Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings

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1 J Physiol (2003), 553.2, pp DOI: /jphysiol The Physiological Society Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings T. J. Searl and E. M. Silinsky Department of Molecular Pharmacology and Biological Chemistry (MPBC), Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high-frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 nm) and adenosine (50 mm) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release (m) =np. We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n; neither agent affected the parameter p. PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high-frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium-charged primed vesicles. (Resubmitted 14 July 2003; accepted after revision 10 September 2003; first published online 12 September 2003) Corresponding author E. M. Silinsky: Department of Molecular Pharmacology and Biological Chemistry (MPBC), Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA. e-silinsky@northwestern.edu Phorbol esters have been shown to be potent stimulators of neurotransmitter secretion at many different synaptic loci (for reviews see Brose & Rosenmund, 2002; Silinsky & Searl, 2003). Until recently, these effects have been attributed to activation of protein kinase C (PKC) (Shapira et al. 1987; Stevens & Sullivan, 1998; Oleskivich. & Walmsley, 2000). However, current evidence suggests that another phorbol ester receptor, Munc-13, might be the primary target for the effects of phorbol esters on neurotransmitter release (Redman et al. 1997; Betz et al. 1998; Searl & Silinsky 1998; reviewed in Tokumaru & Augustine, 1999; Brose & Rosenmund, 2002; Silinsky & Searl, 2003). Briefly, synaptic vesicles docked at the nerve terminal membrane must undergo a maturational process termed priming to become competent to release neurotransmitter. Priming occurs by the intimate association of the core complex of three presynaptic proteins (the SNARES) into a tight helical bundle that brings the vesicle into very close proximity to the plasma membrane (Sutton et al. 1998; Jahn & Sudhof, 1999). Phorbol ester binding to Munc-13 facilitates the priming process by activating the SNARE protein syntaxin (Richmond et al. 2001; for more detailed reviews of the proteins involved in this process, see Brose & Rosenmund, 2002 and Silinsky & Searl, 2003). Given this putative priming role of Munc-13, it might be expected that phorbol esters would augment neurotransmitter release by increasing the number of fusioncompetent synaptic vesicles in the nerve terminal. Specifically, the binding of phorbol esters to Munc-13 would be expected to increase the number of vesicles in the readily releasable pool of neurotransmitter. Indeed we have found that at the frog neuromuscular junction, phorbol esters produced roughly equivalent increases in ACh release at all calcium concentrations (Redman et al. 1997) such that the maximal level of evoked ACh release was increased. This result is consistent with an increase in

