Phosphorylation regulates spontaneous and evoked transmitter release at a giant terminal in the rat auditory brainstem

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1 Keywords: 0158 Journal of Physiology (2000), 526.2, pp Phosphorylation regulates spontaneous and evoked transmitter release at a giant terminal in the rat auditory brainstem Sharon Oleskevich and Bruce Walmsley The Synaptic Structure and Function Group, Division of Neuroscience, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT 0200, Australia (Received 28 September 1999; accepted after revision 10 April 2000) 1. The role of phosphorylation in synaptic transmission was investigated at a large glutamatergic terminal, the endbulb of Held, on bushy cells in the rat anteroventral cochlear nucleus (AVCN). 2. Whole-cell recordings of excitatory postsynaptic currents (EPSCs) were used to examine the effects of kinase inhibitors and activators on low-frequency (baseline) evoked release, spontaneous release, paired-pulse facilitation (PPF) or depression (PPD), repetitive stimuli and recovery from depression. 3. Application of the kinase inhibitor H7 (100 ìò) reduced low-frequency evoked EPSC amplitude (by 15 %) and simultaneously increased PPF (or reduced PPD), with no significant change in other aspects of transmission. H7 did not affect the amplitude or frequency of spontaneous miniature EPSCs. 4. Phorbol esters increased EPSC amplitude (by 50 %) with a concomitant decrease in PPF (or increase in PPD), and reduced the final EPSC amplitude during repetitive stimuli. The effect of phorbol esters was due exclusively to protein kinase C (PKC) activation, as the specific PKC inhibitor bis-indolylmaleimide (Bis) completely blocked the potentiating effect of phorbol esters on EPSC amplitude. 5. Significantly, phorbol esters did not increase the evoked EPSC amplitude at connections in which release was maximized using high extracellular calcium concentrations (4 6 mò). 6. Phorbol esters increased the frequency of spontaneous miniature EPSCs in physiological calcium (by 275 %), and in high extracellular calcium (by 210 %) when phorbol esters did not increase the evoked EPSC amplitude. 7. Our results are most consistent with the actions of H7 to decrease low-frequency release probability and phorbol esters to increase low-frequency release probability at the endbulb bushy cell synaptic connection in the AVCN. The effects of H7 and phorbol esters on paired-pulse responses and tetanic depression appear to be largely consequential to these changes in low-frequency release probability. The probability of vesicular transmitter release at central synapses is a major determinant of synaptic efficacy (Walmsley et al. 1998). Many different mechanisms have been proposed to regulate release probability. At the structural level, release probability may depend on the number of docked vesicles at a release site and the density and location of presynaptic calcium channels (Walmsley, 1991; Schikorski & Stevens, 1997; Walmsley et al. 1998). At the molecular level, protein and membrane interactions underlie the mobilization of vesicles to the release site, and the subsequent priming and docking steps which ultimately lead to vesicle fusion (Augustine et al. 1994; Schweizer et al. 1995; Broadie, 1996). The actions of many proteins involved in the events leading to exocytosis may, in turn, be regulated by phosphorylation (Elferink & Scheller, 1995; Augustine et al. 1996; Liu, 1997; Majewski & Iannazzo, 1998). These proteins include the synapsins, synaptobrevin, synaptophysin, synaptotagmin, rabphilin-3a, Munc-18, neuromodulin and myristoylated alanine-rich protein kinase C substrate (Broadie, 1996; Liu, 1997). Phosphorylation of nerve terminal proteins through the action of protein kinases such as calcium calmodulindependent protein kinase II (CaMPKII), protein kinase A (PKA) and protein kinase C (PKC), has been shown to

