, we decided to manipulate the time course of [Ca]res and test its effect on facilitation. As described in Materials and Methods and the Appendix (see Fig. 10), by introducing EGTA into presynaptic boutons we were able to speed the decay of [Ca]res (Adler et al., 1991
Fig. 10. Simulations of the effect of EGTA on [Ca]res dynamics. A, Calcium transients were simulated as described in the text for control conditions and after the addition of 0.3, 1.3, and 10 mM EGTA. The t1/2 of simulated transients is 40, 23, 10, and 2 msec, respectively. Peak amplitudes were reduced to 98, 92, and 67% of control, respectively. The t1/2 for Ca transients was similar to the half-rise times (21, 9, and 2 msec) of the Ca-EGTA complex (B) for low, medium, and high concentrations of EGTA. The
[View Larger Version of this Image (16K GIF file)]
Figure 4 shows a series of experiments in which successively larger concentrations of EGTA-AM, the membrane-permeant form of EGTA, were bath-applied for 15 min to parallel fibers. The time courses and amplitudes of [Ca]res elevations were monitored with magnesium green (Fig. 4, left panels), with averages of fluorescence transients before and after application of EGTA-AM shown in the right panels; 1 µM EGTA-AM accelerated the t1/2 of [Ca]res transients from 36 msec to 16 msec with no significant effect on peak [Ca]res levels (Fig. 4A). Increasing the concentration of presynaptic EGTA with 20 µM EGTA-AM led to a more pronounced acceleration of the t1/2 of [Ca]res (32 msec to 6 msec), with little if any effect on peak [Ca]res levels (Fig. 4B). Higher concentrations of EGTA-AM (100 µM) sped the t1/2 from 33 msec to 2 msec (Fig. 4C) and reduced peak
Fig. 4. Speeding the decay of calcium with EGTA-AM. The effects of 1 µM (A), 20 µM (B), and 100 µM EGTA-AM (C) on the peaks (solid circles) and time constants of decay (open circles) of the stimulus-evoked magnesium green
[View Larger Version of this Image (32K GIF file)]
A summary of the effects of 15 min applications of EGTA-AM on parallel fiber [Ca]res transients is shown in Figure 5. The t1/2 for control, 1 µM, 20 µM, and 100 µM were 39 ± 1 msec (n = 20), 23 ± 2 msec (n = 5), 10 ± 1 msec (n = 5), and 2 ± 0.2 msec (n = 5), respectively; 20 µM EGTA-AM caused a small 8 ± 4% decrease in peak amplitude, whereas 100 µM EGTA-AM decreased peak amplitude by 45 ± 4%. These experiments demonstrate that EGTA accelerates the decay of [Ca]res transients in a dose-dependent manner and at higher concentrations begins to attenuate the peak of the [Ca]res transient.
Fig. 5. Summary of the effect of EGTA loading on calcium transients.
[View Larger Version of this Image (25K GIF file)]
Effect of rapid presynaptic calcium dynamics on facilitation
We then assessed the effect of altered calcium dynamics on facilitation. Parallel fibers were stimulated every 15 sec while the interpulse interval
Fig. 6. Altering calcium dynamics changes the time course of facilitation. Facilitation before (open circles) and after (solid circles) 15 min applications of 0.1% DMSO and 0 EGTA-AM (A), 1 µM EGTA-AM (B), 20 µM EGTA-AM (C), and 100 µM EGTA-AM (D). Each point is the mean of two to four trials. The fits shown are to functions of the form C0 + C1e
[View Larger Version of this Image (31K GIF file)]
Although EGTA-AM will load all structures within the slice, including presynaptic terminals and Purkinje cells, it is highly likely that the effect of EGTA-AM on facilitation is a consequence of altered granule cell presynaptic calcium dynamics. Measurements of synaptic currents were made with whole-cell patch pipettes, which dialyzed the contents of the Purkinje cells with 10 mM EGTA; thus, EGTA-AM loading is unlikely to change significantly calcium buffering within recorded Purkinje cells.
