The finding that synaptic terminals of the same L2/3 pyramidal cell can display either frequency-dependent facilitation or frequency-dependent depression depending on the type of postsynaptic neurone that they contact, provides a unique opportunity to study these two forms of short-term synaptic plasticity in comparable experimental conditions. Previous experiments on peripheral and central synapses (Kuno, 1964; Charlton et al. 1982; Swandulla et al. 1991; Debanne et al. 1996) have shown that facilitation and depression are interrelated in a complex way. Manipulations that increase or decrease the fraction of vesicles released by an AP shift a given synapse from facilitating to depressing and vice versa (Stevens & Wang, 1995). This is readily understandable if facilitation is considered to represent an increase in the fraction of vesicles released caused directly or indirectly by residual Ca2+ and depression is interpreted as the depletion of release-ready vesicles (Elmqvist & Quastel, 1965; Betz, 1970). In the present experiments we manipulated the [Ca2+] in terminals by intracellular loading of Ca2+ buffers. Thereby we altered both residual Ca2+ and the release-ready fraction of vesicles and, therefore, we have to consider in detail the interactions between facilitation and depression. In the following discussion we try to do so on the basis of a simple model.
The basic description of transmitter release at the neuromuscular junction assumes that the postsynaptic signal is a product of the number of quanta released during an AP (quantal content) m and the mean quantal size q (Katz, 1969). Furthermore, statistical analysis indicates that the number of quanta released can be considered as a product of the number of release-ready vesicles n and the probability p. If we assume that the same basic scheme holds for central synapses and consider possible mechanisms by which added buffers will change release, the most direct and likely effect is a reduction in p due to a reduction of [Ca2+]RS, the concentration of Ca2+ at the release site during an AP. The extent to which such a reduction diminishes p, however, is not only a function of the relative reduction in [Ca2+]RS, but it also depends on the degree of saturation of the Ca2+ sensor during one or several APs. A terminal in which Ca2+ influx during an AP drives the Ca2+ sensor at the release site relatively far into saturation will have a high p value. In such a synapse, added exogenous buffer will reduce p only if it reduces [Ca2+]RS substantially. Our data indicate that those terminals of L2/3 pyramids contacting multipolar cells represent such ‘high p terminals’. A synapse with a lower p, on the other hand, in which the Ca2+ sensor is not saturated during an AP, will be very sensitive to changes in [Ca2+]RS, since its p value is steeply dependent on [Ca2+]RS. Our data indicate that the terminals contacting bitufted cells behave in this way (‘low p terminals’).
Facilitation and depression.
A similar argument applies to the mechanism underlying the facilitation of release. Differences in [Ca2+]RS between the second and the first AP in a pair, due to residual Ca2+, will have stronger effects in the terminal with low p as compared to the high p terminal, because in the former, the relationship between p and [Ca2+]RS is steeper. Release probability, however, influences facilitation in a second way: in a high p terminal a large fraction of release-ready vesicles will be consumed during the first of a pair of APs, such that the number of vesicles available for release in response to the second AP will be reduced. This effect can be interpreted as depression superseding facilitation. Depression will be more pronounced the higher the p value of a given terminal. The [Ca2+]o dependence of release (Fig. 4) shows that close to the physiological [Ca2+]o of 2 mm there is a pronounced difference in the degree of saturation of the Ca2+ sensor during a single AP between terminals on bitufted and multipolar cells.
Using this information we want to determine how differences in saturation of the Ca2+ sensor are related to the effectiveness of exogenous buffer and synaptic facilitation. Specifically, we want to calculate, on the basis of the simplest possible model, what relative changes in [Ca2+]RS occur after buffer loading and during facilitation, assuming that p and the saturation of Ca2+ sensors might be different in the two types of terminal.