2 350 S. Oleskevich and B. Walmsley J. Physiol modulate transmitter release at a variety of nerve terminals (Llinas et al. 1985; Malenka et al. 1986; Fossier et al. 1990; Llinas et al. 1991; Ghirardi et al. 1992; Capogna et al. 1995; Liu, 1997; Carroll et al. 1998; Majewski & Iannazzo, 1998; Minami et al. 1998). This modulation may be due to changes in the voltage-activated channels underlying the presynaptic action potential and calcium entry, or by direct effects on the exocytotic or vesicle recycling pathways (Llinas et al. 1985; Madison et al. 1986; Malenka et al. 1986; Storm, 1987; Doerner et al. 1988; Fossier et al. 1990; Robinson, 1991; Parfitt & Madison, 1993; Swartz et al. 1993; Bielefeldt & Jackson, 1994; Stea et al. 1995; Gillis et al. 1996; Hirling & Scheller, 1996; Papp, 1996; Fomina & Levitan, 1997; Liu, 1997; Stevens & Sullivan, 1998; Hilfiker & Augustine, 1999). Different aspects of transmission may be affected by protein phosphorylation, from immediate effects on the release machinery to more remote or longer term influences on vesicle mobilization and recycling. In the present study, we have investigated the possible role(s) of phosphorylation in synaptic transmission at a large nerve terminal, the endbulb of Held, in the rat auditory brainstem (Oertel, 1997). The endbulb of Held contains hundreds of synaptic specializations and forms a powerful glutamatergic connection with the soma of bushy cells in the anteroventral cochlear nucleus (Lenn & Reese, 1966; Cant & Morest, 1979; Ryugo & Fekete, 1982; Liberman, 1991; Ryugo & Sento, 1991). Our previous electrophysiological studies have demonstrated that nerve stimulation at the endbulb bushy cell connection results in a large, brief excitatory postsynaptic current (EPSC), which rapidly depresses on repetitive stimulation (Isaacson & Walmsley, 1995a, b, 1996; Bellingham & Walmsley, 1999). We have used the general kinase inhibitor H7 and phorbol ester activators and inhibitors of protein kinase C to investigate which aspects of transmission may be modulated by phosphorylation at this synapse. Our stimulus protocol was designed to reveal changes in low-frequency (baseline) transmission, spontaneous release, paired-pulse facilitation (PPF) or depression (PPD), tetanic depression (depletion), and recovery from depression of release. METHODS Electrophysiology Parasaggital slices (150 ìm) were made of the anterior ventral cochlear nucleus (AVCN) of 10- to 12-day-old Wistar rats, following decapitation in accordance with local ethical guidelines (Isaacson & Walmsley, 1995a,b, 1996; Bellingham & Walmsley, 1999). Wholecell patch electrode recordings were performed at room temperature (22 25 C) from bushy cells visualized in thin slices using infra-red differential interference contrast (DIC) optics. Slices were superfused with an artificial cerebro-spinal fluid (ACSF) containing (mò): 130 NaCl, 3 0 KCl, 1 3 MgSOÚ, 2 0 CaClµ, 1 25 NaHµPOÚ, 26 2 NaHCO, 10 glucose, equilibrated with 95 % O2 5 %COµ. Patch electrodes (3 6 MÙ resistance) contained (mò): 97 5 potassium gluconate, 32 5 KCl, 10 Hepes, 1 MgClµ, 2 Mg-ATP, 0 5 GTP-Tris and 5 EGTA or 120 CsCl, 4 NaCl, 4 MgClµ, CaClµ, 10 Hepes, 2 Mg-ATP, 0 2 GTP-Tris, and 10 EGTA (ph 7 3). Series resistance, which was < 10 MÙ, was compensated by > 80 %. QX- 314 (2 mò) was added to the patch electrode to block voltageactivated sodium channels. Excitatory postsynaptic currents (EPSCs) were evoked by focal stimulation of single auditory nerve fibres (0 1 ms; 4 60 V; 0 1 Hz), delivered via a glass microelectrode (5 10 ìm tip) filled with ACSF. Trains of stimuli consisted of 10 pulses at 100 Hz, 1 min apart. The evoked EPSCs were identified as endbulb AMPA receptor-mediated currents by their amplitude, fast kinetics and all-or-none response to graded stimulation intensities (Isaacson & Walmsley, 1995a). The synaptic currents were recorded and filtered at 10 khz with an Axopatch 200B amplifier (Axon Instruments) before being digitized at 20 khz. Mean peak amplitudes were measured as the mean of single evoked responses or 20 trains of evoked responses. In some cases, there was a small consistent run-down in the amplitude of the evoked EPSC. Under these conditions, a sloping baseline (linear regression) was applied to correct for the run-down during control versus test conditions. This correction did not affect the interpretation or the statistical