HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yawo, H. Search for Related Content PubMed PubMed Citation Articles by Yawo, H.

Two components of transmitter release from the chick ciliary presynaptic terminal and their regulation by protein kinase C

Hiromu Yawo

MS 8840 Received 9 October 1998; accepted after revision 11 January 1999.

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. A study was made of the effects of phorbol ester (phorbol 12-myristate 13-acetate, PMA, 0·1 µM) on the two components of evoked transmitter release, namely the fast synchronous and the slow asynchronous components, from the giant presynaptic terminal of the chick ciliary ganglion. The excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp of the postsynaptic neuron.

2. The decay time constant of the slow component was prolonged by replacing Ca²⁺ with Sr²⁺. In 5 mM [Sr²⁺]_o the fast component decayed with a time constant of 2·6 ± 1·4 ms whereas the slow component decayed with a time constant of 19 ± 7 ms.

3. When stimulated with twin pulses with a short interpulse interval, the fast component of the second EPSC was often depressed whereas the slow component was usually facilitated. Both components were positively dependent on [Sr²⁺]_o in a saturable manner, but the fast component approached its maximum at a lower [Sr²⁺]_o than the slow component.

4. PMA potentiated both the fast and slow components to a similar extent and with a similar time course. For each component, the effect of PMA was less potent at high [Sr²⁺]_o than at low [Sr²⁺]_o. For either the fast or the slow component the PMA-induced potentiation was accompanied by a reduction in the paired-pulse ratio (PPR).

5. Despite the different dissociation constant for dextran-conjugated fura-2, the fluorescent ratio for intraterminal [Sr²⁺] ([Sr²⁺]_i) decayed to the baseline after the nerve-evoked increment with a time course similar to that for [Ca²⁺]_i, suggesting that intraterminal Sr²⁺ is buffered less efficiently than Ca²⁺. PMA did not increase the [Sr²⁺]_i transients produced by stimulation of the presynaptic oculomotor nerve.

6. It is suggested that protein kinase C (PKC) modulates both the fast and slow components through common molecular mechanisms that upregulate the Sr²⁺ sensitivity of the vesicle fusion probability.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Nerve-evoked transmitter release is induced by a synchronous Ca²⁺ influx through voltage-gated Ca²⁺ channels at presynaptic release sites (Llinás et al. 1976, 1982; Borst & Sakmann 1998). However, the latency of release (synaptic delay) can be variable among quanta (Kuno, 1964; Katz & Miledi, 1965a,b; Isaacson & Walmsley, 1995; Borst & Sakmann, 1996); most of the quanta are released in a short time window after a presynaptic action potential, but later release of quanta occurs under certain conditions, leading to the notion that nerve-evoked transmitter release consists of two or more components which are kinetically distinct (Barrett & Stevens, 1972a,b; Goda & Stevens, 1994). Goda & Stevens (1994) have characterized two components of evoked transmitter release at hippocampal synapses: a fast synchronous component which is reduced by replacing Ca²⁺ with Sr²⁺ and a slow asynchronous component which is enhanced by the Sr²⁺ substitution (see also, Meiri & Rahamimoff, 1971; McLachlan, 1977). Geppert et al. (1994) recently showed that the fast component is impaired by genetic suppression of synaptotagmin I which is a candidate for the low-affinity Ca²⁺ sensor, whereas the slow asynchronous component and spontaneous transmitter release were unaffected. Therefore, it is suggested that the transmitter release mechanisms differ for the fast and slow components.

It has been previously reported (Yawo, 1999) that activation of protein kinase C (PKC) induces the potentiation of transmitter release from the giant presynaptic terminal of chick ciliary ganglion by upregulating the Ca²⁺ sensitivity of the synaptic vesicle fusion probability. Are different molecules involved in the Ca²⁺-triggered vesicle fusion for the two components of the release? Does PKC differentially modulate the fast and slow components? In this study, these questions have been addressed and the results obtained indicate that both the fast and slow components were potentiated to a similar extent by phorbol ester and with a similar time course. The present results are consistent with the hypothesis that there are two releasable pools of vesicles. Furthermore, PKC appears to upregulate the fusion probability of the vesicles from both pools by the same or by a similar molecular mechanism.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Recordings of excitatory synaptic currents (EPSCs)

The experimental preparation and electrophysiological recording techniques were as described previously (Yawo, 1999). Briefly, chick embryos of day 14 (Stage 39-40) were decapitated, and the ciliary ganglion was removed with the oculomotor nerve. The ganglion was superfused with standard saline (mM): NaCl, 132; KCl, 5; CaCl₂, 2; MgCl₂, 1; Hepes, 10; NaOH, 5; glucose, 11; pH 7·4 adjusted with HCl. The Sr²⁺-containing solution was made by replacing CaCl₂ with SrCl₂.

A conventional whole-cell patch clamp recording was made from a postsynaptic ciliary neuron (Yawo & Chuhma, 1994) using an EPC-7 patch clamp amplifier (List Electronic, Darmstadt-Eberstadt, Germany). Patch pipettes were coated with silicon resin (KE106, Shin-Etsu, Tokyo, Japan), and fire polished, reducing the tip diameter to 2 µm. The pipettes had a resistance of 2·5-3 M when filled with the internal solution containing (mM): CsCl, 130; MgCl₂, 1; Na₂EGTA, 10; Hepes, 10; MgATP, 5; pH 7·4 adjusted with NaOH. The capacitative transient was minimized by compensating the series resistance and the input capacitance. The series resistance was usually smaller than 10 M throughout the experiment and was compensated for by 50-70 %. The whole-cell currents were low-pass filtered at 2 kHz (-3 dB, 8-pole Bessel filter, NF Electronic Instruments, Yokohama, Japan), digitized at 10 kHz (ADX-98E, Canopus, Kobe, Japan) and stored on a computer (PC-9801FA, NEC, Tokyo, Japan). All the experiments were carried out at room temperature (25°C).

