Re-evaluation of phorbol ester-induced potentiation of transmitter release from mossy fibre terminals of the mouse hippocampus

Preparation

All expriments were carried out in accordance with the guiding principles of the Physiological Society of Japan. Postnatal mice (21-28 days old, BALB/c strain) were ether anaesthetized, and the hippocampi were quickly removed from both hemicerebrums. Transverse slices of the hippocampus (0.4 mm thickness) were prepared using a standard technique described previously (Kamiya & Ozawa, 1999), and were incubated for 1 h or more at 33°C in standard artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 126; NaHCO₃, 26; NaH₂PO₄, 1; KCl, 3; CaCl₂, 2; MgCl₂, 1; glucose, 11 (pH 7.4 with 95 % O₂ and 5 % CO₂). In all experiments [Ca²⁺]_o was increased by adding CaCl₂ without changing other ions and was reduced by adding Na₂EGTA, calculating free [Ca²⁺]_o from the dissociation constant. The following experiments were done at 30-33°C unless otherwise noted.

Extracellular recordings

Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum lucidum of the CA3 region using glass microelectrodes with tip diameters of less than 10 μm, which were filled with standard ACSF. The silver bipolar stimulating electrode was placed in the granule cell layer. In each experiment, the positions of both the stimulating and recording electrodes were adjusted until the fEPSP met criteria described previously (Zalutsky & Nicoll, 1990). Twin pulses of 0.2 ms duration and an interpulse interval of 40 ms were applied at 0.1 Hz. The fEPSP slope was calculated by least-squares linear regression over a 1 ms bin period. The minimum value during the first falling phase of the fEPSP was adopted as the representative fEPSP slope. For comparison, the fEPSP slope was normalized to the control mean. The change of the presynaptic volley was negligible throughout the experiments.

Measurement of intraterminal Ca²⁺ concentration

A glass pipette with an approximately 100 μm tip diameter was dipped in a solution of fura-2-conjugated dextran (fura dextran, MW 3000, Molecular Probes Inc.) at a concentration of 10 mg ml⁻¹, dried at 4°C and stored at -20°C. A transverse slice of the hippocampus was mounted in an interface chamber. The fura dextran-containing glass pipette was inserted into the granule cell layer of the slice for 10 min. Similar results were obtained from some experiments in which the pipette was inserted in the stratum lucidum at the CA4 region instead of the granule cell layer. The slice was then incubated for 3-6 h at 33°C in standard ACSF in order to allow the dye to be transported to the mossy fibre terminals. The stimulating bipolar electrode was positioned at the injected site. A conventional epifluorescence system equipped with a water-immersion objective (× 40, NA 0.7, Olympus, Tokyo, Japan) and Xenon lamp (150 W) was focused on the mossy fibre terminals in the stratum lucidum at the CA3 region. Fluorescence was excited alternately at wavelengths of 340 and 380 nm through the minimum iris orifice (diameter, 50 μm). As there is a window (diameter, 50 μm) in front of the photomultiplier tube (OSP-3, Olympus), the background fluorescence behind the focal plane was reduced. The intraterminal Ca²⁺ was calculated from the ratio of fluorescence intensities at wavelengths of 340 and 380 nm (Grynkiewicz et al. 1985) using the dissociation constant (K_D) determined by the manufacturer (389 nM) and the system-dependent parameters determined by a conventional in situ calibration method. This experimental value was represented as [Ca²⁺]_pre, the variation of which must be discriminated from the real time- and space-dependent variation of [Ca²⁺]_i. The signal was sampled at 10-100 Hz by a computer (PC-9801RS, NEC) with software for measuring intracellular Ca²⁺ (MiCa, provided by Drs K. Furuya & K. Enomoto, National Institute of Physiological Science, Japan). The mean fluorescence intensity during 1 s just before stimulation was used for the calculation of basal [Ca²⁺]_pre. The mean fluorescence intensity during 300 ms just after the nerve stimulation-dependent increment was used for calculating the peak [Ca²⁺]_pre, and the difference between the peak and basal [Ca²⁺]_pre was defined as Δ[Ca²⁺]_pre. For comparisons, the Δ[Ca²⁺]_pre was normalized to the control mean.

