Partial Uncoupling of Neurotransmitter Release From [Ca2+]i by Membrane Hyperpolarization

R. Ravin, H. Parnas, M. E. Spira, and I. Parnas

The Otto Loewi Minerva Center for Cellular and Molecular Neurobiology, Department of Neurobiology, The Hebrew University, Jerusalem 91904, Israel


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ravin, R., H. Parnas, M. E. Spira, and I. Parnas. Partial uncoupling of neurotransmitter release from [Ca2+]i by membrane hyperpolarization. The dependence of evoked and asynchronous release on intracellular calcium ([Ca2+]i) and presynaptic membrane potential was examined in single-release boutons of the crayfish opener neuromuscular junction. When a single bouton was depolarized by a train of pulses, [Ca2+]i increased to different levels according to the frequency of stimulation. Concomitant measurements of evoked release and asynchronous release, from the same bouton, showed that both increased in a sigmoidal manner as a function of [Ca2+]i. When each of the depolarizing pulses was immediately followed by a hyperpolarizing pulse, [Ca2+]i was elevated to a lesser degree than in the control experiments, and the rate of asynchronous release and the quantal content were reduced; most importantly, evoked quantal release terminated sooner. The diminution of neurotransmitter release by the hyperpolarizing postpulse (HPP) could not be entirely accounted for by the reduction in [Ca2+]i. The experimental results are consistent with the hypothesis that the HPP reduces the sensitivity of the release machinery to [Ca2+]i, thereby not only reducing the quantal content but also terminating the quantal release process sooner.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The Ca2+-voltage hypothesis (CVH) (Parnas and Parnas 1994; Parnas et al. 1990) asserts that in fast synapses two voltage-dependent factors are required to evoke release of neurotransmitter, the opening of Ca2+ channels and the ensuing increase in intracellular Ca2+ concentration ([Ca2+]i) and the voltage-dependent transformation of a putative inactive release apparatus, T, to its active state, S. According to the CVH, the quantal content depends on both [Ca2+]i and S, whereas the time course of release depends on the T right-left-arrows S transformation (Lustig et al. 1989; Parnas and Parnas 1994; Parnas et al. 1986a,b). Other investigators support the notion that it is the kinetics of entry and removal of Ca2+ that determine the time course of release. In particular, transient local elevation of Ca2+ to hundreds of micromolar concentration initiates release, and the subsequent collapse of such Ca domain terminates release. This hypothesis, termed the Ca2+ microdomain hypothesis for neurotransmitter release, is described in several reviews (Smith and Augustin 1988; Zucker 1996).

Numerous findings support the conclusion that it is not the influx of Ca2+ that triggers release, and it is not the removal of Ca2+ from the microdomain of the transmitter release site that terminates release. Experimental manipulations known to affect Ca2+ influx or its removal did not affect the time course of release (Andrew and Barrett 1980; Arechiga et al. 1990; Datyner and Gage 1980; Hochner et al. 1991; Parnas et al. 1984, 1989; Van der Kloot and Molgo 1994). In other experiments, conditions were such that no significant influx of Ca2+ was possible. In one case, the external solution included EGTA with no added Ca2+ (Silinsky et al. 1995). In another case, Ca2+ was omitted from the circulation solution (Mosier and Zengel 1994), or Ca2+ entry was blocked by increasing [Mg2+]o (Hochner et al. 1989). In these experiments, [Ca2+]i was increased by means other than entry via voltage-gated Ca2+ channels. Evoked release was obtained only when the presynaptic terminal was depolarized, although there was little or no influx of Ca2+. Recently, Mochida et al. (1998) demonstrated that depolarization directly enhances neurotransmitter release and that the Ca2+ channel itself is a voltage sensor connected via the synprint protein to the exocytic machinery.

The third line of experiments tested whether the quantal content and time course of release are affected by the rate of presynaptic repolarization after a test depolarizing pulse. The CVH predicts that if S transforms faster to the T state, the quantal content will be smaller, and termination of release will occur sooner. This was tested experimentally by Dudel (1984), Parnas et al. (1986b), and Arechiga et al. (1990). These authors compared synaptic delay histograms obtained by a control depolarizing test pulse with histograms obtained by the same stimulus followed by a brief hyperpolarizing postpulse (HPP). Indeed, when the test pulse was followed by an HPP, quantal content was reduced, and the duration of evoked release was shortened. These results indicated that the HPP caused a faster S right-arrow T transformation. However, as suggested by Zucker (1987), the reduction in quantal content can well be explained if the HPP merely causes a faster closure of Ca2+ channels (Klockner et al. 1989; Matteson and Armstrong 1986; Swandulla and Armstrong 1988).