The analysis presented below is an attempt to interpret the effects of BAPTA and EGTA on release as well as on the facilitation ratio during paired-pulse stimulation in terms of changes in [Ca2+]RS. It does so based on the steepness of the relationship between synaptic efficacy and [Ca2+]o and by inferring from that the degree of saturation of the Ca2+ sensor under control conditions. It also attempts to correct for the consequences of depletion of release-ready vesicles during the first AP in a pair. Simplifying assumptions are as follows. (i) There are no effects of buffers other than changing [Ca2+]RS. (ii) [Ca2+]RS is proportional to [Ca2+]o both with and without added buffers. The effect of buffers is to change the proportionality constant between [Ca2+]RS and [Ca2+]o. (iii) The relationship between release and [Ca2+]RS is a fourth power Hill equation. (iv) No changes occur during facilitation other than changes in [Ca2+]RS. (v) There is no recruitment of new vesicles between two APs. (vi) Release sites are identical in their properties.
We would like to point out that the saturation of release, observed in Fig. 4, may have reasons other than saturation of the Ca2+ sensors. One mechanism limiting maximum release, which has been discussed extensively in the literature, is the finding that in some types of synapse a given active zone seems not to be able to release more than one vesicle per action potential (Korn & Faber, 1987; Stevens & Wang, 1995). Such terminals may have many docked vesicles per active zone. Saturation of release may occur already at low [Ca2+], when there is a good chance that one out of several vesicles will be released, although the occupancy of Ca2+ sensors of any individual vesicle is moderate. Such an interpretation, however, fails to explain why such terminals readily depress, since they should have plenty of vesicles in reserve and the ‘lateral inhibition’ postulated to prevent subsequent releases is supposed to subside on the time scale of 20 ms (Stevens & Wang, 1995). We therefore think that our simpler model is worth pursuing.
A minimum model
Following the conventional description of transmitter release (Zucker, 1973) the minimum model assumes that release y is proportional to the number of release-ready vesicles n and to the release probability p of a given vesicle according to:
where q is a proportionality factor reflecting quantal size.
We assume that release probability follows a Hill equation for the simultaneous occupancy of a Ca2+ sensor by four Ca2+ (Parnas & Segel, 1981; Reid et al. 1998). The release probability of a given vesicle during an AP can then be written as:
where pmax is the maximum value of p at high [Ca2+]RS and K1/2 is the concentration at which p is one-half of pmax. The value of pmax can approach 1, but may be smaller than 1, because the time of complete (fourfold) occupancy of the release site with Ca2+ may not be long enough during an AP for release to occur. Moreover, the number of release-ready vesicles for the second AP, n2, is different from the number, n1, for the first AP (Tsodyks & Markram, 1997). If, for the minimum model, it is assumed that negligible recruitment of release-ready vesicles occurs in between two APs of a pair then:
where the second term is the number of vesicles released during the first AP. Then we obtain for the PPF ratio y2/y1:
This can be interpreted as the product of an ‘intrinsic’ facilitation ratio (a2/p1), which reflects the increase in release probability due to some facilitation mechanism (see below), and a correction factor (1 - p1), which reflects the fact that fewer vesicles are available for release during the second AP, as compared to the first one. This term can be considered as a factor representing ‘depression’.
The failure rate for the first AP in the pyramidal to bitufted cell connection was found to be 42 % (Fig. 2B). The number of putative synaptic contacts may be up to 10 (authors’ unpublished observation). Assuming that all terminals in each individual pair of cells are identical, and all terminals have at least one release-ready vesicle, then the failure rate for a single facilitating terminal might be 92 % (a is about 0.08). Depletion of the release-ready vesicles should then be negligible. On the other hand, analysis of the effect of variations in [Ca2+]o suggests a release probability p for a release-ready vesicle of 0.17 (for pmax= 0.8, see below). In this case depletion would exert some influence.
The data in Fig. 4 allow us to estimate the ‘relative release probability’pn= p/pmax and the ‘relative calcium concentration at the sensor’[Ca2+]n:
for a given terminal under the assumption that [Ca2+]RS is proportional to [Ca2+]o and that synaptic efficacy (the size of the mean unitary EPSP) is proportional to the release probability p (eqn (2)). Least-square fits of eqn (2) to the data of Fig. 4 yield for the standard [Ca2+]o of 2 mm the following values (with pmax= 1): pn= 0.93 and [Ca2+]n= 1.83 for terminals contacting multipolar cells and pn= 0.21 and [Ca2+]n= 0.72 for terminals contacting bitufted cells.
Influence of buffers on phasic release.