significance of the results. Spontaneous miniature excitatory postsynaptic currents (mepscs) were recorded on videotape with a VCR (Vetter) and digitized off_line. Data acquisition and analysis were performed using Axograph 4.0 (Axon Instruments). The amplitudes of spontaneous EPSCs were measured using a semi-automated detection procedure (Axograph 4.0) in which a sliding template detects all spontaneous events with amplitudes greater than 2 5 standard deviations of the background noise. The template has a time course typical of a synaptic event and, as it slides along the current trace, it is optimally scaled to fit the trace at each position. The event detection criterion is proportional to the scaling factor and inversely proportional to the goodness-of-fit between the template and the current trace at each position. An event is detected when this criterion exceeds a specified threshold level (Clements & Bekkers, 1997). The phorbol esters phorbol 12-myristate 13-acetate (PMA; 0 1 ìò; Sigma) and phorbol 12,13-dibutyrate (PDBu; 0 5 ìò; RBI), bisindolylmaleimide (Bis; 1 ìò; Sigma), ( )-2-amino-5-phosphonopentanoic acid (d-ap5; 30 ìò; Tocris), bicuculline methochloride (10 ìò; Tocris), strychnine hydrochloride (1 ìò; Sigma) and H7 di_hydrochloride (100 ìò; Tocris) were added as indicated to the Ringer solution and applied by bath perfusion. Lidocaine N-ethyl bromide (QX-314; 2 mò; RBI) was added to the patch electrode solution. Calcium is expressed as Ca and magnesium as Mg throughout the text. Results are reported as means ± standard error of the mean (s.e.m.) and statistical tests of significance were determined with a parametric t test (Statview). RESULTS H7 reduces evoked EPSC amplitude and increases paired-pulse ratio Whole-cell patch recordings were made from bushy cells in 150 ìm thick AVCN slices (n = 21). Following focal stimulation, glutamatergic AMPA receptor-mediated currents were isolated by addition of strychnine (1 ìò), bicuculline (10 ìò) and ª_AP5 (50 ìò) to block glycinergic, GABAergic and glutamatergic NMDA receptor-mediated currents, respectively (Fig. 1A). The amplitude of the AMPA receptor-mediated EPSCs in normal calcium and magnesium conditions (Ca 2 mò, Mg 1 3 mò) at 60mV varied from 0 6 to 5 8 na with a mean of 2 7 ± 0 3 na (n = 18). The evoked current was all-or-none at the stimulation

3 J. Physiol Phosphorylation and transmitter release 351 Figure 1. H7 reduces evoked EPSC amplitude and increases paired-pulse ratio A, evoked AMPA receptor-mediated EPSCs recorded from an AVCN bushy cell in the presence of strychnine, bicuculline and ª_AP5. The kinase inhibitor H7 (100 ìò) decreased the response amplitude to 83 % of control in this cell. Average of 20 traces. B, EPSCs evoked during pairedpulse stimuli with an interval of 10 ms. H7 decreased the first EPSC (S1) but increased the paired-pulse ratio (S2:S1) to 119 % of control in this cell. C, H7 significantly decreased the evoked EPSC in all cells to a mean of 85 % of control (n = 7;*P< 0 05). D, H7 significantly increased the paired-pulse ratio of EPSC amplitudes to a mean of 117 % of control (n =7;** P < 0 005). threshold and remained stable for stimulation intensities up to 2 times the threshold. Stimulation intensity was set at 1 5 times the threshold for the experiments. The kinase inhibitor H7 was bath applied at a concentration of 100 ìò for 10 min. At this concentration, H7 inhibits several kinases including camp-dependent kinase, protein kinase C and calmodulin-dependent kinase II (Jiang & Abrams, 1998). Addition of the kinase inhibitor, H7, significantly decreased the evoked EPSC in all cells to a mean of 85 % of the control response (P < 0 05;Fig. 1A and C). Two consecutive stimuli delivered 10 ms apart evoked paired-pulse facilitation (PPF; n = 3 of 7 cells) or pairedpulse depression (PPD; n = 4 of 7 cells). The paired-pulse ratio of the amplitude of the second response (S2) compared with the first (S1) was quite variable from cell to cell, ranging from 0 4 to 1 8 (mean 1 22 ± 0 21, n = 11) in normal ACSF (Ca 2 mò, Mg 1 3 mò). Bath application of H7 (100 ìò) increased the paired-pulse ratio (S2:S1) in all