Estimation of the rate of quantal transmitter release

The rate of quantal transmitter release was analysed by synaptic current integration as described by Goda & Stevens (1994) with minor modification. Since the half-decay time of the unit EPSC was on average 1·08 ms (n = 20), the unit EPSC would be expected to decay to 1/10 of its peak within 3·6 ms. The time integration of the EPSC over a 4 ms bin period is thus proportional to the product of the number of released quanta and the quantal size in electrical charge. The time integration was repeated over the 4 ms bin period from a sampling point to the point with a shift of 0·1 ms and was divided by 4 ms. This enabled the quantal release rate to be followed accurately as a function of time. The decay was tentatively fitted to the sum of two exponential functions, which were consistent with most of the experiments, namely:

Time-integrated EPSC = A₁exp(-t/₁) + A₂exp(-t/₂), (1)

where A₁ and ₁ are the initial amplitude and the decay time constant of the fast component, respectively, and A₂ and ₂ are the initial amplitude and the decay time constant of the slow component, respectively. Each product of the initial amplitude and the time constant (A₁₁ or A₂₂) is proportional to the total number of quanta of each component ('total release', T₁ or T₂). In comparing for A₁ and A₂ it was assumed that the quantal charge is the same for the two release components, although this assumption is not necessarily warranted for this preparation.

When the changes in the initial amplitudes were compared quantitatively in an experiment on the same synapse, the decay phase of the time-integrated EPSC was fitted to eqn (1) according to the average time constant of each component during the experiment. This approximation appeared to be permissible because the time constant of each component was relatively stable during the experiment.

Measurement of intraterminal Sr²⁺ concentration ([Sr²⁺]_i)

The method of measuring [Sr²⁺]_i was almost the same as that for [Ca²⁺]_i as described previously (Yawo, 1999). The oculomotor nerve was cut at its exit from the orbital bone in Ca²⁺-free saline containing 1 mM EGTA. Crystals of fura-2-conjugated dextran (Fura-dextran, molecular weight 10000, Molecular Probes Inc.) were applied to the distal stump. After 30 min of incubation at 10°C, the ganglion was superfused with oxygenated standard saline and incubated at 37°C for 1·5 h. The fluorescence-labelled terminal was focused under the microscope and fluorescence excited alternately at wavelenths of 340 (₁) and 380 nm (₂) was measured at a single spot with a diameter of 50 µm by a photomultiplier tube (OSP-3, Olympus, Tokyo, Japan). The signal was integrated for 100 ms and sampled at 10 Hz by a computer (PC-9801RS, NEC, Tokyo, Japan). To reduce the signal-to-noise ratio, 10 records were averaged using the computer-generated stimulating pulse as a trigger.

In the presence of the two interacting cations, Ca²⁺ and Sr²⁺, the fluorescence intensities at wavelengths ₁ and ₂ will be given by the following equation (Grynkiewicz et al. 1985) using the proportionality coefficients, S_f1 (free dye at ₁), S_f2 (free dye at ₂), S_b1 (Ca²⁺-bound dye at ₁), S_b2 (Ca²⁺-bound dye at ₂), S_b1' (Sr²⁺-bound dye at ₁) and S_b2' (Sr²⁺-bound dye at ₂).

F₁ = S_f1c_f + S_b1c_b + S_b1'c_b', (2)

F₂ = S_f2c_f + S_b2c_b + S_b2'c_b', (3)

where c_f is the concentration of free dye, c_b is the concentration of Ca²⁺-bound dye and c_b' is the concentration of Sr²⁺-bound dye. Provided that Ca²⁺ and Sr²⁺ interact with the dye independently:

c_b = c_f [Ca²⁺]/K_d(Ca), (4)

c_b' = c_f [Sr²⁺]/K_d(Sr), (5)

where K_d(Ca) and K_d(Sr) are the dissociation constants for Ca²⁺ and Sr²⁺, respectively. The fluorescence ratio R = F₁/F₂ is rewritten as follows by using S_f1' = S_f1 + S_b1[Ca²⁺]/K_d(Ca) and S_f2' = S_f2 + S_b2[Ca²⁺]/K_d(Ca).

Solving for [Sr²⁺] yields the following equation.

where = (S_f2'/S_b2'). Note that S_b1'/S_b2' is the limiting value R_max that the ratio has at saturating [Sr²⁺]. Putting R_Ca = S_f1'/S_f2' which is the fluorescence ratio in the absence of [Sr²⁺]_i, the above equation may therefore be rewritten as:

If the [Ca²⁺]_i is little affected by the [Sr²⁺]_i in the range of experiments, the relative change of [Sr²⁺]_i during nerve stimulation, [Sr²⁺]_i, is dependent on R at rest (R_r) and R during stimulation (R_s). That is:

The [Sr²⁺]_i was usually measured in a nominally Ca²⁺-free solution. Therefore, the fluorescence intensity and R_Ca were measured in a nominally Ca²⁺- and Sr²⁺-free solution, and then the minimum fluorescence at ₂ and R_max were measured in a nominally Ca²⁺-free solution containing 10 mM Sr²⁺ and 0·1 mM ionomycin (Calbiochem, La Jolla, CA, USA). Experimentally, = 4·5, R_max = 2·8 and R_Ca = 0·85. The value adopted for K_d(Sr) was 7·6 µM (Kwan & Putney, 1990; see also Schilling et al. 1989).

All the above experiments were carried out in accordance with the guiding principles of the Physiological Society of Japan. The values in the text and figures are means ± S.E.M. (number of experiments). Statistically significant differences between various parameters were determined using Student's two-tailed t test unless otherwise noted.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Properties of the fast and slow components of transmitter release

When [Ca²⁺]_o was replaced with equimolar Sr²⁺, the EPSC amplitude was markedly depressed; in 5 mM [Ca²⁺]_o the EPSC amplitude was 2106 ± 317 pA whereas it was 524 ± 95 pA in 5 mM [Sr²⁺]_o (n = 8, P< 0·001). Therefore, Sr²⁺ supports transmitter release from the chick ciliary giant presynaptic terminal but with less efficiency than Ca²⁺ (Meiri & Rahamimoff, 1971; McLachlan, 1977; Goda & Stevens, 1994).