Because Ravin et al. (1997) have since developed a technique to monitor [Ca2+]i and quantal release from a single-release bouton simultaneously, we reinvestigated the effect of a HPP on [Ca2+]i and on evoked and asynchronous release. We found that in addition to accelerating closure of Ca2+ channels, HPP directly affects the release apparatus, probably by facilitating the S right-arrow T transformation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We used the opener neuromuscular preparation of the first walking leg of the crayfish Procambarus clarkii. Crayfish (3-4 cm long) were purchased from Atchafalaya Biological Supply (Raceland, LA). Animals were kept in aquaria with running and filtered fresh water. The animals were fed with fish fillets twice a week. The preparation was constantly superfused (Gilson minipulse 3, France) with Van Harreveld solution containing (in mM) 220 NaCl, 5.4 KCl, 13.5 CaCl2 (unless otherwise stated), 2.5 MgCl2, and 10 Tris buffer. The pH was adjusted to 7.4 by adding NaOH. Intra-axonal injection of fura-2 (Molecular Probes, Eugene, OR) enabled visualization of single-release boutons and the macropatch electrode placed over a single bouton under visual control. TTX (Sigma, St. Louis, MO and RBI, Natick, MA) 5.10-7 M was added to prevent sodium excitability. The bath temperature was kept at 11 ± 0.5°C.

Ca2+ imaging

[Ca2+]i was measured after fura-2 injection into one of the secondary branches of the excitatory axon and with ratiometric imaging techniques (Grynkieviewicz et al. 1985; Ziv and Spira 1993). The final intraaxonal fura-2 concentration was 50-100 µM (Ravin et al. 1997). For digital video imaging (×40 upright microscope, Nikon, Optiphot 2) we used an intensified charge-coupled device video camera (model 2400-77, Hamamatsu, Hamamatsu City, Japan). The images taken before, during, and after the stimulation period were stored on 3/4-inch videotape. Thus, althouh release was measured after each impulse of the train, Ca2+ imaging reflected the overall Ca2+ concentration during the stimulation period. For further technical details, the reader should consult Ravin et al. (1997) where detailed procedures are provided.

Stimulation and recording

We used the macropatch technique (Dudel 1981), which enables depolarization of a single-release bouton and simultaneous recording of single quanta events with the same electrode (Ravin et al. 1997).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Basic experiment

Figure 1 shows results of a "typical" experiment. Figure 1A shows Ca2+ images of a single-release bouton injected with fura-2. In Fig. A, first row, the bouton was stimulated with a train of depolarizing pulses (-0.7 µA, 0.6 ms) at 100 Hz for 8 s (control). The test depolarizing pulse appears as a negative current pulse on the right. In the second row, a similar train was delivered (7 min later), but now each test depolarizing pulse was followed immediately (0 delay) by an HPP (+0.6 µA, 0.5 ms) (see stimulation protocol on right). In the third row, the train of the test pulse was administered again (7 min later) as a second control. Clearly, the HPP reduced Ca2+ accumulation.



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Fig. 1. Effects of hyperpolarizing postpulses (HPPs) on intracellular calcium [Ca2+]i and on the time course of release. A: fura-2 ratio images of a single-release bouton. Note the macropatch electrode over the bouton. First row, control: bouton stimulated with a train of depolarizing pulses (-0.7 µA, 0.6 ms) at 100 Hz for 8 s (right insert). Second row: similar train delivered, but now each depolarizing pulse was followed by an HPP (+0.6 µA, 0.5 ms, right insert). Third row: control train administered again. Time between trains was 7 min. Scale bar: 12 µm. B: temporal changes in [Ca2+]i, ---: first control; · · · : with HPP; - - - : second control. [Ca2+]i at the plateau was 4.4, 2.37, and 3.9 µM, respectively. C: delay histograms established during the plateau of [Ca2+]i. ---: first control, · · · : with HPP; - - - : second control. Quantal contents were 1.19, 0.39, and 1.1 with the corresponding medians of 2.43, 2.1, and 2.4 ms, respectively. D: samples of recordings. Left column: responses recorded during first control test pulse. Right column: responses recorded for test pulses followed by HPP. Note the change in stimulus artifact.