Experiments such as those presented in Figure 6 demonstrate the effects of EGTA-AM on recordings from single cells. Because of the length of these experiments (
1 hr), we were unable to avoid small increases in access resistance. To minimize the effects of changes in access resistance, we used very low resistance electrodes (0.9-1.5 M
To quantitate better the effect of EGTA-AM on facilitation, we also recorded facilitation curves in cells that had been exposed to EGTA-AM, without measuring the control facilitation curves in the same cells. Such experiments had the advantage that more trials could be performed at a constant series resistance, allowing a better estimate of the time course of facilitation. To overcome cell-to-cell variability, we recorded facilitation curves for many cells, with averages shown in Figure 7. The amplitude of facilitation went from 153 ± 11% (n = 15) in control conditions to 159 ± 25% (n = 14), 74 ± 8% (n = 9), and 70 ± 10% (n = 10), respectively, for applications of 1 µM, 20 µM, or 100 µM EGTA-AM. As is seen most clearly in Figure 7B, the time constant of decay of facilitation was also affected, declining from a control value of 203 ± 18 msec to 138 ± 19 msec, 85 ± 11 msec, and 52 ± 4 msec for 1 µM, 20 µM, or 100 µM EGTA-AM, respectively.
Fig. 7. Summary of the effect of EGTA-loading on facilitation. Averages of non-normalized (A) and normalized (B) facilitation in control solution and after treatment with 1, 20, or 100 µM EGTA-AM. For normalized curves, the facilitation curves from each cell were fit with a single exponential (which was not constrained to decay to 0), normalized to decay from 1 to 0, and then averaged with the other cells. Inset in B shows the same curves on an expanded time scale. Fit parameters for control, 1, 20, and 100 µM EGTA-AM in A were {2, 160, 184}, {5, 162, 114}, {
[View Larger Version of this Image (26K GIF file)]
Figure 8 compares the time course of [Ca]res transients with the time course of facilitation for control conditions and after altering [Ca]res dynamics. For all of our experimental conditions, [Ca]res decayed more rapidly than did facilitation (Fig. 8A). As shown in Figure 8B, increasing the decay rate of [Ca]res in turn speeds the decay of facilitation.
Fig. 8. Comparison of calcium transients and facilitation. A, Time course of calcium transients (solid traces) and facilitation (open circles) for control conditions and after altering calcium dynamics with 15 min applications of 1, 20, and 100 µM EGTA-AM. Both calcium transients and facilitation from EGTA-AM experiments have been normalized to peaks from control conditions. Smooth curves are fits to the average facilitation. B, The time constant of decay of facilitation is plotted as a function of the t1/2 of calcium decay. Each point is the average ± SEM determined from 15, 14, 9, and 10 experiments, respectively, for facilitation, and 20, 5, 5, and 5 experiments for calcium.
[View Larger Version of this Image (16K GIF file)]
Simulations of calcium-activated facilitation
In considering the mechanisms underlying facilitation, we explored the following general reaction scheme, in which calcium binds to a receptor, X, to produce CaX, whose concentration is directly proportional to facilitation, where k+ and kThe relationship between [Ca]resand facilitation could be accounted for by a simple model (Fig. 9, solid traces) that assumes facilitation is the result of a second-order calcium-dependent reaction with one-to-one binding, a k+of 1.5 × 108M
(3) 
Fig. 9. Comparison of experimental and simulated facilitation. Experimentally determined and simulated time courses of facilitation for control conditions (solid circles, solid line) and after altering calcium dynamics with 15 min applications of 1 µM EGTA-AM (open circles, dotted line), 20 µM EGTA-AM (solid squares, solid line), and 100 µM EGTA-AM (open squares, dotted line). The simulations used the following properties for the facilitating molecule: k
[View Larger Version of this Image (17K GIF file)]
The purpose of these simulations was to examine the feasibility of the involvement in facilitation of a high-affinity calcium binding site, and to test the range of parameters that adequately relate the observed calcium transients and facilitation. The strategy was to impose a [Ca]res transient with an amplitude of 200 nM for control conditions (as estimated previously) (Regehr and Atluri, 1995
(
Considerations of models of calcium transients within cells suggested a likely explanation for the inadequacies of the simulations described above. In models that take into account the spatial gradients of calcium on rapid time scales, it is apparent that calcium levels can be extremely high near open calcium channels (Simon and Llinas, 1985
DISCUSSION
Facilitation at the granule cell
Our experiments indicate that [Ca]res plays an important role in facilitation. Speeding the decay of [Ca]res levels with EGTA-AM (Figs. 4, 5) accelerated the decay of facilitation (Figs. 6, 7) in a dose-dependent manner. The application of 1 µM EGTA-AM increased the decay rate of both [Ca]res and facilitation without affecting their peak levels. Higher concentrations of EGTA-AM (20 µM) also left peak [Ca]res largely unaffected, whereas they further accelerated the decay of [Ca]res. For these conditions the peak amplitude and duration of facilitation both decreased. We have shown previously that a single stimulus results in a peak increase in [Ca]res of 200-300 nM (Regehr and Atluri, 1995There is also a component of facilitation that persists after [Ca]res has returned to resting levels. This is most apparent after treating presynaptic terminals with high (100 µM) concentrations of EGTA-AM, which speeds the decay of [Ca]res to such an extent that it becomes a brief impulse lasting only a few milliseconds. Facilitation is still present for such conditions, and persists for tens of milliseconds after a conditioning pulse.