In the following we assume that the only effect of adding buffers is a reduction in [Ca2+]RS. Then it follows from eqns (1), (2) and (5) that the buffering ratio RB (defined as the ratio of the mean amplitude of the first EPSP measured in the presence of a buffer (EPSP1B) to that in control (EPSP1C) is given by:
Subscripts o and b denote values before and after addition of buffers, respectively; the subscript n denotes [Ca2+]RS, normalized to the K1/2 of the sensor. Here, it is assumed that the number of available vesicles, quantal size and pmax do not change with addition of buffer, and that the only effect of the loaded buffer is a change in the proportionality constant between [Ca2+]RS and [Ca2+]o.
Equation (6) allows calculation of [Ca2+]n,b in terms of [Ca2+]n,o, according to:
It can be seen from eqns (5) and (7) that in the case of terminals contacting bitufted cells, where [Ca2+]RS is smaller or comparable to the dissociation constant of the Ca2+ sensor ([Ca2+]n,o < 1), the second term in the denominator in eqn (7) is small. Then [Ca2+]n will be reduced simply by the fourth root of the buffering ratio, as expected from a fourth power relationship between release and [Ca2+]n. For terminals on multipolar cells, however, [Ca2+]n,o is 1.83 (see above), such that for a twofold reduction of efficacy (aB= 0.58; for 0.5 mm BAPTA) the second term in the denominator of eqn (7) is 5.14. In this case, for a given value of RB, [Ca2+]n is reduced more strongly, requiring more buffer.
Figure 11A plots the data of Fig. 7 in terms of [Ca2+]n,b as calculated from eqn (7), after normalization with respect to [Ca2+]n,o. It can be seen that inferred [Ca2+] values drop by about 30 % with very little addition of BAPTA or EGTA. The sensitivity towards these buffers is somewhat greater for depressing terminals than for facilitating terminals, which is exactly the opposite to the order of sensitivity of the original EPSP data. For both types of terminal BAPTA is about sixfold more efficient in reducing [Ca2+]RS than EGTA (as judged from the initial slopes of the two curve pairs). The analysis indicates that a given type of buffer at low concentrations produced very similar reductions in [Ca2+]n in the two classes of terminal, and that the different effectiveness of the buffers seems to reside in a difference in saturation of the Ca2+ sensor during APs at physiological [Ca2+].
Figure 11. Inferences on [Ca2+] at release sites during buffer action and facilitation
A, postulated reduction of [Ca2+]RS by external buffers. The ratio [Ca2+]n,b/[Ca2+]n,o, which is the inferred relative reduction in [Ca2+]RS in the presence of buffer, is calculated according to eqn (7). Individual values correspond to data points of Fig. 7. Squares and circles represent data from facilitating and depressing terminals contacting bitufted and multipolar cells, respectively. B, postulated increase in [Ca2+]RS during facilitation. Values (for terminals on bitufted cells only) are calculated according to eqn (9a). RF values are from Figs 8 and 9; [Ca2+]n,1 is the product of the corresponding value in A and the value of [Ca2+]n,o, which is 0.72 in facilitating terminals on bitufted cells. pmax was assumed to be 0.8.
Download figure to PowerPoint
A lower degree of saturation of the Ca2+ sensor in terminals on bitufted cells can be explained either by a lower affinity of the sensor or by a lower Ca2+ channel density, implying a longer diffusional distance between RS and Ca2+ channels. Unfortunately, the analysis described above cannot discriminate between these two possibilities. However, if the only difference between the two types of terminal resided in different dissociation constants of their Ca2+ sensors, then [Ca2+] at the sensors and its reduction by buffers would be the same, i.e. the two curves for a given buffer in Fig. 11A should superimpose over the whole buffer concentration range, since they are normalized with respect to the corresponding dissociation constant. The fact that they deviate at buffer concentrations above 1 mm (Fig. 11A) indicates that there must be differences other than the affinities of Ca2+ sensors.