Figure 1B shows the temporal change in intracellular Ca2+ concentration for the stimulation regimes described. The HPP reduced the rate of [Ca2+]i accumulation and its plateau level. Under conditions of the first control, [Ca2+]i reached a plateau level of 4.4 µM, and with the HPP the plateau level of [Ca2+]i fell to 2.37 µM (56% of control) and rose again to 3.9 µM during the second control.

During the plateau levels of [Ca2+]i, quantal release (presented as delay histograms) after individual pulses was measured. The results are shown in Fig. 1C. Quantal content of the first control was 1.2. Quantal content declined to 0.4 with the HPP and recovered in the second control to 1.1. The shape of the delay histograms (time course of release) (Katz and Miledi 1965a,b) was actually the same for the first and second controls. The delay histogram corresponding to the HPP condition clearly shows that release stopped sooner than in the two controls. The median for the two controls was 2.4 ms, and with the HPP it fell to 2.1 ms (similar to the findings of Arechiga et al. 1990). In the delay histogram with the HPP, the minimal delay seems to be longer. This is an artifact resulting from the longer duration of saturation of the amplifier when both the depolarizing and hyperpolarizing pulses were administered in comparison with the period of saturation under control (compare stimulation artifacts in Fig. 1D, left and right columns). During the stimulation artifacts, no recording of quanta is possible. This introduces a small error at the beginning of the histogram but not at the decline of the histogram, the relevant phase for termination of release.

Figure 1D shows samples of quantal events recorded while [Ca2+]i remained at the plateau level. In the left column, the traces show responses obtained for the test pulse. In the right column, the test pulse was followed by HPP (ascending stimulus artifact).

Is the reduction in Ca2+ accumulation due to reduced Ca2+ entry or to accelerated removal?

Hyperpolarizing pulses might reduce Ca2+ accumulation by facilitating closure of Ca2+ channels and thereby reducing Ca2+ influx. Alternatively, HPP may predominantly accelerate Ca2+ removal by facilitating the Na+-Ca2+ exchange at the more-negative membrane potentials (Blaustein 1988; Carafoli 1987). In the first case, HPP is expected to reduce Ca2+ accumulation only if administered immediately or very soon after the depolarizing pulse. In the second case, reduction in Ca2+ accumulation is expected to be independent of the interval between depolarization and the HPP.

Figure 2 shows that at 11°C, when the HPP was administered 2 ms or even 1 ms after the end of the depolarizing test pulse, it did not affect Ca2+ accumulation (Fig. 2, B and C). In contrast, when the postpulse was administered with zero delay after the test pulse, Ca2+ accumulation was reduced by 36%, from 3.23 to 2.06 µM (Fig. 2A). In seven such experiments, HPP with zero delay reduced Ca2+ accumulation by 24.7 ± 9.7% (SD). HPP after a 1- or 2-ms delay had no effect on Ca2+ accumulation (0.85 ± 6.4% and 1 ± 4%, respectively, n = 7). The difference between Ca2+ accumulation in the control and accumulation where an HPP was administered at zero delay is very significant (P = 0.0005). These results are consistent with the assumption that HPP accelerates closure of Ca2+ channels, consequently reducing Ca2+ influx.



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Fig. 2. Effect of time interval between the depolarizing pulse and HPP on [Ca2+]i and on the synaptic delay histogram. [Ca2+]i was increased by stimulating bouton with a test pulse (-0.7 µA, 0.6 ms) at 100 Hz for 7 s. HPP was +0.6 µA, 0.5 ms. First control (---) and second control (- - -) were taken at the beginning and end of each experiment. [Ca2+]i levels during the plateau phase of the controls were 3.23 and 3.18 µM, respectively. A: HPP (· · ·) was administered with 0 delay. B: HPP delivered with 1-ms delay. C: HPP given with 2-ms delay. For 3 HPP conditions, Ca2+ concentrations during the plateau levels were 2.06, 3.4, and 3.16 µM, respectively. D-F: corresponding delay histograms for the [Ca2+]i profiles given in A-C. Note in E and F incomplete histograms because of the saturation of the amplifier when the HPP was given.

Figure 2, D-F, shows the corresponding synaptic delay histograms for the Ca2+ profiles given in Fig. 2, A-C. In Fig. 2D, when an HPP was given with zero delay, the quantal content declined from a control value of 0.74 to 0.19 (a decline of 75%). The median of the control histogram was 2.4 ms, and with the HPP it became 2.15 (a shift of 11%). In Fig. 2, E and F, because of the stimulus artifact and saturation of the amplifier, the histograms are incomplete. Yet it is clear that the decay of the histograms is the same with and without HPP. Thus termination of release was not accelerated when the HPP was given with a delay of 1 or 2 ms. Similar results were obtained in seven additional experiments.