cells (Fig. 1B), regardless of initial PPF or PPD. H7 enhanced PPF or attenuated PPD. The paired-pulse ratio was significantly increased to a mean of 117 % of control (P < 0 005; Fig. 1D). Effect of H7 on tetanic depression and recovery A train of ten stimuli was delivered to monitor tetanic depression, followed by a single stimulus to measure recovery (Fig. 2A). Depression and recovery were observed under control conditions and in the presence of the kinase inhibitor H7 (n = 7; Fig. 2A). The rate of depression during the tetanus was defined as the ratio of the tenth response to the first response (S10:S1) for a given time period (Ät = 100ms). The level of depression was calculated by comparison of the tenth response in control conditions versus H7 conditions (S10:S10). The rate of recovery was defined as the ratio of the eleventh response to the tenth Figure 2. Effect of H7 on tetanic depression and recovery A, EPSCs evoked during a stimulation protocol of 10 pulses at 100 Hz followed by a single stimulus (delay 100 ms), to monitor the effect of H7 on tetanic depression and recovery. B, H7 did not significantly change the rate of depression, measured as the ratio of the amplitude of the tenth EPSC compared with the first EPSC in the train of stimuli (S10:S1) over a given time period (Ät = 100 ms). The level of depression was unaffected by H7 as no difference was observed between the amplitude of the tenth EPSC (S10) in control versus H7 conditions. C, the rate of recovery (S11:S10) over a given time period (Ät = 100 ms) and the level of recovery (S11:S1) in the evoked EPSCs were not significantly affected by H7. All values have been normalized to a control value of 100 % (n = 7).

4 352 S. Oleskevich and B. Walmsley J. Physiol H7 does not affect spontaneous mepsc amplitude Spontaneous miniature excitatory postsynaptic currents (mepscs) were recorded in AVCN bushy cells in the presence of strychnine (1 ìò), bicuculline (10 ìò) and ª_AP5 (50 ìò; n = 9 cells; Fig. 3A). The mean amplitude of mepscs was 48 1±4 7pA at a membrane potential of 60 mv (Fig. 3A, inset). Exposure to H7 (100 ìò) did not affect mean mepsc amplitude or mepsc frequency (Fig. 3B). This result indicates that H7 did not affect the postsynaptic AMPA channel properties. Figure 3. H7 does not affect spontaneous mepsc amplitude or frequency A, spontaneous miniature excitatory postsynaptic currents (mepscs) recorded in an AVCN bushy cell in the presence of strychnine, bicuculline and ª_AP5. Inset: the averaged mepsc recorded in this cell (mean amplitude 48 pa at a membrane potential of 60 mv). B, H7 did not significantly affect mean mepsc amplitude or mepsc frequency (n = 9). response (S11:S10) for a given time period (Ät = 100 ms) and the level of recovery was defined as the ratio of the eleventh response to the first response (S11:S1). (Although there may be multiple rate constants defining the entire time course of depression and recovery, we have performed measurements of these processes at a single time interval, which allows a direct comparison between control and test conditions.) The kinase inhibitor did not have a significant effect on these ratios, and therefore appeared to have little, if any, effect on depression and recovery under this protocol (Fig. 2B and C). Phorbol esters increase evoked EPSC amplitude and decrease paired-pulse ratio The phorbol ester phorbol 12,13-dibutyrate (PDBu; 0 5 ìò) was bath applied to monitor the potential effects of protein kinase C (PKC) activation on baseline transmission. Phorbol esters caused a substantial increase in the amplitude of the EPSC (n = 5; Fig. 4A) in normal ACSF (Ca 2 0 mò, Mg 1 3 mò). EPSC amplitude was increased to a mean of 153 % of control (P < 0 05;Fig. 4C). The effect of phorbol esters on paired-pulse depression was examined in five cells. Figure 4B illustrates paired-pulse depression of an evoked EPSC at an interval of 10 ms. Phorbol esters significantly enhanced the paired-pulse depression in four of five cells. The paired-pulse ratio was decreased to 70 % of control in the presence of phorbol esters (P < 0 05; Fig. 4D). Phorbol esters did not affect the amplitude of