The time course of the EPSC was compared in 1 mM [Ca²⁺]_o and in 5 mM [Sr²⁺]_o solution in which the EPSC amplitudes were comparable. The top trace in Fig. 1A shows an overlay of 10 successive records in 1 mM [Ca²⁺]_o. Although the onset times of EPSC appeared synchronous, a small but significant slow component was revealed by analysing the synaptic current integration (Fig. 1A, bottom). In 5 mM Sr²⁺, small EPSCs appeared with variable latencies (Fig. 1B, top) after the capacitative electrical coupling response originated from an action potential in the presynaptic terminal (Yawo & Chuhma, 1994). The Sr²⁺ substitution did not affect the decay time constant of the fast component (₁), whereas it prolonged the decay time constant of the slow component (₂) (Fig. 1B, bottom). In summary, neither ₁ nor ₂ were dependent on [Ca²⁺]_o (Fig. 1C and D). In 11 experiments in 5 mM [Sr²⁺]_o, ₁ was on average 2·3 ± 0·4 ms, which was not significantly different from that in [Ca²⁺]_o solution (1·7 ± 0·2 ms, n = 14, P > 0·2). On the other hand, ₂ measured in [Sr²⁺]_o solution (18 ± 1 ms, n = 11) was significantly longer than that measured in [Ca²⁺]_o solution (9·2 ± 0·5 ms, n = 14, P< 0·001).

		View larger version [in this window] [in a new window]
Figure 1. Analysis of the fast and slow components of transmitter release A, top, sample records of EPSCs in solutions containing 1 mM Ca2+. Each is the overlay of 10 successive records from one preparation. Bottom, the EPSCs were averaged, time-integrated over 4 ms bin periods, normalized to their peak values, and plotted on a semi-logarithmic scale. The dotted line shows the best fits to the sum of the two exponential functions (eqn (1) in the text); A1exp(-t/1) + A2exp(-t/2), where A1 = 60·5 pA, 1 = 1·03 ms, A2 = 24·2 pA and 2 = 7·1 ms. B, top, the overlay of 10 successive records of EPSCs in solutions containing 5 mM Sr2+. Bottom, the 4 ms-integrated EPSC was plotted as shown in A and fitted to the sum of the two exponential functions (dotted line); A1 = 111 pA, 1 = 1·25 ms, A2 = 30·9 pA and 2 = 20·3 ms. C, the effect of Sr2+ substitution on 1. Each column and bar (from left to right) represent mean ± S.E.M. of 1 at 1 mM [Ca2+]o (n = 7), at 5 mM [Ca2+]o (n = 7) and at 5 mM [Sr2+]o (n = 11). The effect of Sr2+ substitution was not significant (P > 0·3 for 1 mM [Ca2+]o and P > 0·1 for 5 mM [Ca2+]o). D, similar to C but the Sr2+ substitution significantly prolonged 2 (P < 0·001 for both 1 and 5 mM [Ca2+]o).

In the experiment shown in Fig. 2A, the presynaptic nerve was stimulated with twin pulses (40 ms interval) in a Sr²⁺-substituted solution. The time integration of either the first or the second EPSC was fitted to eqn (1) with similar ₁ (the first EPSC, 1·02 ms; the second EPSC, 1·28 ms) and ₂ (the first EPSC, 22·1 ms; the second EPSC, 33·8 ms) (Fig. 2B). For the second EPSC, the initial amplitude of the fast component (A₁) was strongly depressed compared with the first EPSC (first EPSC, 218 pA; second EPSC, 120 pA) and the paired-pulse ratio (PPR; A₁ of the second EPSC divided by A₁ of the first EPSC) was 0·551. In contrast, the initial amplitude of the slow component (A₂) was slightly facilitated (first EPSC, 82·9 pA; second EPSC, 99·3 pA) (Fig. 2A and B) and the PPR (A₂ of the second EPSC divided by A₂ of the first EPSC) was 1·20. In a few experiments, the fast component was almost abolished at the second EPSC. In nine experiments in 5 mM [Sr²⁺]_o, the PPR with a pulse interval of 40 ms was 0·90 ± 0·20 (range, 0·0-2·29) for the fast component and 1·95 ± 0·22 (range, 1·28-3·33) for the slow component, and the difference was significant (P < 0·002).

		View larger version [in this window] [in a new window]
Figure 2. Differentiation of two components by paired-pulse protocol A, average of 5 successive records of paired EPSCs with an interval of 40 ms in a solution containing 5 mM Sr2+. B, semi-logarithmic plot of 4 ms integration of the first EPSC (thick line) and the second EPSC (thin line) shown in A. Values were normalized to the peak amplitude of the first EPSC. The dotted lines show the best fits to the sum of the two exponential functions (eqn (1) in the text) as in Fig. 1B. For the first EPSC, A1 = 218 pA, 1 = 1·02 ms, A2 = 82·9 pA and 2 = 22·1 ms. For the second EPSC, A1 = 120 pA, 1 = 1·28 ms, A2 = 99·3 pA and 2 = 33·8 ms.

The depression of A₁ could not be explained by the desensitization of the postsynaptic acetylcholine (ACh) receptors because A₂ was facilitated at the same time and the postsynaptic ACh receptor desensitizes in the order of seconds in the chick ciliary ganglion (Ogden et al. 1984). If the depression of A₁ is attributed to the partial depletion of the readily releasable pool of vesicles (Yawo, 1999), the vesicle pool for the slow component is different from that for the fast component: the fast component originates from a smaller pool than the slow component (see later section and Discussion). Alternatively, the [Sr²⁺]_i inactivates the fast component more effectively than the slow component.