Throughout all experiments, D(-)2-aminophosphonopentanoic acid (AP5; 25 μM) was used to block possible contamination from NMDA-dependent processes. At the end of each experiment a type 2 metabotropic glutamate receptor agonist, DCG-IV (1 μM), was bath applied to stimulate the metabotropic glutamate receptor (mGluR2/3) which is selectively expressed in the mossy fibre terminal (Kamiya & Ozawa, 1999). If this treatment did not diminish the fEPSP slope to less than 50 % of that before the application of DCG-IV, the data were excluded from the following analyses. Most experiments fitted these criteria. In standard ACSF DCG-IV diminished Δ[Ca²⁺]_pre to 72 ± 4 % of control (n = 6) as expected from the non-linear relationship between the Δ[Ca²⁺]_pre and fEPSP (Kamiya & Ozawa, 1999). Once the basal [Ca²⁺]_pre exceeded 500 nM, the data were excluded from the following analyses. Repeat experiments were made in another laboratory using a membrane-permeant Ca²⁺ indicator, rhod-2 AM (Dojindo Laboratory, Kumamoto, Japan), which was loaded into the mossy fibre terminals without injuring the granule cells (Kamiya & Ozawa, 1999). The results were similar to those with fura dextran (H. Kamiya, unpublished observations).

Recording of miniature EPSCs

Whole-cell recordings were made from CA3 pyramidal neurons as described (Kamiya & Ozawa, 1999). The membrane currents were recorded at -70 mV in the presence of 0.5 μM tetrodotoxin and 100 μM picrotoxin. Patch pipettes were filled with an internal solution (pH 7.2) containing (mM): caesium gluconate, 150; EGTA, 0.2; NaCl, 8; Hepes, 10; MgATP, 2; and QX-314, 5 (Sigma-Aldrich). Detection criteria included an amplitude of 8 pA and an area threshold of 10 fC. The effect of PDAc in the presence of BIS-I on either the amplitude or inter-event interval was assessed using the Kolmogorov-Smirnov test.

Reagents

Pharmacological agents were usually bath applied through a perfusion line at a constant flow rate. The solution in the chamber (ca 1 ml) was completely replaced in less than 2 min. Agents used in this study and their sources were as follows: D(-)-2-amino-5-phosphonopentanoic acid (AP5, Tocris Cookson, Bristol, UK); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris Cookson); (2S,2′R,3′R)2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV, Tocris Cookson); phorbol 12,13-diacetate (PDAc, Wako, Osaka, Japan); 4α-phorbol (Calbiochem, La Jolla, CA, USA); bisindolylmaleimide I (BIS-I, Calbiochem); forskolin (Research Biochemicals International, Natick, MA, USA); 3-isobuty-1-methylxanthine (IBMX, Sigma-Aldrich, St Louis, MO, USA); ω-conotoxin GVIA (ω-CgTx_GVIA, Peptide Institute Inc., Minoh, Japan); ω-agatoxin IVA (ω-AgTx_IVA, Peptide Institute Inc.); nimodipine (Research Biochemicals International). The effects of BIS-I were assessed after 1 h preincubation which had little effect on the first fEPSP amplitude (data not shown). CNQX, PDAc, 4α-phorbol, BIS-I, forskolin and nimodipine were dissolved in DMSO, then diluted. The concentration of DMSO did not exceed 0.2 %, and by itself had no effect on the fEPSP and [Ca²⁺]_pre. The stock solutions of ω-CgTx_GVIA and ω-AgTx_IVA were each prepared at a concentration of 1 mM and 0.1 mM in an extracellular solution containing 100 mg ml⁻¹ bovine serum albumin (Sigma-Aldrich). The stock solution was added directly to the recirculating perfusion solution saturated with 95 % O₂ and 5 % CO₂.

The values in the text and figures are means ±s.e.m. (n = number of experiments). Unless otherwise noted, statistically significant differences between various parameters were determined using Student's two-tailed t test between raw data for the paired experiments and the Mann-Whitney U test for unpaired experiments. P < 0.05 was considered significant.