Time course of release is not affected by experimental manipulations that affect [Ca2+]i

The time course of release was found to be insensitive to treatments known to affect Ca2+ entry or removal (Andrew and Barrett 1980; Datyner and Gage 1980; Hochner et al. 1991). We examined whether the average level of [Ca2+]i that was found to significantly affect the quantal content (Ravin et al. 1999) affects also the time course of release. As shown in Fig. 3, the normalized delay histograms were the same for different levels of [Ca2+]i, whether [Ca2+]i was increased by stimulation at different frequencies (Fig. 3D) or by application of ionomycin (Fig. 3B). When [Ca2+]i was increased by ionomycin (Fig. 3, A and B), the medians of the delay histograms were 2.02, 1.97, 2.02, 1.97, and 2.02 ms for [Ca2+]i levels of 0.5, 0.87, 1, 1.3, and 1.45 µM, respectively. Thus, with a threefold change in [Ca2+]i, the median of the histograms did not change. Similarly, when [Ca2+]i was increased by stimulation at different frequencies (a different bouton, a different animal Fig. 3, C and D), the medians of the delay histograms were 2.45, 2.42, 2.45, 2.42, and 2.52 ms for [Ca2+]i levels of 0.68, 1.43, 1.84, 2.58, and 3.25 µM. Here with a fourfold change in [Ca2+]i the medians were almost the same. Altogether, in 14 boutons (different preparations) where [Ca2+]i was raised by repetitive stimulation, the average change in the median for 35 histograms was 3.3 ± 5.3% for [Ca2+]i, ranging between a few hundred nanomoles and a few micromoles. In 10 experiments where [Ca2+]i was raised by ionomycin, the average change in the median for 93 histograms was 1.4 ± 4.7% for [Ca2+]i, ranging between a few hundred nanomolars and a few micromolars.



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Fig. 3. Level of [Ca2+]i does not affect the time course of release. A: [Ca2+]i was elevated by application of 1.75 µM ionomycin. To establish synaptic delay histogram, the bouton was stimulated, and delay histograms were established as [Ca2+]i increased in time. Synaptic delay histograms shown were obtained by drawing a continuous line through the middle point of bins of 0.25 ms, during which the number of quanta was counted. The quantal contents were 0.49, 1.03, 1.3, 1.63, and 1.84, and the medians of the delay histogram were 2.02, 1.97, 2.02, 1.97, and 2.02 ms. B: same data as in A, but each histogram is normalized to its peak. C: different bouton. [Ca2+]i was elevated by a train of 1,000 pulses of 1 ms, -0.9 µA at frequencies of 20, 40, 60, 90, and 100 Hz. [Ca2+]i concentrations were 0.68, 1.43, 1.84, 2.58, and 3.25 µM, respectively. Synaptic delay histograms shown were obtained for each frequency. Quantal contents were 0.08, 0.25, 0.56, 1.04, and 1.57, respectively. Medians of the delay histograms were 2.45, 2.42, 2.45, 2.42, and 2.52 ms, respectively. D: same data as in C, but each histogram is normalized to its peak.

Is reduced Ca2+ entry responsible for the faster termination of release?

Because we found that the HPP caused a reduction in Ca2+ entry, it is possible that this reduction is responsible for shortening the delay histograms. One way to test this question is to reduce Ca2+ entry by means other than a faster closure of the voltage-dependent Ca2+ channels and study the effect on the synaptic delay histogram. In the experiment depicted in Fig. 4, we compared the consequences of reduction in [Ca2+]i under two conditions, HPP and increased extracellular Mg2+ concentration ([Mg2+]o). For both treatments, on the same release bouton, the reduction in [Ca2+]i was similar (compare Fig. 4, A and D). In Fig. 4A (with HPP), Ca2+ accumulation declined from a level of 4.3 to 3.0 µM with recovery to a level of 4.1 µM (a reduction of 29%). The quantal content was reduced from 1.45 to 0.58 (a reduction of 60%) with recovery to 1.26. The corresponding delay histograms show once again that with HPP release terminated sooner (Fig. 4B). The median was 2.35 ms for both controls, and it declined to 2.05 for the postpulse (a shift of 13%). Normalization (Fig. 4C) shows that with the postpulse the peak of the histogram shifted to the left, and release clearly terminated sooner. Figure 4, D-F, shows the effects of increasing [Mg2+]o to 8.5 mM. In the control, [Ca2+]i was 4.1 µM (this is the second control value from Fig. 4A). It declined to 2.9 µM (in comparison with 3.0 µM with HPP in Fig. 4A) and recovered to 3.9 µM after removal of the excess Mg2+ (a reduction of 28%). With increased [Mg2+]o, the quantal content declined from 1.26 to 0.9 (a reduction of 28%), and now the peaks of the delay histograms were in the same place (medians being 2.35 ms, both for the first control and for elevated [Mg2+]o; the median of the second control was 2.45 ms). Normalization (Fig. 4F) shows that the histograms overlap completely. It should be noted that for a similar reduction in [Ca2+]i the HPP reduced the quantal content by ~30% more than the reduction obtained with increased [Mg2+]o.