the second response (Fig. 4C). Effect of phorbol esters on tetanic depression and recovery The effect of phorbol esters on tetanic depression and recovery was investigated using a train of ten stimuli at 100 Hz (S1 through to S10) and a single stimulus after a recovery interval of 100 ms (S11; Fig. 5A). Phorbol esters significantly increased the rate of depression (S10:S1) to Figure 4. Phorbol esters increase evoked EPSC amplitude and paired-pulse ratio A, AMPA receptor-mediated EPSCs evoked in the presence of strychnine, bicuculline and ª_AP5. The PKC activator PDBu (0 5 ìò) increased the evoked EPSC amplitude to 204 % of control in this cell. Average of 20 traces. B, PDBu significantly increased paired-pulse depression to 56 % of control in this cell. C, PDBu significantly enhanced the evoked EPSC amplitude of the first response (S1) to a mean of 153 % of control (* P < 0 05, n = 5) without affecting the amplitude of the second response (S2). D, PDBu significantly decreased the paired-pulse ratio to 70 % of control (* P < 0 05, n = 5).

5 J. Physiol Phosphorylation and transmitter release 353 Figure 5. Effect of PDBu on tetanic depression and recovery of evoked EPSCs A, evoked EPSCs during a stimulation protocol of 10 pulses at 100 Hz followed by a single pulse (100 ms delay) to monitor the effect of PDBu on tetanic depression and recovery. B, PDBu significantly increased the rate of depression of evoked EPSC amplitudes (S10:S1) to 53 % of control (** P < 0 005; Ät = 100 ms). The level of depression was significantly enhanced by PDBu as the amplitude of the tenth EPSC (S10) was reduced in PDBu by a mean of 74 % compared with the amplitude of the tenth EPSC in control conditions (* P < 0 05). C, the rate of recovery (S11:S10; Ät = 100 ms) and the level of recovery (S11:S1) of evoked EPSC amplitudes were not significantly affected by PDBu. All values have been normalized to a control value of 100 % (n = 5). 53 % of control (n = 5; P <0 005; Fig. 5B). The level of depression of the response to the tenth stimuli (S10:S10) was also significantly greater in the presence of phorbol ester than in control conditions. The depressed EPSC amplitude (S10) in phorbol esters was 74 % of the control value (P < 0 05; Fig. 5B). Phorbol esters did not significantly affect the rate of recovery (S11:S10) or the level of recovery (S11:S1) as compared with control conditions (Fig. 5C). Effect of phorbol esters in the presence of elevated external calcium At elevated calcium concentrations (> 4 mò), release appears to reach maximal levels at some endbulb bushy cell connections (Bellingham & Walmsley, 1999; Oleskevich et al. 2000). If the effect of phorbol esters was to increase the size of the total pool available for release, then this effect may be apparent under elevated calcium conditions in which most of the available pool is released (see Discussion). The effect of phorbol esters was therefore investigated in the presence of high external calcium (4 6 mò). For each cell, saturation was determined by a lack of significant increase in EPSC amplitude following an increase in extracellular calcium concentration by 2 mò (either from 2 to 4 mò, or from4to6mò). Six out of nine cells examined exhibited a saturation of release in high calcium, and the effects of phorbol ester application on one of these cells is illustrated in Fig. 6A and B. No significant increase in EPSC amplitude Figure 6. Lack of phorbol ester enhancement of evoked EPSC amplitude in high extracellular calcium A and B, phorbol ester (PMA) did not affect the evoked EPSC amplitude or paired-pulse depression in high extracellular calcium (4 mò). Average of 100 traces. C, summary data showing no effect of PMA on evoked EPSC amplitude in high extracellular calcium (Hi Ca ; 4 6 mò; n = 6). D, PMA significantly enhances miniature EPSC frequency in physiological calcium (* P < 0 05; n = 4) and in high extracellular calcium where phorbol esters did not affect the evoked EPSC amplitude (Hi Ca ; 4 6 mò; n = 4). The miniature EPSC frequency was not significantly different between PMA and Hi Ca + PMA. All values have been normalized to a control value of 100 %.