[Ca²⁺]_pre in the mossy fibre terminal

When injected into the granule cell layer in the hippocampal dentate gyrus (Fig. 1A1), the Ca²⁺-sensitive dye fura dextran diffused into the injured neurons and was allowed to accumulate in the terminals during an incubation time of 3-6 h. Figure 1A2 shows a fluorescence image of fura dextran in the hippocampal slice. The fluorescence-labelled axons were all in a narrow bundle (Fig. 1A2), as long as 1.0 mm, in the stratum lucidum from the injected site to the terminal boutons (Fig. 1A2 and A3), indicating that the mossy fibres were specifically labelled. The large mossy fibre terminals were easily recognizable like bright stars in a galaxy (Fig. 1B). We never observed neuronal somata retrogradely labelled with fura dextran in the CA3 region. A photomultiplier was focused in the CA3 on a small circular region with a diameter of 50 μm which contained several large fluorescence terminals (Fig. 1C). The fluorescence signals were monitored by alternating the excitation wavelength between 340 and 380 nm, and the [Ca²⁺]_pre was quantified by the ratio of their fluorescence intensities (Fig. 1D). Although mossy fibres contain small terminals at GABAergic interneurons, the volume ratio suggests that at least 80 % of the fluorescence should come from the large terminals at the CA3 pyramidal cell dendrites (Acsády et al. 1998). The results were almost identical from a smaller area (10 μm in diameter) containing only a single large bouton (n = 9). Upon invasion of the action potential, voltage-dependent Ca²⁺ channels were activated, leading to an increase in [Ca²⁺]_pre (Fig. 1D). With standard ACSF containing 2 mM Ca²⁺ and 1 mM Mg²⁺, the resting [Ca²⁺]_pre was 139 ± 30 nM (n = 20); [Ca²⁺]_pre increased by 14.9 ± 1.5 nM (n = 7) following a single nerve stimulus.

Although [Ca²⁺]_i transients should be much larger and faster in the vicinity of Ca²⁺ channel clusters (Zucker, 1996; Neher, 1998), the volume-averaged fura-2 signal is related to the magnitude of Ca²⁺ influx through Ca²⁺ channels (Sinha et al. 1997). In fact, the stimulation-dependent accumulation of [Ca²⁺]_pre (Δ[Ca²⁺]_pre) was dependent on the number of pulses in a train with high frequency (Fig. 1E). At 50 Hz, Δ[Ca²⁺]_pre was almost proportional to the number of pulses in a train when it was below 20, but became non-linear above 20. In Fig. 1F, the dependence of Δ[Ca²⁺]_pre on [Ca²⁺]_o was also investigated. Although the relationship was non-linear, Δ[Ca²⁺]_pre was sensitive to [Ca²⁺]_o between 1-4 mM. Unless otherwise indicated, all other experiments were performed in 2 mM [Ca²⁺]_o. In addition, Δ[Ca²⁺]_pre was reversibly suppressed by a GABA_B receptor agonist, baclofen (data not shown), which inhibits transmitter release by reducing Ca²⁺ influx into the presynaptic terminal (Wu & Saggau, 1997). These results suggest that Δ[Ca²⁺]_pre is sensitive to the change in Ca²⁺ influx during this type of neuromodulation.

Mechanisms of phorbol ester-induced potentiation

A phorbol ester, phorbol 12,13-diacetate (PDAc), potentiated the field excitatory postsynaptic potential (fEPSP) recorded from the stratum lucidum of the CA3 region, as shown in Fig. 2A and B. A sustained potentiation was observed 20 min or more after removal of PDAc. The potentiation was usually accompanied by a reduction of the paired-pulse ratio (PPR) (Fig. 2A and C), consistent with previous studies indicating that PDAc-dependent potentiation is presynaptic at the mossy fibre-CA3 synapse (Yamamoto et al. 1987). On average the addition of 10 μM PDAc significantly potentiated the fEPSP slope to a value 463 ± 57 % of that at 2 mM [Ca²⁺]_o (Fig. 2B; P < 0.001) and significantly reduced the PPR from 2.55 ± 0.18 to 0.92 ± 0.03 (Fig. 2C; P < 0.001). Even when the stimulation strength was attenuated in order to evoke fEPSP at a comparable amplitude, the reduction of the PPR was again significant (1.18 ± 0.06, P < 0.001).