Also, if the KD of the Ca2+ sensor in a facilitating terminal were significantly higher than that in a depressing one, the release from the facilitating terminal should be more synchronized compared to that from the depressing terminal, since [Ca2+]RS would drop below the higher threshold level within a shorter time. The alternative hypothesis that the lower degree of saturation of the Ca2+ sensor in one type of terminal is due to a longer diffusional distance between the RS and Ca2+ channels might imply that Ca2+ needs more time to diffuse over longer distances, making release less synchronized. Latency distributions of synaptic responses measured in both pyramid-to-bitufted cell and pyramid-to-multipolar cell connections differ from each other, being significantly wider for the former (Fig. 5A), thus favouring the second hypothesis. Moreover, assuming that Ca2+ sensors in the two types of terminal have the same high affinity, but that the sensor is located more distally from Ca2+ channels in pyramid-to-bitufted cell contacts, we can explain the rest of our data including the effect of buffers on facilitation (see below, and Fig. 12).
Figure 12. Schematic representation of the possible geometry of Ca2+ channels and vesicle release sites in different boutons of pyramidal cell axon collaterals
A, bouton of a pyramidal cell axon contacting a bitufted cell. B, bouton of a pyramidal cell axon contacting a multipolar cell. Voltage-dependent calcium channels (VDCCs) can be linked to a vesicle release site or non-linked.
Download figure to PowerPoint
Remarkably, BAPTA at high concentrations becomes less effective in reducing [Ca2+]RS. One might consider two alternative explanations. First, the steep portion of the curves (Fig. 11A) may reflect the fact that there are Ca2+ channels that are located distally from the RS (Fig. 12). Their contribution might be readily intercepted by a low concentration of buffer. The shallow portion of the curves (Fig. 11A) may represent the contribution of Ca2+ channels closely linked to release sites. Added Ca2+ buffers would be very inefficient in intercepting the Ca2+ entering through such nearby channels (Neher, 1998b). Second, Ca2+ entering through a channel might preferentially diffuse over some distance in a surface layer close to the negatively charged membrane, in which unbound BAPTA (being fourfold more negatively charged) is excluded due to surface charge (Schumaker & Kentler, 1998). Under this assumption BAPTA at low concentrations may effectively chelate the bulk of the Ca2+ producing the steep part of the curve. However, even at high concentrations of BAPTA, Ca2+ would still be able to diffuse over some distance within the BAPTA-free perimembrane layer producing the shallow portion of the curve. To distinguish between these alternatives more specific experiments are required. However, irrespective of the exact mechanism by which some channels are functionally more tightly linked to the sensor than others, the plot of Fig. 11A suggests that low concentrations of BAPTA are able to reduce [Ca2+]n by a larger relative amount in depressing terminals than in facilitating terminals. As a consequence the two curves for a given buffer are reversed relative to those of Fig. 7A and B.
As a third interpretation regarding the differences in efficacy between the two terminal types one could assume that their morphology is exactly the same (e.g. equal channel densities) and that the higher [Ca2+]n in terminals on multipolar cells is due to a lower concentration of endogenous buffer. In this case, however, one would expect a steeper dependence of [Ca2+]n,b/[Ca2+]n,o upon adding buffer in these terminals. This is because smaller amounts of exogenous buffers are required to compete successfully with the endogenous one. This is contrary to the data of Fig. 7 and 11A. Also, such an interpretation would face the problem that mobile endogenous buffers (mobile ones are most effective in reducing [Ca2+]RS) should be present in terminals of the same axon at different concentrations.
Mechanisms of facilitation
Based on the minimum model discussed above, one can make a number of inferences on the facilitation data. From eqns (2), (4) and (5) the paired-pulse ratio (a) in general can be calculated:
In the case where changes in the size of a readily releasable pool of vesicles due to depletion during the first AP are negligible, eqn (8a) may be simplified to eqn (8b).
where RF is the paired-pulse ratio for facilitating terminals.
This assumes that the only change during a second AP (subscript 2) with respect to the first (subscript 1) is a higher value of [Ca2+]n at the release site. Such changes in [Ca2+]n can be calculated from eqn (8a) for general eqn (9a) and from eqn (8b) for simplified eqn (9b) situations as follows:
Note, that in order to use eqn (9a) for the terminals loaded with exogenous buffers, [Ca2+]n,1 should be replaced by the quantity [Ca2+]n,b (see eqn (7)).