Taken together, our results establish that the time course of facilitation is set in part by the decay of [Ca]res and in part by the slow kinetics of either calcium binding or of the calcium-activated process that produces facilitation.
Simulations of facilitation
The simulations of facilitation (Fig. 9) demonstrate that a model for facilitation based on a calcium-driven reaction with second-order kinetics provides an adequate description of facilitation at this synapse. One particularly interesting aspect of these simulations is that it is possible to think in terms of two components of facilitation, one driven by residual calcium and another driven by locally high levels of calcium that our calcium measurement techniques cannot detect (see Materials and Methods). Facilitation curves in conditions in which residual calcium decays extremely rapidly (Fig. 8A) (100 µM EGTA-AM experiments) allow us to estimate the intrinsic time constant of facilitation for our experimental conditions to be ~40 msec. According to reaction scheme (3) above, this intrinsic time constant reflects the reverse rate constant of calcium binding, which is given by (In this model, facilitation decay lags behind [Ca]res decay for two reasons. The first reason is the slow kinetics of calcium dissociation, which determines the time course of facilitation when residual calcium decays very rapidly. The second reason to expect facilitation to lag behind Ca is saturation of its receptor X, which significantly slows the decay of facilitation in control conditions. When X is nearly saturated, an increase in [Ca]res produces a very small increase in [CaX]. Conversely, for a given decrease in [Ca]res, a much smaller decrease in [CaX] occurs if X is nearly saturated than would occur if X were far from saturation. If the KD of X is comparable in magnitude to the 200-300 nM Ca elevations produced in these terminals by single action potentials, then the saturation of X may contribute to the observed differences between the time course of facilitation and that of [Ca]res transients. In this model, the slow decay of facilitation owing to saturation of X is analogous to the slow decay of the fluorescence transients reported by high-affinity dyes such as fura-2 (Fig. 2C and Materials and Methods) (Regehr and Atluri, 1995
According to the model presented at the end of the Results section, there is significant calcium binding to the receptor X even at resting calcium levels, because the KD for the calcium binding site is just 167 nM. In the simulations of Figure 9, which assumed a resting calcium of 40 nM, [CaX]/[Xtotal] went from 0.19 to 0.48, an increase of 150%. The amplitude of this increase is about the same as the magnitude of facilitation for these conditions, suggesting that unfacilitated release may also depend on calcium binding to X.
It must be stressed that the working model based on a calcium-driven reaction with simple kinetics (Fig. 9) is still preliminary and that additional experiments will be required to unravel some of the details of the role of calcium in facilitation. One deficiency of the model is that for control conditions elevations of [Ca]res seem to outlive facilitation (Fig. 3A for interpulse intervals >500 msec). This may reflect small contributions of additional processes, such as depression, to use-dependent changes in synaptic strength. In addition, other models, which are slightly more complex, can also describe our data. For example, consider the following general reaction scheme, in which calcium first binds to its receptor X to produce CaX, which then undergoes an activation reaction to produce CaX*, a species directly proportional to facilitation:
where k+1, k
(4) Comparison to other synapses
It is instructive to compare the role of [Ca]res in facilitation at the granule cell to Purkinje cell synapse with that described previously at other synapses. It seems that a high-affinity calcium-binding site distinct from that involved in triggering release is involved in facilitation for the crayfish NMJ and frog tectal synapses (Yamada and Zucker, 1992Important features of facilitation, however, are not universal. The relative importance of calcium dynamics and the kinetics of the calcium-activated process in determining the time course of facilitation depend on the unique properties of a synapse. In one extreme, calcium-activated reaction kinetics dictate the time course of facilitation when they are much slower than calcium dynamics. This is consistent with the conclusion that [Ca]res dynamics does not play a prominent role in determining the time course of facilitation at the crayfish NMJ (Blundon et al., 1993
Our results differ greatly from the only other direct examination of calcium dynamics and facilitation in mammalian brain of which we are aware (Wu and Saggau, 1994
Comparison to the role of calcium in augmentation and PTP
The role of [Ca]res in facilitation as described here is reminiscent of that proposed for [Ca]res in augmentation and PTP. These forms of use-dependent synaptic enhancement, which last for tens of seconds, are observed during and after periods of elevated firing that increase [Ca]res. In augmentation and PTP, modest elevations of [Ca]res (tens to hundreds of nanomolar) enhance release by binding to high-affinity sites (Delaney et al., 1989Here we have used a stimulus protocol that emphasizes a rapidly decaying form of enhancement. With longer stimulus trains, additional forms of use-dependent plasticity such as PTP are also present at this synapse, but as yet the details of the action of calcium in such processes have not been described.