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Fig. 4. Effect of HPP and [Mg2+]o on [Ca2+]i accumulation and on evoked release. A: changes in [Ca2+]i during stimulation with a train of depolarizing pulses (-0.7 µA, 0.6 ms) at 100 Hz for 7 s with and without HPP. ---: first controls; - - - : second controls. The plateau levels of [Ca2+]i of the first and second control were 4.3 and 4.1 µM, respectively. HPP of +0.6 µA, 0.5 ms with a 0 delay (· · ·) reduced plateau level of [Ca2+]i to 3.0 µM. B: delay histograms obtained during the first and second controls and with HPP. Medians of the first and second controls were 2.35 ms. Corresponding quantal contents were 1.45 and 1.26. HPP with 0 delay (· · ·) reduced the quantal content to 0.58 and shifted the median to 2.05 ms. C: normalization of the delay histograms shown in B to their peak. With HPP the peak shifted to the left and release terminated sooner. Symbols as in A. D: same bouton as in A. Changes in [Ca2+]i for the same pulse trains (as in A) at 2 levels of [Mg2+]o; 2.5 mM at the 2 controls (--- and - - - ) and 8.5 mM. Corresponding [Ca2+]i plateau levels for the controls were 4.1 and 3.9 µM. Increasing [Mg2+]o to 8.5 mM (· · ·) reduced the [Ca2+]i plateau level to 2.9 µM. E: corresponding delay histograms for controls and elevated [Mg2+]o. The quantal content declined from 1.26 to 0.9 without a change in the medians, which remained 2.35 ms. F: normalization to the peak shows overlapping of the delay histograms.

In five experiments of this type, we measured the medians of delay histograms under the three experimental conditions (control, HPP, and increased [Mg2+]o). In each experiment, measurements were made from the same release bouton under the three experimental conditions. Because the medians of the control histograms varied in the different boutons, we present the data as percent shift of medians from the control value (taken as 100%). In all five experiments, the HPP reduced the median. The average value of reduction was 8.8 ± 2.37%. On the other hand, when Ca2+ accumulation was reduced by increasing [Mg2+]o, a slight increase or decrease in the values of the median was seen. The average change in the medians was 1.1 ± 0.98%. The difference between the "HPP" and "Mg" values is significant (P = 0.05).

These results show that the effect exerted by the HPP differs from the effect obtained by increasing [Mg2+]o. The HPP reduces [Ca2+]i and in addition accelerates the termination of release; increasing [Mg2+]o only reduces [Ca2+]i.

Effect of HPP does not depend on the level of [Ca2+]i

Ravin et al. (1997) showed that repetitive stimulation at various frequencies generates different levels of [Ca2+]i, with the highest level corresponding to the highest frequency of stimulation. Furthermore, Ravin et al. (1999) showed that quantal content depends steeply on this average level of [Ca2+]i.

We repeated the same experimental protocol described for Fig. 1 at frequencies of 100, 60, and 20 Hz. Figure 5 shows that at 100 Hz [Ca2+]i reached a plateau level of 4.7 µM. The corresponding quantal content was 1.64. With the HPP, [Ca2+]i dropped to 2.36 µM (~50% reduction), and quantal content was reduced to 0.36 (a reduction of ~78%). [Ca2+]i in the second control reached a level of 4.6 µM, and the quantal content recovered to 1.45 (Fig. 5B). For the lower frequencies of 60 and 20 Hz, [Ca2+]i reached plateau levels of 2.49 µM (Fig. 5C) and 1.08 µM (Fig. 5E), respectively. After an HPP, [Ca2+]i declined to 1.83 µM at 60 Hz (a reduction of 27%) and to 0.71 µM at 20 Hz (a reduction of 34%). The corresponding quantal content was 0.74 in the control at 60 Hz, and it fell to 0.16 after an HPP (a reduction of 78%) (Fig. 5D). For 20 Hz, the quantal content was 0.17 in the control and declined to 0.04 after HPP (a reduction of 77%) (Fig. 5F).