6 354 S. Oleskevich and B. Walmsley J. Physiol was evident when the extracellular calcium concentration was raised from control (2 mò) to high calcium (4 mò) conditions. In this cell, addition of the phorbol ester PMA (0 1 ìò) in the presence of high calcium produced no further increase in the evoked EPSC amplitude nor a change in paired-pulse depression. Identical results were obtained in other cells exhibiting saturation of release (Fig. 6C). This result suggests that phorbol esters do not increase the total amount of transmitter release above the maximal level achieved under conditions of elevated calcium entry. Phorbol esters increase mepsc frequency Phorbol esters did not affect the mean amplitude of mepscs (n = 5) suggesting that phorbol esters do not affect the postsynaptic AMPA channel properties and instead act presynaptically to modify transmitter release. However, phorbol esters significantly increased the frequency of mepscs by a mean of 273 % in physiological calcium conditions (n = 4; P < 0 05; Fig. 6D). In high extracellular calcium concentrations, where phorbol esters did not affect the evoked EPSC amplitude, PMA still increased the frequency of mepscs (210 %; n = 4; Fig. 6D). Figure 7. Protein kinase C inhibitor blocks phorbol ester enhancement of evoked EPSC amplitude A, phorbol ester (PMA) did not potentiate the evoked EPSC amplitude in the presence of the PKC inhibitor bisindolylmaleimide (Bis). Bis alone did not affect the evoked EPSC amplitude. B, summary data showing no significant differences in evoked EPSC amplitude following the addition of Bis alone or Bis and PMA (n = 5). All values have been normalized to a control value of 100 %. Phorbol esters affect evoked and miniature EPSCs via activation of PKC Phorbol esters may enhance transmitter release via mechanisms other than PKC activation (Hori et al. 1999). The action of phorbol esters was investigated with the selective protein kinase C inhibitor bis-indolylmaleimide (Bis; Fig. 7). Bath application of Bis alone (1 ìò for 10 min) did not significantly affect the amplitude of the evoked EPSC (Fig. 7A). However, Bis blocked the potentiating effect of the phorbol ester PMA (0 1 ìò). Summary data in Fig. 7B show no effect of Bis alone on the evoked EPSC amplitude(97%ofcontrol)andalackofpotentiationby PMA in the presence of Bis (92 % of control) for all five cells. Bis was also found to block the phorbol ester enhancement of mepsc frequency. The amplitude and frequency of mepscs were not affected by Bis alone or PMA in the presence of Bis (n = 3; data not shown). DISCUSSION Phosphorylation of nerve terminal proteins has been implicated in the regulation of a variety of processes underlying transmitter release. Phosphorylation may play a role in vesicle mobilization and recycling, or directly affect the priming, docking and fusion of synaptic vesicles (Liu, 1997). The properties of the voltage-activated sodium and potassium channels underlying the presynaptic action potential, and the calcium channels responsible for release, may be modulated by phosphorylation. These regulatory mechanisms may be evident in different aspects of synaptic transmission, affecting properties such as the total number of vesicles available for release, the depression of release during repetitive stimulation, and the rate of recovery from transmitter depletion or depression. At the large endbulb synapse, application of the kinase inhibitor H7 caused a reduction in the amplitude of the evoked EPSC, and a simultaneous increase in paired-pulse facilitation. Despite a large tetanic depression of the EPSC during repetitive stimulation, no significant changes were observed in the amount or rate of depression, or in the subsequent recovery, following application of H7. We have recently demonstrated an approximately linear inverse relationship between mean release probability and the paired-pulse ratio at the endbulb bushy cell connection (Oleskevich et al. 2000). These results show that a reduction in mean release probability, achieved by lowering the extracellular calcium concentration, leads to a one- to twofold increase in paired-pulse facilitation. In the present experiments, H7 application led to a reduction in the EPSC of approximately 15 % and an increase in paired-pulse ratio of 17 %, consistent with the inverse relationship. This suggests that the effect of H7 is to reduce release probability and increase paired-pulse ratio through a mechanism similar to the effects of reducing calcium entry. This argues against