To determine whether PDAc potentiates transmitter release by increasing Ca²⁺ influx into the presynaptic terminal during presynaptic stimulation, the effects of PDAc on fEPSP and Δ[Ca²⁺]_pre were compared. Figure 3A shows a sample record of [Ca²⁺]_pre in response to a single electrical stimulus. Exposure to 10 μM PDAc increased the Δ[Ca²⁺]_pre, reaching a steady state within 10 min. On average PDAc enhanced the Δ[Ca²⁺]_pre to a value 153 ± 5 % of control (n = 7, P < 0.001). In the following experiments, the Δ[Ca²⁺]_pre was evoked by a 10-pulse train at 50 Hz in order to increase the signal:noise ratio. PDAc again enhanced the Δ[Ca²⁺]_pre to a mean value 140 ± 4 % of control (Fig. 3B; P < 0.002). The results were not significantly different from those using a single electrical pulse as a stimulus. This is consistent with the observation that the enhanced Δ[Ca²⁺]_pre was also proportional to the number of pulses in a train when it was less than 10 (data not shown). The mean time course of Δ[Ca²⁺]_pre enhancement during exposure to PDAc (Fig. 3B) was somewhat faster than that of the mean fEPSP slope potentiation (Fig. 2B). The reason for this difference is unknown.

Since about 5-10 boutons were included in the 50 μm diameter recording area, the apparent increase of Δ[Ca²⁺]_pre might have resulted from an increased number of active terminals in the recording area. To test this, we recorded the fluorescence from a small recording area 10 μm in diameter which contained only a single large bouton. In all seven boutons tested at 2 mM [Ca²⁺]_o, PDAc faithfully increased the Δ[Ca²⁺]_pre on average to 136 ± 7 % of control (P < 0.002). The results were almost the same as those from the 50 μm diameter recording area.

Following the stimulus-induced increase, [Ca²⁺]_pre decayed slowly as shown in Fig. 3A. The falling phase between 0-5 s was fitted to a single exponential function. At 2 mM [Ca²⁺]_o, the falling phase time constant τ was 939 ± 81 ms. If PDAc decelerates intracellular Ca²⁺ sequestration, an increase of τ would be expected. However, as shown in Fig. 3C, τ was actually significantly reduced in PDAc (P < 0.005). In addition, the resting [Ca²⁺]_pre was not affected by PDAc (n = 7). There is, thus, no evidence of a deceleration of intracellular Ca²⁺ buffering or removal.

The difference between the effects of PDAc on the fEPSP slope and on Δ[Ca²⁺]_pre may be attributed to the non-linear relationship between presynaptic Ca²⁺ influx and transmitter release (Dodge & Rahamimoff, 1967) and/or to an additional modulation of the exocytotic machinery downstream of Ca²⁺ influx (Yawo, 1999). Actually, when [Ca²⁺]_o was varied with constant [Mg²⁺]_o, the fEPSP slope changed with a non-linear relationship (Fig. 4A) which was well fitted to the Dodge-Rahamimoff equation as follows:

(1)

where y_max is the maximum value of the normalized fEPSP slope (5.94) and K_D’ is the apparent dissociation constant (1.57 mM). The least-squares approximation of the co-operativity integer, n, was 3. According to the relationship between [Ca²⁺]_o and Δ[Ca²⁺]_pre (Fig. 1F), the relationship between the fEPSP slope and Δ[Ca²⁺]_pre was obtained as in Fig. 4B, which could be approximated by a power function (Kamiya & Ozawa, 1999):

(2)

The power (m) was estimated to be 2.5 for [Ca²⁺]_o between 1 and 8 mM. When the experimental values of the fEPSP slope and Δ[Ca²⁺]_pre were plotted after exposure to PDAc on the same graph of Fig. 4B, they deviated markedly at both 0.5 and 10 μM from those obtained by changing [Ca²⁺]_o. Therefore, the potentiating effects of PDAc might not be simply the result of the enhancement of Ca²⁺ influx (Wu & Saggau, 1997; Kamiya & Ozawa, 1999).

The PPR was positively dependent on [Ca²⁺]_o below 4 mM whereas it was negatively dependent on [Ca²⁺]_o above 4 mM. As a result, the relationship between Δ[Ca²⁺]_pre and PPR was biphasic as shown in Fig. 4C. Although it is premature to discuss this relationship, the positive phase might be explained by the fact that facilitation is upregulated by residual [Ca²⁺]_i (Kamiya & Zucker, 1994; Delaney & Tank, 1994) and the negative phase could be attributed to depression either through depletion of the readily releasable pool of vesicles or through refractoriness of release sites (Neher, 1998; Dittman et al. 2000). When the experimental values of PPR and Δ[Ca²⁺]_pre were plotted after exposure to PDAc on the same graph of Fig. 4C, they deviated markedly at both 0.5 and 10 μM from those obtained by changing [Ca²⁺]_o, even when considering the effect of a fEPSP size of 133 % (Fig. 2C). These results also suggest that PDAc potentiates the fEPSP through mechanisms other than by enhancing Ca²⁺ influx. Since the difference between 0.5 and 10 μM PDAc was quantitative rather than qualitative, we usually applied PDAc at 10 μM in the following experiments to see the clear effects of the phorbol ester.