In principle, eqn (9a) allows one to predict the relative increase in [Ca2+] at the RS during the second AP compared with that during the first ([Ca2+]n,2/[Ca2+]n,1). However, the accuracy of such predictions depends quite strongly on the assumption regarding the value of pmax. In fact, eqn (9a) can be solved only if:
This restriction is particularly critical in the case of depressing terminals, where [Ca2+]n,1 is large, such that most of the vesicles might be released during the first AP. Hence, in order to estimate the release probability during the second AP, one needs to know the precise number of remaining vesicles. Also, any heterogeneity of release probability will complicate all steps in the analysis and lead to large errors when part of the vesicle population has a release probability close to 1. We, therefore, restricted this analysis mainly to data on facilitating terminals, in which [Ca2+]n,1 is small and the size of the readily releasable pool of vesicles is almost unaffected by the first AP.
The analysis presented in Fig. 11B indicates that to induce facilitation under control conditions [Ca2+]n should be 25-40 % higher during the second AP compared to the first. This also holds for low concentrations of BAPTA (0.02–0.5 mm). Further elevation of the BAPTA concentration (≥ 1 mm) decreased the inferred ratio of [Ca2+]n,2/[Ca2+]n,1, as plotted in Fig. 11B. Here pmax was set to 0.8 but varying pmax between 0.7 and 1 had little influence on the numbers. However, EGTA even at relatively low concentrations (≥ 0.2 mm) abolished facilitation, reducing the [Ca2+]n,2/[Ca2+]n,1 ratio close to 1. This can be interpreted in the sense that EGTA quite efficiently chelates residual free Ca2+, or competes with endogenous sites for residual bound Ca2+.
To explain facilitation at low BAPTA concentrations, it should be considered that BAPTA very efficiently reduces [Ca2+]RS during the first AP and may remain partially saturated for tens of milliseconds. Thus, it will not be as effective in limiting the increase in [Ca2+]RS during the second AP, resulting in a facilitated response. This mechanism will be called ‘pseudofacilitation’ below. In order to avoid saturation of BAPTA and the resulting pseudofacilitation, an amount of BAPTA much larger than the total amount of Ca2+ entry is required.
An interesting question is why EGTA interferes with endogenous facilitation, but not with pseudofacilitation, when it is present in a mixture with BAPTA (Fig. 10A). The ‘non-effect’ of EGTA on pseudofacilitation can readily be understood on the basis of the slowness of the Ca2+ exchange reaction between BAPTA and EGTA. A given slow buffer at free concentration [B] will take up Ca2+ in the presence of one or several fast buffers with an apparent first order binding rate:
where the sum is that of the Ca2+-binding ratios (κυ) of all competing fast buffers. In the presence of 0.2 mm BAPTA this sum is 200 or more. Thus, with kon,EGTA= 2.5 x 106m−1 s−1 and [EGTA]= 0.2 mm the transition of Ca2+ from BAPTA to EGTA will happen in a time range of 1/kapp, longer than 400 ms. In the absence of BAPTA, however, when only endogenous buffer (probably a fixed one with κ< 50) is present, the time required for uptake of Ca2+ by EGTA is expected to be about 100 ms. This means that EGTA can efficiently chelate some of the residual Ca2+ in between two APs separated by 100 ms.
When varying [Ca2+]o in the presence of internal BAPTA we found that facilitation changed as expected for the partial buffer saturation mechanism, i.e. more buffer (BAPTA) was saturated during the first AP at high [Ca2+]o causing more facilitation. In native terminals, however, the opposite trend was observed. Two explanations may be given for this finding. (i) The mechanism of facilitation in native terminals is similar to that under BAPTA (i.e. is caused by partial saturation of an endogenous buffer) but is suppressed by depletion of release-ready vesicles, since release is much larger in the absence of BAPTA. (ii) The mechanism of facilitation is not influenced by saturable buffers and residual Ca2+ is sequestered faster into organelles at higher [Ca2+]o. Indeed, Ca2+ extrusion and mitochondrial Ca2+ uptake depend on [Ca2+]i in a supralinear fashion (Regehr & Atluri, 1995; Xu et al. 1997; Ohnuma et al. 1999). Therefore, the ratio between residual Ca2+ and Ca2+ entering the terminal with the second AP would be larger at lower [Ca2+]o and smaller at higher [Ca2+]o resulting in increased and decreased facilitation, respectively.