7 J. Physiol Phosphorylation and transmitter release 355 a specific additional effect of kinase inhibition on the pairedpulse ratio. Application of phorbol esters causes an increase in the baseline EPSC amplitude of 53 %, and a decrease in pairedpulse ratio of 30 %. This result is consistent with the effect of phorbol esters to increase mean low-frequency release probability, with a concomitant reduction in paired-pulse ratio. Our recent study using variance mean analysis has shown that the mean release probability (Pr) at this synapse is 0 55 in physiological calcium (Oleskevich et al, 2000). Therefore, our present results indicate that phorbol esters raise release probability to a very high value (Pr > 0 83). Several recent studies have investigated the mechanisms of action of phorbol esters in enhancing transmitter release. At cultured hippocampal synapses, Stevens & Sullivan (1998) concluded that phorbol esters increased the size of the readily releasable pool, using hypertonic solutions to deplete the pool, and optical measurements of fluorescently labelled vesicles to estimate pool size. In contrast, Yawo (1999) suggested that, at the chick ciliary ganglion giant terminal, phorbol esters did not change the size of the releasable pool. Yawo (1999) used fluorescence methods to show that phorbol esters did not produce an increase in the amount of calcium entering the terminal following the arrival of a nerve impulse, and concluded that phorbol esters upregulate the calcium sensitivity of the process that regulates release probability. Recently, Hori et al. (1999) have shown by direct recordings at the calyx of Held in the medial nucleus of the trapezoid body (MNTB), that phorbol esters do not change the amplitude of the presynaptic calcium current at this synapse. They also observed that the specific PKC inhibitor Bis only partially blocked the actions of phorbol esters. Hori et al. (1999) concluded that phorbol esters enhance release at a target downstream from calcium influx, possibly involving both protein kinase C and Doc2a Munc13-1 interaction. In contrast with this result, but in agreement with Yawo (1999), our results at the endbulb of Held synapse demonstrate an exclusively PKCmediated enhancement of release, since Bis completely blocked the effect of phorbol esters on the evoked EPSC amplitude. Under conditions of high extracellular calcium concentration (4 6 mò), some endbulb connections exhibit maximal release, such that an increase in the amplitude of the evoked EPSC is not evident in higher calcium concentrations (Bellingham & Walmsley, 1999; Oleskevich et al. 2000). If the effect of phorbol esters was to increase the total size of the releasable pool of vesicles, then an increase in release should be observed under these calcium-saturated release conditions. However, we observed that phorbol ester application did not produce a further increase in the amplitude of the evoked EPSC at connections exhibiting saturated release in high calcium. A similar result has also been observed at the chick ciliary presynaptic terminal, under conditions of high extracellular calcium and phorbol ester application (Yawo, 1999). We also observed an increase in the rate of depression during repetitive stimulation. This suggests a more rapid depletion of a fixed-size vesicle pool, following phorbol ester enhancement in the initial amount of release. Our results are most consistent with an enhancement of transmission by phorbol esters at the endbulb of Held due to an increase in the probability of release, without an apparent change in the size of the total pool of available vesicles. At physiological calcium concentration (2 mò), phorbol esters increase the frequency of mepscs in AVCN bushy cells. From their studies in hippocampal CA1 cells, Parfitt & Madison (1993) concluded that the phorbol ester-induced increase in mepsc frequency was due to modulation of a presynaptic L-type calcium channel that does not participate in evoked transmitter release. Our results demonstrate that phorbol esters increase mepsc frequency under conditions where there is no effect on the evoked EPSC (saturated release in high extracellular calcium), consistent with such a differential modulation of spontaneous and evoked release. 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