Sr²⁺ sensitivity of two components of transmitter release

The A₁/A₂ ratio was negatively correlated with [Sr²⁺]_o, e.g. 2·80 ± 0·57 at 2 mM (n = 12), 0·81 ± 0·17 at 10 mM (n = 9); this difference was significant (P < 0·03). Therefore, it is suggested that the fast and slow components of transmitter release differ in their sensitivity to [Sr²⁺]_o. This notion was tested by plotting the total release of each component (T₁ or T₂) against [Sr²⁺]_o (x), which can quantitatively be fitted by the following equation under constant Mg²⁺ (Dodge & Rahamimoff, 1967):

T = T_max(x/(x + K_D'))ⁿ, (10)

where T_max is the maximum capacity and K_D' is the apparent dissociation constant. The co-operativity, n, was tentatively assumed to be 3 (Yawo & Chuhma, 1994). For the fast component, the release-[Sr²⁺]_o relation had a tendency to be saturated at high [Sr²⁺]_o (Fig. 3, ); a twofold change of [Sr²⁺]_o from 5 to 10 mM increased T₁ by 1·2. On the other hand, the slow component is more sensitive to [Sr²⁺]_o, even at high concentrations (Fig. 3, ); a twofold change of [Sr²⁺]_o from 5 to 10 mM increased T₂ by 4·8. As a result, the release-[Sr²⁺]_o relation differed considerably between the two components: K_D' = 3·3 mM and T_max = 8·2 nC for the fast component (T₁) and K_D' = 9·6 mM and T_max = 337 nC for the slow component (T₂) when fitted to eqn (10). Thus, the slow component was apparently less sensitive to [Sr²⁺]_o than the fast component, which is consistent with the fact that the A₁/A₂ ratio decreased when [Sr²⁺]_o increased.

		View larger version [in this window] [in a new window]
Figure 3. The [Sr2+]o dependency of the two components of transmitter release Total release of the fast and slow components (T1, ; T2, ) are plotted against [Sr2+]o of 1 mM (n = 5), 2 mM (n = 11), 5 mM (n = 11) and 10 mM (n = 9). The Mg2+ concentration was constant at 1 mM. The line is the prediction from the Dodge-Rahamimoff equation (eqn (10) in text): T = Tmax(x/(x + KD'))3, where Tmax is the maximal capacity and KD' is the apparent dissociation constant. For the fast component, Tmax = 8·2 nC and KD' = 3·3 mM, whereas for the slow component, Tmax = 337 nC and KD' = 9·6 mM.

The PPR was also dependent on [Sr²⁺]_o for both the fast and slow components. The PPR of the fast component (A₁) was usually over unity in 2 mM [Sr²⁺]_o, and decreased with the increase of [Sr²⁺]_o (Fig. 4A, ). The PPR of the slow component (A₂) also decreased with the increase of [Sr²⁺]_o (Fig. 4B, ), but was usually larger than that for the fast component. This is consistent with the idea that manoeuvres which increase the probability of vesicular exocytosis would decrease PPR as a result of the depletion of releasable vesicles (Debanne et al. 1996; Schulz, 1997).

		View larger version [in this window] [in a new window]
Figure 4. Effects of PMA on the paired-pulse ratio (PPR) A, paired-pulse ratio (PPR) of the fast component of EPSCs at various [Sr2+]o before () and after () treatment with PMA (0·1 µM). The presynaptic oculomotor nerve was stimulated by twin pulses with an interval of 40 ms. Both the first and second EPSCs were fitted to eqn (1) in the text as in Fig. 2B, and the PPR was calculated as A1 of the second EPSC divided by A1 of the first EPSC. Number of experiments was 11 (control) and 10 (PMA) in 2 mM [Sr2+]o, 9 (control) and 10 (PMA) in 5 mM [Sr2+]o, 8 (control) and 3 (PMA) in 10 mM [Sr2+]o. B, PPR of the slow component of EPSCs in various [Sr2+]o before () and after () treatment with PMA (0·1 µM). The PPR was calculated as A2 of the second EPSC divided by A2 of the first EPSC. Number of experiments is the same as in A.

Effect of PMA on the two components of release

Figure 5A shows the effect of 0·1 µM PMA on the fast component of release in a Sr²⁺-substituted solution. A brief application of PMA potentiated the fast component (A₁) in a sustained manner. Similarly, PMA potentiated the slow component (A₂) of the transmitter release (Fig. 5B). In order to determine if PMA potentiates both the fast and slow components with a similar time course, the data in Fig. 5A and B were re-plotted as Fig. 5C. With the application of PMA, the initial amplitudes of both components (A₁ and A₂) were potentiated simultaneously. Although variable during the experiment, ₁ and ₂ were little affected by PMA. The possibility that a change in the time constants caused the appearance of the potentiation was tested by comparing four control records with four records during and after the application of PMA which had similar values of ₁ (1·0 < ₁ < 1·3 ms). Under control conditions, A₁ = 105 ± 4·9 pA, A₂ = 28·9 ± 4·0 pA, ₁ = 1·16 ± 0·04 ms and ₂ = 21·4 ± 1·5 ms. During and after the application of PMA, A₁ = 254 ± 2·1 pA and A₂ = 76·5 ± 3·3 pA, respectively (P < 0·001, compared with their corresponding controls), while ₁ = 1·15 ± 0·04 ms and ₂ = 22·5 ± 0·9 ms, respectively (P > 0·6, compared with their controls). In nine similar experiments with 5 mM [Sr²⁺]_o, PMA potentiated A₁ 2·71 ± 0·82-fold (P < 0·01, between raw data) and A₂ 2·37 ± 0·62-fold (P < 0·01, between raw data).