Alternative models of facilitation.
Atluri & Regehr (1996) have suggested that facilitation involves an additional high-affinity Ca2+-binding site (X-receptor) with a relatively slow Ca2+ binding which operates cooperatively with the main vesicle sensor. According to their hypothesis Ca2+ rapidly binds to this site during the first AP and remains bound at the time of the second AP, thus increasing the amount of release in response to the second AP. The evidence for such a mechanism is that 0.1 mm EGTA-AM accelerates the decay of free Ca2+ concentration more strongly than that of facilitation so that a component of facilitation still remains, while free Ca2+ drops to the initial basal level.
In our experiments at 10 Hz stimulation EGTA at 0.2 mm and above abolished facilitation by chelating residual free Ca2+. Under similar conditions BAPTA must be more effective at reducing free Ca2+, but in 0.2 mm BAPTA facilitation was even larger than in control. Assuming the existence of the X-receptor to be responsible for facilitation, BAPTA as a faster buffer should be able to abolish facilitation even more efficiently than EGTA, because it could compete with the X-receptor for Ca2+ entering during the first AP. However, facilitation still occurs with BAPTA over a concentration range of 0.05–0.7 mm. We explain this by saturation of the added buffer (see above). Likewise, one can assume that in the experiments described by Atluri & Regehr (1996) partial saturation of EGTA during the first AP may underlie the short-lasting facilitation. Indeed, in their experiments 0.1 mm EGTA-AM decreased the Ca2+ peak amplitude by 45 % whereas 0.02 mm EGTA-AM was almost ineffective (8 %). Thus a relatively small decrease in free buffer concentration during the first AP may increase the Ca2+ available for the RS following the second AP to produce noticeable facilitation. In this view facilitation is determined by the peak [Ca2+], which is higher during the second AP due to saturation of the buffer. The decay of facilitation, which is slower than the fast decay of free Ca2+, may reflect the kinetics of the equilibration of bound and unbound buffer near the RS. Noteworthy in our experiments is the fact that facilitation still exists with 0.1 mm EGTA, presumably due to EGTA oversaturation during the first AP. It should be pointed out, though, that none of our data in the absence of an added buffer would be incompatible with a special fast Ca2+ sensor for facilitation (Kamiya & Zucker, 1994) particularly if it had a relatively high affinity, as suggested by Ravin et al. (1997), Delaney & Tank (1994) and Tang et al. (2000).
Calcium concentrations at the sensor.
We would like to discuss the question of the absolute value of [Ca2+] reached at the release site. Our experimental data provide only numbers that are relative to the K1/2 of the sensor and the relative increase during facilitation (which was found to be between 25 and 40 %). Under the simplest assumption that [Ca2+]RS during the first AP has some value [Ca2+]RS,1 and that [Ca2+]RS,2 is the sum of residual Ca2+ and the same [Ca2+]RS,1, it is possible to calculate [Ca2+]RS,1 from the relative increase, if residual [Ca2+] is known (in analogy with Ravin et al. 1999). Depending on assumptions about residual [Ca2+] one can obtain estimates for [Ca2+]RS,1 of between 0.5 μm and several micromolar. This range of values is much lower than generally assumed to hold at the RS. Recent reports (Bollmann et al. 2000; Schneggenburger & Neher, 2000) have shown that in glutamatergic calyx-type synapses the concentration of Ca2+ at release sites sufficient to produce transmitter release is in the range 10–25 μm. Thus, it should be considered that residual Ca2+ and [Ca2+]RS,1 may add non-linearly (Neher, 1998b). On the other hand, the latency distribution of release events (Fig. 5) shows that the turn-on and turn-off of release is not as rapid in terminals of pyramidal cells as it is in the case of the calyx of Held. Furthermore, the frequency of release events is only about a factor of 5–10 higher during peak release than it is for the early asynchronous release. Therefore, one cannot exclude the interpretation of facilitation as a result of linear summation of residual calcium and the Ca2+ increment during an action potential. Indeed, Koester & Sakmann (2000) have shown that AP-evoked Ca2+ transients produce an increase in the steady-state [Ca2+]i level in terminals of rat L2/3 pyramidal neurones during stimulation by a train of APs at 10 Hz (monitored by a low affinity Ca2+ indicator).