Implications for the properties of molecules involved in facilitation
Our findings constrain the properties of the molecule or molecules that govern the time course of facilitation at the granule cell to Purkinje cell synapse. The calcium sensor for facilitation must have a high calcium affinity and must be activated rapidly (i.e., have a fast on-rate). Furthermore, the molecular mechanism responsible for facilitation introduces a delay between calcium and facilitation, either through slow kinetics of calcium binding or of a long-lived product of a calcium-driven reaction. Calmodulin and synaptotagmin III, both of which are present in parallel fibers and sensitive to moderate levels of calcium (Ullrich et al., 1994
FOOTNOTES
Received May 10, 1996; revised July 2, 1996; accepted July 3, 1996.
This work was supported by National Institutes of Health Grant R01-NS32405-01, a McKnight Scholars Award, and a Klingenstein Fellowship Award in the Neurosciences to W.R. We thank Bernardo Sabatini, Jeremy Dittman, A. Vyshedskiy, and J.-W. Lin for comments on this manuscript.
Correspondence should be addressed to Dr. Wade G. Regehr, Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115.
APPENDIX
Simulations of the effect of EGTA on calcium transients
We sped the decay of [Ca]res by introducing EGTA to presynaptic terminals (Figs. 4, 5, 6, 7, 8). Bath-applied EGTA-AM, a membrane-permeant form of the chelator, freely entered cells, where it was deesterified and rendered cell-impermeant, and accumulated within parallel fibers (Tsien, 1981To understand how EGTA speeds the decay of calcium, we simulated calcium dynamics (Fig. 10A) with a single-compartment model using methods similar to those described previously (Tank et al., 1995
The key to understanding the actions of EGTA on this time scale lies in realizing that because it is a slow buffer, EGTA takes some time to equilibrate with free calcium. After calcium entry and rapid equilibration with the fast endogenous buffer, calcium begins to bind to EGTA, resulting in a reduction of free calcium levels. The time course of [Ca]res decay, until free calcium equilibrates with EGTA, is determined mostly by the time course of calcium binding to EGTA (compare Fig. 10, A and B). Extending reasoning similar to that of Tank et al. (1995)
Once calcium has equilibrated with both the endogenous buffer and EGTA, and calcium has been reduced to low levels, the small remaining [Ca]res decays slowly with a time constant approximated by the following equation (Neher and Augustine, 1992
![&tgr;<SUB>fast</SUB>=<FR><NU><FENCE>[B]/K<SUB>B</SUB></FENCE></NU><DE>(V<SUB>max</SUB>/K<SUB>P</SUB>)+k<SUP>+</SUP><SUB>EGTA</SUB> [EGTA]</DE></FR>.](/content/vol16/issue18/fulltext/5661/img005.gif)
(5) It should be noted that although the control [Ca]res transients we detect experimentally are not perfect monoexponentials, the model described above seems to capture the major features of the speeding of [Ca]res transients by EGTA.
![&tgr;<SUB>slow</SUB>=<FR><NU><FENCE>[B]/K<SUB>B</SUB></FENCE>+<FENCE>[EGTA]/K<SUB>EGTA</SUB></FENCE></NU><DE>(V<SUB>max</SUB>/K<SUB>P</SUB>)</DE></FR>.](/content/vol16/issue18/fulltext/5661/img006.gif)
(6) Note added in proof: Bertram et al. (1996)
REFERENCES
- Adler EM, Augustine GJ, Duffy SN, Charlton MP (1991) Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse. J Neurosci 11:1496-1507 . [Abstract]
- Augustine GJ, Neher E (1992) Neuronal Ca2+ signalling takes the local route. Curr Opin Neurobiol 2:302-307 . [Medline]
- Bain AI, Quastel DM (1992) Multiplicative and additive Ca2+-dependent components of facilitation at mouse end plates. J Physiol (Lond) 455:383-405 .