Ca²⁺ channel toxin sensitivities of the phorbol ester-induced potentiation

If PDAc preferentially upregulates a subtype of Ca²⁺ channels, the PDAc sensitivity of Δ[Ca²⁺]_pre would be expected to increase or decrease after treatment with a channel subtype-specific toxin. As shown in Fig. 5A, the Δ[Ca²⁺]_pre was not clearly reduced by 10 μM ω-CgTx_GVIA (N-type specific channel blocker) in 5 of 13 experiments. In eight other experiments, ω-CgTx_GVIA inhibited Δ[Ca²⁺]_pre to a steady-state level of 72-93 % of control in 10 min. In summary, the effect of 10 μM ω-CgTx_GVIA on the Δ[Ca²⁺]_pre was inconsistent. On the other hand, Δ[Ca²⁺]_pre was clearly reduced by ω-AgTx_IVA (P/Q-type specific channel blocker) as shown in Fig. 5A and B, with the maximum effect at 200 nM. The remaining component was sensitive to neither nimodipine (10 μM, 91 ± 5 % of value before treatment, n = 6) nor Ni²⁺ (50 μM, 98 ± 3 % of value before treatment, n = 7). Kamiya & Ozawa (1999) reported that ω-CgTx_GVIA reduced Δ[Ca²⁺]_pre to 60-70 % of control in the hippocampal mossy fibre terminals of younger mice (10-18 days old). Therefore, the toxin sensitivity of mossy fibre terminals changes with development (see Iwasaki et al. 2000). Exposure to 10 μM PDAc enhanced the ω-CgTx_GVIA-resistant component of Δ[Ca²⁺]_pre to 137 ± 8 % of the control amplitude, the ω-AgTx_IVA-resistant component to 146 ± 6 % of the control amplitude and the residual component to 138 ± 7 % of the control amplitude, respectively (Fig. 5C). Therefore, PDAc effects did not select between the AgTx_IVA-sensitive and -resistant components.

Effects of PKC inhibitors

Although PDAc as low as 0.5 μM consistently potentiated the fEPSP and enhanced the Δ[Ca²⁺]_pre, the inactive analogue 4α-phorbol (10 μM) did not enhance the fEPSP slope (101 ± 6 % of control; n = 5), did not affect the PPR (from 2.40 ± 0.08 to 2.43 ± 0.15; n = 5), and had no effect on Δ[Ca²⁺]_pre (100 ± 2 % of control; n = 6).

To determine whether the PDAc-induced potentiation is mediated by PKC or non-PKC phorbol ester receptors, we investigated the effects of a PKC-selective inhibitor, bisindolylmaleimide I (BIS-I), which is structurally similar to staurosporine and acts as a competitive inhibitor for the kinase domain of PKC (Toullec et al. 1991). As shown in Fig. 6A, after 1 h preincubation in ACSF containing 10 μM BIS-I, the fEPSP was potentiated by exposure to 10 μM PDAc in the presence of BIS-I. Although PDAc potentiated the fEPSP to a mean of 258 ± 34 % of control in the presence of BIS-I, this effect was significantly less potent than in the presence of vehicle alone (0.1 % DMSO, Fig. 6B). The PPR was significantly reduced by PDAc in the vehicle-alone experiments, but was not reduced in the BIS-I experiments (Fig. 6A and C). The effects of PDAc were negligible at a concentration of 0.5 μM in the BIS-I-treated slice: the fEPSP slope after exposure to 0.5 μM PDAc was 97 ± 2 % of control (n = 6). BIS-I almost abolished the PDAc-dependent enhancement of Δ[Ca²⁺]_pre (Fig. 6D). Therefore, the BIS-I-resistant component does not appear to be accompanied by an enhancement of Δ[Ca²⁺]_pre. When BIS-I was removed, both the fEPSP slope and the Δ[Ca²⁺]_pre showed a tendency to be potentiated, suggesting that the effects of BIS-I are reversible.