Complexity in short-term plasticity due to Ca2+ buffers.
One goal of this study was to understand better the cellular mechanisms underlying PPF. Based on the analysis of our data we can conclude that facilitation in terminals of L2/3 pyramidal cells that form synapses with bitufted interneurones is tightly linked to the accumulation of residual free Ca2+. Our data are compatible with the idea that the same Ca2+ sensor mediates both phasic release and facilitation. In addition we have found that partial saturation of fast acting buffers leads to facilitating responses, when a buffer such as BAPTA is present in a terminal at a high enough concentration. We call this phenomenon pseudofacilitation.
The mechanism underlying pseudofacilitation is likely to also contribute to facilitation in intact terminals in cell types that express high affinity Ca2+-binding proteins (Baimbridge et al. 1992). However, this seems not to be the case for the facilitating terminals studied here, as addition of minute amounts of BAPTA strongly reduced transmission, indicating that it does not compete with endogenous buffers. In addition, our finding that 0.2 mm EGTA supresses endogenous facilitation, but has no influence on pseudofacilitation (included by loading with 0.05-0.2 mm BAPTA) indicates that endogenous (natural) facilitation is different from pseudofacilitation in these terminals.
Our study suggests that it may be possible to distinguish between synapses that employ residual Ca2+ and partial buffer saturation mechanisms of facilitation. The residual Ca2+ mechanism of facilitation would apply to synapses where residual free Ca2+ is in equilibrium with low affinity endogenous buffers at the time of the second AP without substantially influencing the concentrations of the unbound buffer species. Alternatively, a significant fraction of the buffers (either mobile or immobile) that limit the [Ca2+] rise during the first AP may be saturated, resulting in reduced buffer action during the second AP. The ‘residual’ Ca2+ mechanism, which may apply to the case of native terminals on bitufted cells, is characterized as follows: (i) EGTA at given concentrations blocks facilitation; (ii) facilitation can be higher at lower [Ca2+]o and lower at higher [Ca2+]o; (iii) the endogenous Ca2+-binding ratio is low and release is very sensitive to exogenously added buffers. The synapses employing the partial buffer saturation mechanism probably resemble terminals on bitufted cells after addition of BAPTA, displaying the following properties: (i) EGTA should not affect facilitation; (ii) within a certain concentration range of [Ca2+]o facilitation should be small at lower [Ca2+]o and increase at higher [Ca2+]o; (iii) endogenous Ca2+ buffer capacity should be high and release should be less sensitive to exogenously added buffers.
A third type of synapse might have an accumulation of high affinity fixed buffers at the active zones, with properties other than those conferred by the mobile buffer BAPTA.
Target cell specificity of synaptic efficacy and frequency-dependent short-term plasticity in the mammalian CNS have been reported also for connections between cortical L5 pyramid cells and different interneurones (Markram et al. 1998) and for hippocampal pyramid-to-interneurone connections (Ali & Thomson, 1998; Ali et al. 1998) as well as for the connections between CA3 pyramidal cells formed in hippocampal slice cultures (Scanciani et al. 1998). It will be necessary to establish whether the tentative interpretation given here for the differences between the low efficacy, facilitating and the high efficacy, depressing terminals of layer 2/3 pyramids - where it is assumed that the geometry of the Ca2+ channels and vesicle release sites and/or the density of Ca2+ channels strongly influences spatiotemporal Ca2+ dynamics - may also be valid for the other connections.
Direct imaging of Ca2+ fluorescence transients in single boutons of L2/3 pyramid axon collaterals has indicated that the size of single AP-evoked Ca2+ transients in different boutons varies over an almost tenfold range (Koester & Sakmann, 2000). Tentatively one could attribute the boutons with smaller Ca2+ influx to low efficacy, facilitating connections, and those with larger Ca2+ influx to connections that have a high efficacy and show depression of release. Equally important will be to identify the retrograde signals that, presumably, act on a long time scale, which are responsible for the target-specific differences of Ca2+ dynamics of pyramid cell nerve terminals.