		View larger version [in this window] [in a new window]
Figure 5. Effects of PMA on the two components of transmitter release A, representative data showing the effect of PMA on the fast component in a solution containing 5 mM Sr2+. The initial amplitude (A1) of eqn (1) was plotted against time. PMA (0·1 µM) was perfused during the indicated period. B, effect of PMA on the slow component of the same experiment in A. Initial amplitude (A2) of eqn (1) was plotted against time. PMA (0·1 µM) was perfused during the indicated period. C, the 5th to 11th data points from the same experiment as in A and B are plotted to reveal the correlation between the fast component and the slow component. The correlation coefficient was 0·94.

PMA decreased the PPR of both the fast (Fig. 4A, ) and slow components (Fig. 4B, ). After treatment with PMA in 5 mM [Sr²⁺]_o, the PPR with a pulse interval of 40 ms was 0·52 ± 0·14 (n = 10, P < 0·02 compared with the control) for the fast component and 1·46 ± 0·15 (n = 10, P < 0·002) for the slow component.

Sr²⁺ dependency of PMA-induced potentiation

Is the PMA-induced potentiation of each component dependent on [Sr²⁺]_o? Figure 6A shows a summary of the PMA-induced potentiations of the fast and slow components in various [Sr²⁺]_o. In 2 mM [Sr²⁺]_o PMA (0·1 µM) consistently potentiated the fast component (A₁) (range, 1·5-7·2 of control, n = 9), and the magnitude of potentiation was significant (P < 0·01). In 10 mM [Sr²⁺]_o the same concentration of PMA did not potentiate A₁ significantly (P > 0·8, n = 9). The effects of PMA on the slow component of transmitter release (A₂) were also dependent on [Sr²⁺]_o (Fig. 6A); PMA did not show significant effects on A₂ (P > 0·1, n = 9) in 10 mM.

		View larger version [in this window] [in a new window]
Figure 6. Sr2+ dependency of PMA-induced potentiation A shows that PMA-induced potentiation of EPSC was dependent on [Sr2+]o. Each EPSC amplitude was normalized to the control value before the application of PMA. The columns are (from left to right) the effect of 0·1 µM PMA on the fast component (A1) with [Sr2+]o of 2 mM (n = 9), 5 mM (n = 11) and 10 mM (n = 9), and the effect of 0·1 µM PMA on the slow component (A2) in [Sr2+]o of 2 mM (n = 9), 5 mM (n = 11) and 10 mM (n = 9). For the fast component, the effect of PMA was significantly less in 10 mM [Sr2+]o than in 2 mM (P < 0·03). For the slow component, the effect of PMA was significantly less in 10 mM [Sr2+]o than in 2 mM (P < 0·03) and in 5 mM (P < 0·01). There was no significant effect of PMA in 10 mM [Sr2+]o for both the fast (P > 0·2) and slow components (P > 0·09). B, effects of PMA on EPSC in a solution containing 20 mM Sr2+ and the effects of changing the solution to 10 mM Ca2+. Each trace is an average of 5 consecutive records evoked at 0·05 Hz. The traces are (from left to right) the control record in 20 mM [Sr2+]o, after 3-5 min application of 0·1 µM PMA, and 3-5 min after switching to 10 mM [Ca2+]o.

Figure 6B shows another experiment in which [Sr²⁺]_o was 20 mM. Since the fast component was extremely small compared with the slow component, and the slow component peaked with a delay of 3-4 ms after the fast component, it was difficult to fit the time integration of EPSC into a model as simple as eqn (1). The addition of PMA only slightly potentiated the EPSC, suggesting that the slow transmitter release had approached its maximum in 20 mM [Sr²⁺]_o. In six similar experiments, the mean peak EPSC was 1·40 ± 0·14 of control in the presence of PMA, and the effect of PMA was not significant (P > 0·1). It appears unlikely that release of ACh from the nerve terminal in 20 mM [Sr²⁺]_o was so great as to be near the saturation of the postsynaptic ACh receptors, because significantly larger EPSCs could be evoked at the same synapse when the extracellular solution was switched to one containing 10 mM Ca²⁺ (Fig. 6B). Very similar results were observed in five other experiments (mean EPSC = 3·16 ± 0·43 of control, n = 6). The influence of substitution of Ca²⁺ for Sr²⁺ on the amplitude of single EPSCs, if any, did not appear to be sufficient to explain the marked increase in EPSC amplitude (McLachlan, 1977).

Presynaptic Sr²⁺ influx

It has previously been shown (Yawo, 1999) that PMA did not affect the [Ca²⁺]_i transients in the giant presynaptic terminals during stimulation of the presynaptic oculomotor nerve. Thus, it is assumed that PMA-induced potentiation is accompanied by no changes in net Sr²⁺ influx, buffering or removal. To test this notion, presynaptic [Sr²⁺]_i transients were measured. As shown in the top traces in Fig. 7A, in 2 mM [Sr²⁺]_o, the increase in [Sr²⁺]_i in response to a single stimulus was 0·36 µM, whereas it was 3·44 µM in response to a train of 10 stimulation pulses at 50 Hz. In the same calyx, the intraterminal transient of [Ca²⁺]_i was much smaller than that of Sr²⁺ (Fig. 7A, bottom). On average, in 2 mM [Sr²⁺]_o, the [Sr²⁺]_i increment during the train was 3·12 ± 0·31 µM (n = 7), 9·2 ± 0·6-fold greater than the response to a single stimulus (0·31 ± 0·03 µM). Similarly, in 2 mM [Ca²⁺]_o, the [Ca²⁺]_i increment during the train was 365 ± 24 nM (n = 7), which is 9·3 ± 0·5-fold greater than the response to a single stimulus (41 ± 5 nM). Therefore, the accumulation of divalent cations during a train was nearly proportional to the number of pulses at this condition. If [Sr²⁺]_i is buffered as efficiently as [Ca²⁺]_i, one can expect that the fluorescence ratio of fura-2-conjugated dextran would decay more rapidly in the [Sr²⁺]_o-substituted solution than in the [Ca²⁺]_o solution, because the K_d(Sr) (2·6 µM, Schilling, et al. 1989; 7·6 µM, Kwan & Putney, 1990) was about one order of magnitude higher than the K_d(Ca) (350 nM, Yawo & Chuhma, 1994). Actually, the calculated [Sr²⁺]_i decreased to the baseline with a time course similar to that of the calculated [Ca²⁺]_i (Fig. 7A). In seven experiments the half-decay time of the fluorescence ratio after a train was 618 ± 75 ms with 2 mM [Ca²⁺]_o and was 645 ± 45 ms with 2 mM [Sr²⁺]_o (P > 0·8). As a result, the Sr²⁺ concentration remained as high as several hundred nanomolar for seconds after a single action potential in a presynaptic terminal whereas the Ca²⁺ concentration was reduced to less than 100 nM within 1 s. It is thus suggested that intracellular Sr²⁺ is buffered less efficiently than Ca²⁺.