Transmitter release from mossy fibre terminals is dependent on cAMP and potentiated by forskolin (Weisskopf et al. 1994; Huang et al. 1994). Forskolin (50 μM) potentiated the fEPSP slope to 529 ± 100 % of control in the presence of 100 μM IBMX (Fig. 6E). In contrast to PDAc-induced potentiation, forskolin-induced potentiation was not accompanied by a significant reduction of the PPR (Fig. 6F). To investigate the specificity of BIS-I, forskolin-induced potentiation of fEPSP was investigated in the presence of 10 μM BIS-I after 1 h preincubation. As shown in Fig. 6E, the effects of BIS-I on forskolin-induced potentiation were negligible, consistent with the hypothesis that the effect of BIS-I was specific to PKC. Figure 6F indicates that the effect of BIS-I on the PPR was also negligible.

During PDAc-induced potentiation, the PPR was always decreased from its initial value throughout the course of the potentiation (Fig. 2C). However, this was not the case in the BIS-I-treated slice (Fig. 7A). In BIS-I, the PPR was initially 2.29 ± 0.21 (n = 16), in the same range as in the absence of BIS-I, but subsequent exposure to PDAc (10 μM) no longer reduced the PPR (Fig. 7A), although it continued to potentiate the fEPSP slope (Fig. 6B). To show this clearly, the PPR was plotted against the fEPSP slope during a 10 min exposure to PDAc (Fig. 7B). For the control experiment shown in Fig. 2C, the PPR was reduced along with the enhancement of the fEPSP slope (○, Fig. 7B) with a correlation coefficient of -0.97 ± 0.006 (n = 17). However the correlation between the PPR and the fEPSP slope in the BIS-I-treated slice was on average only -0.36 ± 0.13 (n = 16, Fig. 7B), significantly less than in the absence of BIS-I. If PDAc upregulated the fusion probability during synaptic vesicle exocytosis, a reduction of the PPR would be expected (see Discussion). Therefore, the BIS-I-resistant component of fEPSP potentiation appears not to have been accompanied by an upregulation of fusion probability, thereby being different from the BIS-I-sensitive component.

It has been shown that phorbol ester-induced potentiation is presynaptic (Yamamoto et al. 1987). To confirm this in the presence of BIS-I, the effect of PDAc on the amplitude of the miniature excitatory synaptic current (mEPSC) was examined in a CA3 pyramidal neuron under whole-cell voltage clamp at -70 mV. Since axonal action potentials and GABA_A-mediated synaptic responses were blocked in the presence of both tetrodotoxin and picrotoxin, the intermittent inward deflections of current were regarded as mEPSCs mediated by the spontaneous release of glutamate (Fig. 8A, top trace). Consistent with this hypothesis, the AMPA-type glutamate receptor antagonist CNQX completely abolished current deflections above the noise level (Fig. 8A, bottom trace). Exposure to 10 μM PDAc increased the frequency of mEPSCs even in the presence of 10 μM BIS-I without a noticeable change in their amplitude (Fig. 8A, middle trace and Fig. 8B). When cumulative probability plots of mEPSC amplitude were compared before and after exposure to PDAc (Fig. 8C), the difference was insignificant in all 11 cells tested. On the other hand, cumulative probability plots of inter-event intervals (Fig. 8D) showed a significant difference before and after exposure to PDAc (P < 0.05) in all cells. In summary, PDAc had negligible effects on mean mEPSC amplitude, but enhanced mEPSC frequency. These results are consistent with the notion that the BIS-I-resistant component of PDAc-induced potentiation is presynaptic.