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Fig. 5. Effects of HPP on [Ca2+]i and on the delay histograms at different rates of stimulation. Bouton was stimulated with trains of 600 pulses (-0.6 µA, 0.6 ms) at frequencies of 100, 60, and 20 Hz. Two controls at 100 Hz were taken at the beginning and the end of the experiment. At each frequency, an HPP (+0.6 µA, 0.5 ms) with 0 delay was administered, and [Ca2+]i and quantal content were measured. A: at 100 Hz, the HPP reduced the plateau [Ca2+]i from 4.7 µM (---) to 2.36 µM (· · ·) to recover to 4.6 µM (- - -) during the second control. The control quantal content was 1.64, and it was reduced by the HPP to 0.36. The quantal content of the second control was 1.45. The corresponding histograms are shown in B. C: at 60 Hz, the HPP reduced the [Ca2+]i from 2.49 to 1.83 µM, and the quantal content was reduced from 0.74 to 0.16. The corresponding delay histograms are shown in D. E: at 20 Hz, the HPP reduced [Ca2+]i from 1.08 to 0.71 µM, and quantal content was reduced from 0.17 to 0.04. The corresponding delay histograms are shown in F.

These results show that the effect of the HPP on quantal content was fairly constant at the various frequencies, irrespective of the plateau level of [Ca2+]i. Furthermore, the effect of the postpulse on quantal content was also independent of the magnitude of reduction in [Ca2+]i caused by the HPP. Similar results were obtained in five experiments. These results therefore support the possibility that the HPP, in addition to reducing Ca2+ influx, also causes the release machinery to be less sensitive to the level of [Ca2+]i.

At this stage, however, an alternative explanation cannot be excluded. Such an explanation is as follows. The HPP reduces Ca2+ influx. As a result of this reduction, the local (below release sites) Ca2+ concentration is reduced to the same extent at all frequencies, and hence the percentage of reduction in quantal content is always the same. This possibility is not very likely, as Ravin et al. (1999) showed that evoked release is very sensitive to the average level of [Ca2+]i. Nevertheless, in the following section we describe results of experiments designed specifically to distinguish between these two possibilities.

Effect of HPP on asynchronous release

It is well accepted that when asynchronous release occurs the level of Ca2+ concentration below release sites is the same as the average [Ca2+]i in the terminal (Aharon et al. 1994; Miledi 1973; Rahamimoff et al. 1978; Ravin et al. 1997). Therefore if the only effect of HPP is to reduce Ca2+ influx, and hence [Ca2+]i, it is expected that the decline in the rate of asynchronous release will correlate with the decline in [Ca2+]i caused by the HPP. On the other hand, if the HPP also causes the release machinery to be less sensitive to [Ca2+]i, then, as in the case of evoked release, the rate of asynchronous release should be much less sensitive to changes in [Ca2+]i. Figure 6 shows pooled results (·) relating the rate of asynchronous release given as number of quanta/s to [Ca2+]i raised by repetitive stimulation. Asynchronous release was measured as described by Ravin et al. (1997), 5 ms after each depolarizing pulse. Asynchronous release was also measured when each of the depolarizing pulses was followed by an HPP. Figure 6A clearly shows that with HPP the rate of asynchronous release (open circle ) is almost independent of [Ca2+]i. The points with HPP (open circle ) fall well below the control points (·). For example, for [Ca2+]i, ranging between 1.6 and 2.1 µM, the average rate of asynchronous release (average of the values in this range of [Ca2+]i) was 22.12 ± 15.6 quanta/s (n = 36); with postpulses the average was only 8.2 ± 6.6 quanta/s (n = 14). The difference is extremely significant (P << 0.0001). For [Ca2+]i levels in the range of 2.8-4 µM, the average rate of asynchronous release declined from a value of 70.16 ± 58 quanta/s (n = 24) in the control to 18.13 ± 7.5 quanta/s (n = 4) with HPP (P = 0.0001).