[Abstract/Free Full Text] - Bertram R, Sherman A, Stanley EF (1996) Single-domain/bound calcium hypothesis of transmitter release and facilitation. J Neurophysiol 75:1919-1931.
[Abstract/Free Full Text] - Bittner GD (1989) Synaptic plasticity at the crayfish opener neuromuscular preparation. J Neurobiol 20:386-408 . [ISI][Medline]
- Blundon JA, Wright SN, Brodwick MS, Bittner GD (1993) Residual free calcium is not responsible for facilitation of neurotransmitter release. Proc Natl Acad Sci USA 90:9388-9392 .
[Abstract/Free Full Text] - Buonomano DV, Merzenich MM (1995) Temporal information transformed into a spatial code by a neural network with realistic properties. Science 267:1028-1030 .
[Abstract/Free Full Text] - Charlton MP, Smith SJ, Zucker RS (1982) Role of presynaptic calcium ions and channels in synaptic facilitation and depression at the squid giant synapse. J Physiol (Lond) 323:173-193 .
[Abstract/Free Full Text] - Claflin DR, Morgan DL, Stephenson DG, Julian FJ (1994) The intracellular Ca2+ transient and tension in frog skeletal muscle fibres measured with high temporal resolution. J Physiol (Lond) 475:319-325 .
[Abstract/Free Full Text] - Cohen MW, Jones OT, Angelides KJ (1991) Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent
-conotoxin. J Neurosci 11:1032-1039 . [Abstract]
- Delaney KR, Tank DW (1994) A quantitative measurement of the dependence of short-term synaptic enhancement on presynaptic residual calcium. J Neurosci 14:5885-5902 . [Abstract]
- Delaney KR, Zucker RS, Tank DW (1989) Calcium in motor nerve terminals associated with post-tetanic potentiation. J Neurosci 9:3558-3567 . [Abstract]
- Delaney KR, Tank DW, Zucker RS (1991) Presynaptic calcium and serotonin-mediated enhancement of transmitter release at crayfish neuromuscular junction. J Neurosci 11:2631-2643. [Abstract]
- Delbono O, Stefani E (1993) Calcium transients in single mammalian skeletal muscle fibers. J Physiol (Lond) 463:689-707 .
[Abstract/Free Full Text] - Dodge FA, Rahamimoff R (1967) Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J Physiol (Lond) 193:419-432 .
[Abstract/Free Full Text] - Feller MB, Delaney KR, Tank DW (1996) Presynaptic calcium dynamics at the frog retino-tectal synapse. J Neurophysiol, in press.
- Goda Y, Stevens CF (1994) Two components of transmitter release at a central synapse. Proc Natl Acad Sci USA 91:12942-12946 .
[Abstract/Free Full Text] - Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450 .
[Abstract/Free Full Text] - Heidelberger R, Heinemann C, Neher E, Matthews G (1994) Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature 371:513-515 . [Medline]
- Heinemann C, Chow RH, Neher E, Zucker RS (1994) Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+. Biophys J 67:2546-2557 .
[Abstract/Free Full Text] - Herrington J, Bookman RJ (1995) Pulse control V4.5: IGOR XOPs for patch clamp data acquisition. Miami: University of Miami.
- Kamiya H, Zucker RS (1994) Residual Ca2+ and short-term synaptic plasticity. Nature 371:603-606 . [Medline]
- Katz B, Miledi R (1968) The role of calcium in neuromuscular facilitation. J Physiol (Lond) 195:481-492 .
[Abstract/Free Full Text] - Konishi M, Olson A, Hollingworth S, Baylor SM (1988) Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J 54:1089-1104 .
[Abstract/Free Full Text] - Konishi M, Hollingworth S, Harkins AB, Baylor SM (1991) Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra. J Gen Physiol 97:271-301 .