Three components of phorbol ester-dependent potentiation

If the potentiation of the fEPSP slope occurs presynaptically, it could be caused by an increased magnitude of transmitter release and/or an increased synchrony of release. Neurotransmitter release is regulated by the number of docked/primed vesicles that are readily releasable (readily releasable pool) and by the fusion probability (Rosenmund & Stevens, 1996). Depolarization of the nerve terminal activates voltage-gated Ca²⁺ channels, leading to a local increase in [Ca²⁺]_i, and Ca²⁺ binds to the putative ‘Ca²⁺ sensor’, which regulates the fusion probability (Augustine et al. 1987). The present results indicate that the phorbol ester PDAc increased the Δ[Ca²⁺]_pre (Fig. 3) even in a single mossy fibre terminal. However, PDAc potentiated the fEPSP more than expected from the increase of Δ[Ca²⁺]_pre (Fig. 4). PDAc also reduced the PPR to a value less than that expected from the enhancement of Ca²⁺ influx (Fig. 4). A simple explanation is that PDAc also potentiates the fEPSP with a reduction of the PPR through a mechanism other than increasing the action potential-dependent increment of presynaptic [Ca²⁺]_i. This is consistent with a previous finding in a synaptosome preparation enriched with mossy fibre terminals that phorbol esters enhance Ca²⁺-dependent glutamate release without changing the high K⁺-induced [Ca²⁺]_i increment (Terrian, 1995), and with the demonstration that ionomycin-evoked glutamate release is enhanced by PKC activation (Zhang et al. 1996). However, we could not exclude the possibility that one subtype of Ca²⁺ channels is located closer to the vesicles than others and is selectively upregulated by PDAc, thus enhancing the fEPSP to a level higher than would be expected from the bulk increase of Δ[Ca²⁺]_pre.

If the latter is the case, it is expected that the PDAc effect would select between the Ca²⁺ channel toxin-sensitive and -resistant components. The Δ[Ca²⁺]_pre of the mossy fibre terminal consists of a ω-AgTx_IVA-sensitive (P/Q-type) component and a component resistant to both ω-CgTx_GVIA and ω-AgTx_IVA and insensitive to nimodipine and Ni²⁺ (Fig. 5). The effects of ω-CgTx_GVIA were inconsistent. The ω-AgTx_IVA sensitivity of the Δ[Ca²⁺]_pre was less than that expected from the toxin sensitivity of the EPSP (Castillo et al. 1994). Although toxin sensitivity may vary with developmental stage and species, the ω-AgTx_IVA-sensitive Ca²⁺ channels were shown to couple with transmitter release more efficiently than others in hippocampal CA3-CA1 synapses (Wu & Saggau, 1997). In our study the effect of ω-AgTx_IVA was saturated at 200 nM, but there remains a possibility that another channel subtype is also sensitive to ω-AgTx_IVA at micromolar levels. Additional experiments are necessary to clarify these possibilities in this preparation. Since PDAc effects did not select between the AgTx_IVA-sensitive and -resistant components (Fig. 5), we have no evidence for the presence of Ca²⁺ channel clusters preferentially upregulated by PDAc. In two additional experiments we tested the ω-CgTx_GVIA sensitivity after the PDAc-induced enhancement of Δ[Ca²⁺]_pre, but Δ[Ca²⁺]_pre was not sensitive to ω-CgTx_GVIA in either experiment (100 and 104 % of the value before ω-CgTx_GVIA treatment). This argues against the notion that PDAc promotes the facilitation or activation of ω-CgTx_GVIA-sensitive Ca²⁺ channels which are silent before exposure to PDAc.

In contrast to the BIS-I-sensitive components, the BIS-I-resistant component of fEPSP potentiation, which is not dependent on Δ[Ca²⁺]_pre (Fig. 6), was not accompanied by a reduction of the PPR (Figs 6 and 7). Therefore, the BIS-I-sensitive and BIS-I-resistant components of fEPSP potentiation employ distinct mechanisms. In summary, the PDAc-induced potentiation of the fEPSP appears to consist of at least three components: (1) a BIS-I-sensitive component that is accompanied by enhanced Δ[Ca²⁺]_pre; (2) a BIS-I-sensitive component that is independent of Δ[Ca²⁺]_pre; and (3) a BIS-I-resistant component without enhancement of Δ[Ca²⁺]_pre.

The Δ[Ca²⁺]_pre-dependent component (the first component)

Since neither the fEPSP nor Δ[Ca²⁺]_pre was affected by 4α-phorbol, the BIS-I-sensitive component of PDAc-induced potentiation could be explained by activation of PKC. Reduction of Ca²⁺ sequestration by PKC is unlikely because PDAc neither decelerates the falling phase of Δ[Ca²⁺]_pre nor increases the resting [Ca²⁺]_pre. Rather, reduction of the falling phase-time constant could be attributed to Δ[Ca²⁺]_pre-dependent enhancement of Ca²⁺ sequestration (Ohnuma et al. 1999). Since PDAc enhanced Δ[Ca²⁺]_pre in each single mossy fibre terminal, the change would not have resulted from arousal of inactive terminals. PKC may possibly increase the action potential-induced Ca²⁺ influx into each nerve terminal by either upregulating Ca²⁺ channels (Shearman et al. 1989; Swartz, 1993; Stea et al. 1995) or by downregulating K⁺ channels (Doerner et al. 1988; Shearman et al. 1989; Hoffman & Johnston, 1998). This is consistent with the previous observation that the depolarization-evoked [Ca²⁺]_i increment is enhanced by PKC activation in cerebral synaptosomes (Herrero et al. 1992).