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Fig. 6. Effect of HPP and of Mg2+ on the rate of asynchronous release. A: relation between the rate of asynchronous release (quanta/s) and [Ca2+]i at control condition (no HPP; ·) and when HPP was given with 0 delay (open circle ). With the HPP the cloud of empty circles falls well below the cloud of dots. B: rate of asynchronous release and [Ca2+]i for the control condition (·) and when [Mg2+]o was increased to levels between 8 and 12.5 mM (open circle ). Now the cloud of empty circles falls within the cloud of dots.

On the other hand, in 13 experiments when [Ca2+]i was reduced by increasing [Mg2+]o (Fig. 6B), the reduced rate of asynchronous release (open circle ) related fully to [Ca2+]i. For [Ca2+]i ranging between 1.6 and 2.1 µM, the average rate of asynchronous release was similar in the presence of elevated [Mg2+]o and in the control (23.34 ± 14.4 quanta/s (n = 5) and 22.12 ± 15.6 quanta/s, respectively, an insignificant difference, P = 0.58). For [Ca2+]i levels ranging between 2.8 and 4 µM, the average rate of asynchronous release in the presence of elevated [Mg2+]o was 65.12 ± 26.6 quanta/s (n = 4), an insignificant change from the control of 70.16 ± 58 quanta/s.

These results strengthen the conclusion that the HPP exerts two effects. It clearly reduces Ca2+ influx, but in addition to that effect it also affects the release apparatus associated with asynchronous release, rendering it less sensitive to [Ca2+]i.

To further support this conclusion and to test whether the apparatus associated with evoked release is also affected by the HPP, we compared the relationship between the percent of reduction in [Ca2+]i (achieved either by HPP or elevated [Mg2+]o) and the percent of reduction in either asynchronous release or evoked release.

Percent reduction in release is given as (L1 - L2)/L1, and the percent reduction in Ca2+ is given as (C1-C2)/C1. Here, L1 is the control release, and L2 is the release after an HPP or after increasing [Mg2+]o. C1 stands for the control [Ca2+]i, and C2 is the Ca2+ concentration after the treatments. This way of presenting the data was selected as it generates a straight line (for moderate changes in [Ca2+]i, mathematical derivation not shown). The data to consider must be in the range where C2 is not very small in comparison with C1 (hence the same applies also to L2 in comparison with L1) such that the ratio of (L1 - L2)/L1 or (C1 - C2)/C1 is sufficiently smaller than 1. When C2 or L2 is too small, these ratios approach 1, and the existence or lack of correlation between changes in release and changes in [Ca2+]i cannot be resolved. For this reason we limited our analysis to the results where the change in [Ca2+]i did not exceed 25%. The results presented in Fig. 7 show that, when [Ca2+]i was reduced by increasing [Mg2+]o, the lines relating percent change in evoked release (Fig. 7A) or asynchronous release (Fig. 7B) show slopes of 3 and 2.5, respectively. The coefficients of determination (r2) were 0.6 and 0.5, respectively, showing a high correlation between the change in percent release as a function of the change in [Ca2+]i. Furthermore, the intercept of the two lines with the y- and x-axis is near the zero point. In other words, without a change in Ca2+ entry there is no change in the level of evoked release or asynchronous release. On the other hand, with HPP, the slopes relating the percent changes in evoked (Fig. 7C) or asynchronous release (Fig. 7D) as a function of the percent change in [Ca2+]i where 0.5 and 0.4, respectively, and the coefficients of determination were only 0.06 and 0.03, respectively. Thus with the HPP the percent change of release practically does not depend on the changes in [Ca2+]i. Also, the intercept of these two lines with the y-axis is at a level of ~55%. This result shows that not as with Mg2+ the HPP has an appreciable effect on release even when there is no reduction in Ca2+ entry.