[Abstract/Free Full Text] - Kortje KH, Kortje D, Rahmann H (1991) The application of energy-filtering electron microscopy for the cytochemical localization of Ca(2+): ATPase activity in synaptic terminals. J Microsc 162:105-114 . [ISI][Medline]
- Li C, Ullrich B, Zhang JZ, Anderson RG, Brose N, Sudhof TC (1995) Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375:594-599 . [Medline]
- Liu Y, Stanley EF (1995) Calcium binding sites of the transmitter release mechanism: clues from short-term facilitation. J Physiol (Paris) 89:163-166. [ISI][Medline]
- Llano I, Marty A, Armstrong CM, Konnerth A (1991) Synaptic- and agonist-induced excitatory currents of Purkinje cells in rat cerebellar slices. J Physiol (Lond) 434:183-213 .
[Abstract/Free Full Text] - Llinas R, Gruner JA, Sugimori M, McGuinness TL, Greengard P (1991) Regulation by synapsin I and Ca2+-calmodulin-dependent protein kinase II of transmitter release in squid giant synapse. J Physiol (Lond) 436:257-282 .
[Abstract/Free Full Text] - Luther PW, Yip RK, Bloch RJ, Ambesi A, Lindenmayer GE, Blaustein MP (1992) Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations. J Neurosci 12:4898-4904 . [Abstract]
- Magleby KL (1987) Short-term changes in synaptic efficacy. In: Synaptic function (Edelman, GM, Gall, WE, Cowan, WM, eds) , p. 21. New York: Wiley.
- McNaughton BL (1982) Long-term synaptic enhancement and short-term potentiation in rat fascia dentata act through different mechanisms. J Physiol (Lond) 324:249-262 .
[Abstract/Free Full Text] - Mintz IM, Sabatini BL, Regehr WG (1995) Calcium control of transmitter release at a central synapse. Neuron 15:675-688 . [ISI][Medline]
- Naranjo D, Plant C, Dunlap K, Brehm P (1994) Two subcellular mechanisms underlie calcium-dependent facilitation of bioluminescence. Neuron 13:1293-1301 . [ISI][Medline]
- Neher E, Augustine GJ (1992) Calcium gradients and buffers in bovine chromaffin cells. J Physiol (Lond) 450:273-301 .
[Abstract/Free Full Text] - Palay SL, Chan-Palay V (1974) Cerebellar cortex. .
- Palkovits M, Magyar P, Szentagothai J (1971) Quantitative histological analysis of the cerebellar cortex in the cat. III. Structural organization of the molecular layer. Brain Res 34:1-18 . [ISI][Medline]
- Perkel DJ, Hestrin S, Sah P, Nicoll RA (1990) Excitatory synaptic currents in Purkinje cells. Proc R Soc Lond [Biol] 241:116-121 . [Medline]
- Regehr WG, Atluri PP (1995) Calcium transients in cerebellar granule cell presynaptic terminals. Biophys J 68:2156-2170 .
[Abstract/Free Full Text] - Regehr WG, Mintz IM (1994) Participation of multiple calcium channel types in transmission at single climbing fiber to Purkinje cell synapses. Neuron 12:605-613 . [ISI][Medline]
- Regehr WG, Tank DW (1991) Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods 37:111-119 . [ISI][Medline]
- Regehr WG, Delaney KR, Tank DW (1994) The role of presynaptic calcium in short-term enhancement at the hippocampal mossy fiber synapse. J Neurosci 14:523-537 . [Abstract]
- Roberts WM, Jacobs RA, Hudspeth AJ (1990) Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J Neurosci 10:3664-3684 . [Abstract]
- Rosahl TW, Spillane D, Missler M, Herz J, Selig DK, Wolff J, Hammer RE, Malenka RC, Sudhof TC (1995) Essential functions of synapsins I and II in synaptic vesicle regulation. Nature 375:488-497 . [Medline]
- Sabatini BL, Regehr WG (1995) Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice. Neuropharmacology 34:1453-1467 . [ISI][Medline]
- Schulz PE, Cook EP, Johnston D (1994) Changes in paired-pulse facilitation suggest presynaptic involvement in long-term potentiation. J Neurosci 14:5325-5337 . [Abstract]
- Schweizer FE, Betz H, Augustine GJ (1995) From vesicle docking to endocytosis: intermediate reactions of exocytosis. Neuron 14:689-696 . [ISI][Medline]
- Simon SM, Llinas RR (1985) Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release. Biophys J 48:485-498 .
[Abstra
ABSTRACT