The Δ[Ca²⁺]_pre-insensitive and BIS-I-sensitive component (the second component)

Although PDAc increased Δ[Ca²⁺]_pre, this could not explain the marked reduction of PPR (Fig. 4). In contrast, the BIS-I-resistant component of the PDAc effect was not accompanied by reduction of PPR (Fig. 6). As discussed above, these findings strongly suggest the existence of a BIS-I-sensitive component of the PDAc effect which cannot be explained by an increase in Δ[Ca²⁺]_pre.

PKC increases the size of the readily releasable pool of synaptic vesicles in cultured hippocampal neuronal synapses (Stevens & Sullivan, 1998) as well as in chromaffin cells (Gillis et al. 1996). This is consistent with the observation that PKC increases the frequency of miniature synaptic events in hippocampal neurons in a Ca²⁺-independent manner (Capogna et al. 1995). In many synapses, the magnitude of the PPR is negatively correlated with the quantal content of the first response (Debanne et al. 1996; Schulz, 1997). The most plausible mechanism seems to be a depletion of the readily releasable pool for the second release or the refractoriness of release sites as a result of an increase in the fusion probability (Debanne et al. 1996; Dobrunz & Stevens, 1997; O'Donovan & Rinzel, 1997; Yawo, 1999; Dittman et al. 2000). In our preparation, the PPR decreased along with the development of PKC-dependent potentiation of the fEPSP slope (Fig. 7). It is assumed that the second pulse of a paired-pulse protocol admits the same amount of Ca²⁺ as the first, and produces the same elevation of [Ca²⁺]_i as the first before (Fig. 1E) and after exposure to PDAc. Since PPR reduction was correlated with potentiation, PKC could at least upregulate the fusion probability of exocytosis by the first pulse. A plausible mechanism is an enhancement of the Ca²⁺ sensitivity of the exocytotic fusion probability by increasing the local [Ca²⁺]_i in the vicinity of the Ca²⁺ sensor, by locating vesicles closer to the Ca²⁺ channel clusters, by upregulating the Ca²⁺ sensitivity of the Ca²⁺-sensor molecule, or by facilitating the linkage between the Ca²⁺ sensor and the fusion machinery.

The BIS-I-resistant component (the third component)

We could not exclude the possibility that the BIS-I-resistant component of the fEPSP potentiation is postsynaptic. However, this possibility appears unlikely because PDAc increased the mEPSC frequency in BIS-I without affecting the amplitude distribution in the CA3 pyramidal cell, and because the quantal size of the mossy fibre synapse was not increased during PDAc-induced potentiation of the EPSP (Yamamoto et al. 1987). This is in contrast to the observation that BIS-I prevents the phorbol ester-induced increase in mEPSC frequency in cultured hippocampal neuronal synapses (Capogna et al. 1999). Assuming that the BIS-I-resistant component is presynaptic, it is suggested to result from an increased size of the readily releasable pool of vesicles rather than from upregulation of the fusion probability (Gillis et al. 1996) because the fEPSP potentiation was not accompanied by a reduction of the PPR (Figs 6 and 7). It remains to be elucidated whether the BIS-I-resistant component is evoked by a BIS-I-insensitive PKC isoform or by non-PKC C₁ domain-containing molecules, e.g. Munc13-1 which binds phorbol esters with an affinity similar to that of PKC and is distributed preferentially in mossy fibre terminals (Betz et al. 1998). We could not detect BIS-I-resistant fEPSP potentiation when the PDAc concentration was 0.5 μM. This would not necessarily mean that the non-PKC phorbol ester receptor is less sensitive to PDAc than the PKC receptor because responsiveness may be amplified by phosphorylation. However, this observation implies that a local accumulation of activators (e.g. diacylglycerol) would be a prerequisite for activation of non-PKC C₁ domain-containing molecules.