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Fig. 7. Relationship between percent reduction in [Ca2+]i and percent reduction of evoked release when [Ca2+]i was reduced with Mg2+ or with HPP. A and B: effect of increasing [Mg2+]o to levels of 8-12.5 mM on evoked release (A) and asynchronous release (B). Slope of the line in A is 3 ± 1.02 and r2 = 0.6. Slope of the line in B is 2.5 ± 1 and r2 = 0.5. C and D: effect of HPP on evoked release C and asynchronous release D. Slope of the line in C is 0.5 ± 0.4, r2 = 0.06. Slope of the line in D is 0.4 ± 0.6, r2 = 0.03.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of HPPs on evoked release (quantal content and time course of release) was first described by Dudel (1984) in the frog neuromuscular junction and by Parnas et al. (1986b) in the crayfish. These authors investigated the effects of HPP on the time course of release because according to the CVH (see INTRODUCTION) an HPP is expected to accelerate termination of release caused by a faster transition of the active release machinery, S, into its inactive form, T. Indeed, although all other experimental treatments to date, except one (Schweizer et al. 1998), affected only the quantal content, the HPP affected both quantal content and the time course of release, reducing quantal content and accelerating termination of release. These results were interpreted to support the CVH. At the time of these earlier studies, the method for measuring intracellular Ca2+ concentration from single release boutons depolarized by a macropatch electrode (Ravin et al. 1997) was not available. For this reason, an indirect method (test pulse facilitation) (Parnas et al. 1986a) was used to estimate whether an HPP reduced Ca2+ influx (Klockner et al. 1989; Matteson and Armstrong 1986; Swandulla and Armstrong 1988) or accelerated the transition S right-arrow T or did both. Accepting that facilitation depends on residual Ca2+ (Katz and Miledi 1968), which in turn depends on Ca2+ entry and removal, it was assumed that, if the HPP reduces Ca2+ entry, both the magnitude and duration of facilitation will be reduced. Because this was not found, it was concluded that the HPP does not reduce Ca2+ entry but rather facilitates the S right-arrow T transition. This interpretation was criticized by Zucker (1987), who argued that the method of test pulse facilitation is not sensitive enough to detect small changes in Ca2+ entry. Our current results corroborate Zucker's criticism. Indeed we found that as shown by others (Klockner et al. 1989; Matteson and Armstrong 1986; Swandulla and Armstrong 1988), an HPP facilitates closure of Ca2+ channels, thus causing a reduction in Ca2+ influx.

However, our results show that HPP exerts, in addition to the previous effect, an effect on the release machinery itself. These results are consistent with the earlier interpretation of Parnas et al. (1986a) that the HPP increases the rate constant associated with the S right-arrow T transition. As a result, S right-arrow T transition is faster, and a larger fraction of S is transformed to the T state. Consequently, the quantal content is expected to decline, not only because of the reduction in Ca2+ influx but also because of the faster reduction in S. The duration of quantal release is expected to be shorter because of the faster transition S right-arrow T. Finally, for a given concentration of [Ca2+]i, the rate of asynchronous release, immediately after evoked release, is expected to be lower because of the lower level of S at the resting potential. With time, S will eventually reach the steady-state level typical for resting potential. However, in the absence of depolarization to cause T right-arrow S transition, recovery of S will take quite a long time.

The following experimental results support the conclusion that the postpulse affected the release machinery. 1) HPP reduced quantal content and accelerated termination of quantal release, whereas elevated [Mg2+]o only reduced the quantal content and did not affect the time course of quantal release. 2) With elevated [Mg2+]o, a strong correlation was found between the percent reduction in quantal content and percent reduction in [Ca2+]i, whereas with HPP this correlation was poor. The poor correlation observed in the latter case is anticipated by analysis of the complete model of the CVH (Lustig et al. 1989). Accordingly, a faster S right-arrow T transition causes the release machinery to exhibit a lower affinity to Ca2+ at the time when evoked release takes place (see Eq. 19 in Lustig et al. 1989). 3) Asynchronous release retained its control dependence on [Ca2+]i when [Ca2+]i was reduced by elevated [Mg2+]o. In contrast, asynchronous release became less sensitive to [Ca2+]i after administration of an HPP.

In conclusion, with the aid of the technique developed by Ravin et al. (1997) for measuring [Ca2+]i and release directly from single-release boutons, we were able to discern the two effects that HPP exerts and fully distinguish one from the other.


    ACKNOWLEDGMENTS

M. E. Spira is Jacomo De Viali Professor for Neurobiology and I. Parnas is Greenfield Professor for Neurobiology.

This work was supported by Grant SFB 391 from the Deutsche Forschungsgemeinschaft, Germany, to I. Parnas, H. Parnas, and J. Dudel. We thank the Anna Lea Foundation for continuous support to I. Parnas. This work was also supported by Grant I-392-216.01/94 from the German-Israel Foundation to M. E. Spira.


    FOOTNOTES

Address for reprint requests: I. Parnas, Dept. of Neurobiology, Life Sciences, Hebrew University, Jerusalem 91904, Israel.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 November 1998; accepted in final form 1 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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