		View larger version [in this window] [in a new window]
Figure 7. Presynaptic Sr2+ during PMA-dependent potentiation A, sample records of intraterminal Sr2+ concentration ([Sr2+]i) in 2 mM [Sr2+]o (top traces) and those of intraterminal Ca2+ concentrations in 2 mM [Ca2+]o from the same calyx (bottom traces). For each condition, the response to a single stimulus to the oculomotor nerve and that to a train of 10 stimuli at 50 Hz are overlaid. Note the different scale for ion concentrations. B, [Sr2+]o dependence of the train-induced increase of [Sr2+]i. Seven experiments were summarized by normalizing the train-induced increment of [Sr2+]i ([Sr2+]pre) to the value in 1 mM [Sr2+]o. C, summary of the effect of PMA on [Sr2+]pre (n = 7). The values are normalized to the control value in 2 mM [Sr2+]o before the application of PMA (left column). [Sr2+]o was changed from 4 mM (right column) to 2 mM and the effect of PMA (0·1 µM) was examined (middle column). The addition of 0·1 µM PMA significantly reduced [Sr2+]pre (P < 0·03).

In Fig. 7B, the dependence of the [Sr²⁺]_i increment on [Sr²⁺]_o was tested. Since the train-induced increment of [Sr²⁺]_i ([Sr²⁺]_pre) was almost linearly dependent on [Sr²⁺]_o between 1 and 4 mM, [Sr²⁺]_pre in 2 mM [Sr²⁺]_o was used to evaluate the effect of PMA on the Sr²⁺ influx. If PMA potentiates transmitter release by increasing Sr²⁺ influx into the presynaptic terminal, a PMA-dependent enhancement of [Sr²⁺]_pre would be expected. Actually, the addition of 0·1 µM PMA slightly reduced [Sr²⁺]_pre (to 0·85 ± 0·04 of the control; n = 7, P < 0·03; Fig. 7C). Therefore, the PMA-induced potentiation of transmitter release in the Sr²⁺-substituted solution was not accompanied by enhanced Sr²⁺ influx.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Potentiation of two components of transmitter release by PKC

Replacement of the extracellular Ca²⁺ by Sr²⁺ revealed two temporally distinct components of evoked transmitter release: a fast synchronous component and a slow asynchronous component (Fig. 1A and B). These components appear to be independently regulated based on the following three observations. First, the decay time constant of the slow component (₂) was prolonged twofold by Sr²⁺ substitution, whereas the decay time constant of the fast component (₁) was not (Fig. 1C and D). Secondly, when two stimuli were delivered in close succession, the fast component was often depressed while the second component was usually facilitated (Fig. 2). Thirdly, the release-[Sr²⁺]_o relationship differed considerably between the two components (Fig. 3): the fast component had a lower K_D' value than the slow component when fitted to eqn (10). The fact that the A₁/A₂ ratio was negatively correlated with [Sr²⁺]_o indicates that the release-[Sr²⁺]_o relations differ between the fast and slow components without assuming eqn (10). The different T_max values also suggest that the release mechanisms differ between the two components. The total charge carried by the EPSC in 5 mM [Sr²⁺]_o was on average 2·7 ± 0·6-fold (n = 7) of that in 1 mM [Ca²⁺]_o. This value corresponds to 80-160 quanta, if the average EPSC in 1 mM [Ca²⁺]_o is considered to consist of 30-60 quanta (Yawo, 1999). According to eqn (10), 44-88 quanta is the maximum capacity of releasable quanta for the fast component and 160-320 quanta for the slow component, if the quantal charge is assumed to be the same for both components. There must be a mechanism regulating the maximum capacity of releasable quanta for each component.

Does PKC differentiate the two components of transmitter release in a Sr²⁺-substituted solution? PMA potentiated both the fast and slow components to a similar extent and with a similar time course (Fig. 5C). For each component the effect of PMA was dependent on [Sr²⁺]_o and was less potent in high [Sr²⁺]_o than in low [Sr²⁺]_o (Fig. 6). Since PMA did not increase the Sr²⁺ influx (Fig. 7C), the two exocytotic processes, the fast and slow transmitter release, both seem to be potentiated by PKC activation through mechanisms enhancing the sensitivity to Sr²⁺ of the synaptic vesicle fusion probability. This is consistent with the observation that PMA decreased the PPR of each component with an increase in the EPSC amplitude (Fig. 4). This is also consistent with the fact that PMA-induced potentiation is not accompanied by a change in the size of the readily releasable pool of vesicles (Yawo, 1999). In summary, there is no evidence suggesting that PKC differentially modulates the two components.

Mechanisms that disintegrate the transmitter release into two components

How does Sr²⁺ substitution prolong the slow component? Since the intracellular buffering activity may be weaker for Sr²⁺ than for Ca²⁺ (Fig. 7A; see also Proks & Ashcroft, 1995), the increased level of free [Sr²⁺]_i would be maintained for a longer period.

Goda & Stevens (1994) proposed a two-sensor hypothesis: at least two Ca²⁺-sensitive molecules, a low-affinity Ca²⁺ sensor and a high-affinity Ca²⁺ sensor, regulate exocytosis in parallel. Sr²⁺ is less effective than Ca²⁺ on the low-affinity sensor which induces rapid synchronous exocytosis upon binding, whereas Sr²⁺ binding enhances the efficacy of the high-affinity sensor which induces slow asynchronous exocytosis. This hypothesis is consistent with the observation that the fast component is impaired by genetic suppression of synaptotagmin I, which is a candidate for the low-affinity Ca²⁺ sensor (Geppert et al. 1994). This hypothesis predicts that the A₁/A₂ ratio would be positively correlated with [Sr²⁺]_o, but this was not the case in the present experiment. The A₁/A₂ ratio was high in low [Sr²⁺]_o and low in high [Sr²⁺]_o. To account for this discrepancy additional mechanisms are required, e.g. the possible inactivation of the low-affinity sensor in high [Sr²⁺]_o. The same mechanism might depress the fast component at the second stimulus of a twin-pulse protocol. Since PMA potentiated the fast and slow components simultaneously (Fig. 4C), PKC appears to modulate a molecule regulating the Sr²⁺ sensitivity of both the high- and low-affinity Ca²⁺ sensor molecules.

However, it is not necessary to assume the presence of two types of Ca²⁺ sensors, if two fusion-competent mechanisms are proposed: a fast fusion-competent mechanism that is rapidly inactivated in high [Ca²⁺]_i and a slow fusion-competent mechanism that is not inactivated (two-kinetic component hypothesis). Upon binding Ca²⁺/Sr²⁺, the Ca²⁺ sensor triggers the two fusion-competent mechanisms in parallel. For the fast fusion-competent mechanism the conformational transition occurs rapidly, releasing the transmitter with a short latency period. Subsequently, the mechanism is inactivated by Ca²⁺/Sr²⁺, thereby forming the fast component of the transmitter release. For the slow fusion-competent mechanism the transition latency is variable, releasing transmitter asynchronously (the slow component of transmitter release). Since the latter mechanism is not inactivated by divalent cations, the release is enhanced by Sr²⁺ which is weakly buffered. The difference in K_D' values as well as the difference in the PPR between the two components may be attributable to the presence of a Sr²⁺-dependent inactivation of the fast fusion-competent mechanism. In this model, the fast component could be expected to be progressively depressed by repeated stimulation with a short interval. Therefore, additional mechanisms are required to account for the fact that the EPSC approached the steady state during a high-frequency stimulation (Yawo, 1999). If one assumes the two-kinetic component hypothesis, PKC would upregulate the Sr²⁺ sensitivity of a single Ca²⁺ sensor.

Alternatively, the vesicle pool for the fast component and that for the slow component might be different (two-releasable pool hypothesis). One possibility is that the vesicles docked adjacent to N-type Ca²⁺ channels (Yoshida et al. 1992; Sheng et al. 1994) are the pool for the fast component (instantaneously releasable pool). Those vesicles that are docked at the membrane but are not associated with Ca²⁺ channels constitute the pool for the slow component (reserve releasable pool). The Sr²⁺ concentration near the vesicle pool for the fast component is elevated instantaneously and causes synchronous transmitter release. The releasing ability rapidly declines due to the small size of the vesicle pool. For the slow component, the Sr²⁺ concentration near the vesicle pool increases and decreases slowly, releasing transmitters asynchronously. The fast component must arise from a smaller vesicle population and have a higher fusion probability than the slow component because the fast component is more easily depressed. The difference in K_D' values between the two components may be attributable to the topological difference between the two vesicle pools. The vesicle-Ca²⁺ channel interaction would draw vesicles from the reserve releasable pool towards the instantaneously releasable pool. This idea comes from a recent experiment in which presynaptic injection of 'synprint', the syntaxin binding domain of the N-type Ca²⁺ channel, shifted the release-[Ca²⁺]_o relation towards lower [Ca²⁺]_o with no change in the maximum capacity (Rettig et al. 1997; Neher, 1998). In fact, at the frog neuromuscular junction high K⁺ stimulation initially activates exocytosis at the active zones followed by partial inactivation, and subsequently activates ectopic exocytosis (Ceccarelli et al. 1988). In this model, the Ca²⁺-sensing mechanism for the fast and slow components may be identical. If one assumes the two-releasable pool hypothesis, PKC would upregulate the Sr²⁺ sensitivity of a single Ca²⁺ sensor of both vesicle pools. The present results are all consistent with the two-releasable pool hypothesis. However, further studies are necessary in order to discriminate among the two-sensor hypothesis, the two-kinetic component hypothesis and the two-releasable pool hypothesis.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Barrett, E. F. & Stevens, C. F. (1972a). Quantal independence and uniformity of presynaptic release kinetics at the frog neuromuscular junction. The Journal of Physiology 227, 665-689	[Medline]
Barrett, E. F. & Stevens, C. F. (1972b). The kinetics of transmitter release at the frog neuromuscular junction. The Journal of Physiology 227, 691-708	[Medline]
Borst, J. G. G. & Sakmann, B. (1996). Calcium influx and transmitter release in a fast CNS synapse. Nature 383, 431-434	[Medline]
Borst, J. G. G. & Sakmann, B. (1998). Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem. The Journal of Physiology 506, 143-157	[Abstract/Full Text]
Ceccarelli, B., Fesce, R., Grohovaz, F. & Haimann, C. (1988). The effect of potassium on exocytosis of transmitter at the frog neuromuscular junction. The Journal of Physiology 401, 163-183	[Abstract]
Debanne, D., Guéromeai, N. C., Gähwiler, B. H. & Thompson, S. M. (1996). Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release. The Journal of Physiology 491, 163-176	[Abstract]
Dodge, F. A. Jr & Rahamimoff, R. (1967). Co-operative action of calcium ions in transmitter release at the neuromuscular junction. The Journal of Physiology 193, 419-432	[Medline]
Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F. & Südhof, T. C. (1994). Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717-727	[Medline]
Goda, Y. & Stevens, C. F. (1994). Two components of transmitter release at a central synapse. Proceedings of the National Academy of Sciences of the USA 91, 12942-12946	[Medline]
Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450	[Abstract]
Isaacson, J. S. & Walmsley, B. (1995). Counting quanta: direct measurements of transmitter release at a central synapse. Neuron 15, 875-884	[Medline]
Katz, B. & Miledi, R. (1965a). The measurement of synaptic delay, and the time course of acetylcholine release at the neuromuscular junction. Proceedings of the Royal Society B 161, 483-495.
Katz, B. & Miledi, R. (1965b). The effect of temperature on the synaptic delay at the neuromuscular junction. The Journal of Physiology 181, 656-670	[Medline]
Kuno, M. (1964). Quantal components of excitatory synaptic potentials in spinal motoneurones. The Journal of Physiology 175, 81-99.
Kwan, C.-Y. & Putney, W. Jr (1990). Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Journal of Biological Chemistry 265, 678-684	[Abstract]
Llinás, R., Steinberg, I. Z. & Walton, K. (1976). Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate. Proceedings of the National Academy of Sciences of the USA 73, 2918-2922.
Llinás, R., Sugimori, M. & Simon, S. M. (1982). Transmission by presynaptic spike-like depolarization in the squid giant synapse. Proceedings of the National Academy of Sciences of the USA 79, 2415-2419	[Medline]
McLachlan, E. M. (1977). The effects of strontium and barium ions at synapses in sympathetic ganglia. The Journal of Physiology 267, 497-518	[Abstract]
Meiri, U. & Rahamimoff, R. (1971). Activation of transmitter release by strontium and calcium ions at the neuromuscular junction. The Journal of Physiology 215, 709-726	[Medline]
Neher, E. (1998). Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20, 389-399	[Medline]
Ogden, D. C., Gray, P. T. A., Colquhoun, D. & Rang, H. P. (1984). Kinetics of acetylcholine activated ion channels in chick ciliary ganglion neurones grown in tissue culture. Pflügers Archiv 400, 44-50	[Medline]
Proks, P. & Ashcroft, F. M. (1995). Effects of divalent cations on exocytosis and endocytosis from single mouse pancreatic -cells. The Journal of Physiology 487, 465-477	[Abstract]
Rettig, J., Heinemann, C., Ashery, U., Sheng, Z.-H., Yokoyama, C. T., Catterall, W. A. & Neher, E. (1997). Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction. Journal of Neuroscience 17, 6647-6656	[Abstract/Full Text]
Schilling, W. P., Rajan, L. & Strobl-Jager, E. (1989). Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Journal of Biological Chemistry 264, 12838-12848	[Abstract]
Schulz, P. E. (1997). Long-term potentiation involves increases in the probability of neurotransmitter release. Proceedings of the National Academy of Sciences of the USA 94, 5888-5893	[Abstract/Full Text]
Sheng, Z.-H., Rettig, J., Takahashi, M. & Catterall, W. A. (1994). Identification of a syntaxin-binding site on N-type calcium channels. Neuron 13, 1303-1313	[Medline]
Yawo, H. (1999). Protein kinase C potentiates the transmitter release from the chick ciliary presynaptic terminal by increasing exocytotic fusion probability. The Journal of Physiology 515, 169-180	[Abstract/Full Text]
Yawo, H. & Chuhma, N. (1994). -Conotoxin-sensitive and -resistant transmitter release from the chick ciliary presynaptic terminal. The Journal of Physiology 477, 437-448	[Abstract]
Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T. & Takahashi, M. (1992). HPC-1 is associated with synaptotagmin and -conotoxin receptor. Journal of Biological Chemistry 267, 24925-24928	[Abstract]

Acknowledgements

I thank S. Sai for technical support, Drs T. Abe, K. Kawa and M. Umemiya for comments on this manuscript, and Mr B. Bell for reading the revised manuscript. Reviews by Drs E. M. McLachlan and K. Kuba are gratefully acknowledged. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, the Yamanouchi Foundation for Research on Metabolic Disorders and the Gonryou Medical Foundation.

Corresponding author

H. Yawo: Neurophysiology Division, Department of Physiology and Pharmacology, Tohoku University School of Medicine, Sendai 980-8575, Japan.

Email: yawo{at}mail.cc.tohoku.ac.jp

This article has been cited by other articles:

				A. Rodriguez-Contreras, P. Lv, J. Zhu, H. J. Kim, and E. N. Yamoah Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells J Neurophysiol, October 1, 2008; 100(4): 2115 - 2124. [Abstract] [Full Text] [PDF]

				L. B. Silverman-Gavrila, P. M. R. Orth, and M. P. Charlton Phosphorylation-Dependent Low-Frequency Depression at Phasic Synapses of a Crayfish Motoneuron J. Neurosci., March 23, 2005; 25(12): 3168 - 3180. [Abstract] [Full Text] [PDF]

				K. Berglund, M. Midorikawa, and M. Tachibana Increase in the Pool Size of Releasable Synaptic Vesicles by the Activation of Protein Kinase C in Goldfish Retinal Bipolar Cells J. Neurosci., June 15, 2002; 22(12): 4776 - 4785. [Abstract] [Full Text] [PDF]

				M. A. Xu-Friedman and W. G. Regehr Probing Fundamental Aspects of Synaptic Transmission with Strontium J. Neurosci., June 15, 2000; 20(12): 4414 - 4422. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yawo, H. Search for Related Content PubMed PubMed Citation Articles by Yawo, H.