2 446 T. J. Searl and E. M. Silinsky J Physiol the readily releasable pool of neurotransmitter. However, others have found evidence to support the suggestion that the effects of phorbol esters are largely due to increases in the probability of release of primed vesicles (Rhee et al. 2002; Rosenmund et al. 2002). Interestingly, it has been reported that, at the amphibian neuromuscular junction, phorbol esters reduced the potency of the endogenous neuromodulator adenosine, suggesting that these two drugs interact at a common site in the release process (Sebastiao & Ribeiro, 1990). The actions of adenosine at the amphibian neuromuscular junction have been studied in detail over many years (see e.g. Silinsky, 1980, 1981, 1984; Ribeiro & Sebastiao, 1987; Meriney & Grinnell, 1991; Redman & Silinsky, 1994; Silinsky & Redman, 1996; Robitaille et al. 1999). It is known that the target for the inhibitory effect of adenosine at the amphibian neuromuscular junction is a strategic part of the secretory apparatus downstream of calcium entry (Silinsky, 1981, 1984; Robitaille et al. 1999). The characteristics of inhibition of ACh release by adenosine, stimulation independency and rapid onset, suggest that inhibition takes place at a late post-priming stage in the release process (Silinsky et al. 2001). (For a global review of the effects of adenosine at other synapses, see Miller, 1998.) The differing mechanisms of action suggested for phorbol esters (priming) and adenosine (post-priming) to alter evoked neurotransmitter release are thus at odds with the suggestion that both agents have a common site of action (Sebastiao & Ribeiro, 1990). Changes in the mean level of evoked neurotransmitter release from synaptic vesicles (m) can be ascribed to changes in either the number of vesicles available for release (n) or in the average probability of release (p). We employed two different experimental approaches to determine which of these processes were affected by PDBu and adenosine. We first examined the effects of these drugs on the parameters n and p as estimated by the binomial analysis of evoked ACh release. Despite criticisms concerning the application of binomial statistics to evoked neurotransmitter release (Brown et al. 1976; Redman, 1990), detailed analysis of spontaneous release in strontium solutions suggests that binomial analyses may provide an adequate description of the neurotransmitter release process (Searl & Silinsky, 2002). We therefore decided to determine whether evoked ACh release is also amenable to binomial analysis and which parameters of the binomial distribution were affected by PDBu and adenosine. As a second approach we compared the effects of PDBu and adenosine on the pattern of evoked release seen during short, highfrequency trains of nerve stimuli. This second approach provides information on both changes in the size of the readily releasable pool of ACh and changes in the dynamics of the processes involved in the mobilisation of vesicles for release. METHODS General Frogs (Rana pipiens) were killed by anaesthesia with 5 % ether, followed by double pithing, in accordance with guidelines laid down by our institutional animal welfare committee. Isolated cutaneous pectoris nerve muscle preparations were used in all experiments. Following dissection, nerve muscle preparations were paralysed with formamide treatment (2 M formamide, min) (Del Castillo & Motta, 1978) which allows the recording of miniature endplate potentials (MEPPs) and endplate potentials (EPPs) simultaneously from the same endplate. Intracellular recordings were made using microelectrodes filled with 3 M KCl with resistances 3 10 MV. The signal from the microelectrode was fed into a conventional high-input impedance microelectrode pre-amplifier (Axoclamp-2A, Axon Instruments). Responses were fed into a personal computer using either a Digidata 1200 or TL1-125 A/D converter (Axon Instruments). Solutions were delivered by superfusion with a peristaltic pump and removed by vacuum suction. All experiments were carried out at room temperature (22 24 C). Normal recording solutions contained (mm): NaCl, 115; KCl, 12; Hepes, 2; and CaCl 2, 0.35 mm, unless otherwise stated (see Results). For the adenosine concentration-response experiments, EPPs were evoked at Hz in 1.8 mm CaCl 2 Ringer s solution, in the presence of 12 mm tubocurarine. For the estimates of the readily releasable store, short trains of EPPs were evoked at 100 Hz in 5.4 mm calcium and 20 mm tubocurarine. Tubocurarine has long been known to cause a rundown of EPPs in preparations (Magleby et al. 1981). For the purposes of this study, however, it would appear advantageous to have any prejunctional autoreceptors blocked to preclude the possibility that at different levels of release, different degrees of feedback might have confounded the interpretation of the experimental results. Records of MEPPs and evoked EPPs were analysed using CDR, WCP and SCAN programs (DOS versions, Strathclyde University Superior Software; John Dempster). The data were analysed using a modified version of a computer program for evoked release (Mark Longerbeam, BME Systems; see also Christian & Weinreich, 1992), Microsoft Excel, Corel Quattro Pro and Sigma Plot. All drugs used in this study were obtained from Sigma St Louis, USA, except Ro that was obtained from Calbiochem, San Diego, USA. Overview of binomial analysis of evoked neurotransmitter release The simple binomial distribution is given by the following equation: n f(x) = p x q n_x. (1) x In this equation, f(x) reflects the probability of observing 0, 1, 2, 3,... n quanta released, p reflects the average probability of release and q the probability of a failure of release (where q =(1_p)). As mentioned in the Introduction, m = np. (2) In the binomial distribution, the variance (Var) is related to the average probability of release by the following equation: p =1_(Var/m). (3) Briefly, the EPPs and MEPPs were collected with the EPPs corrected for non-linear summation according to the method of

3 J Physiol Changes in ACh pools produced by phorbol esters and adenosine 447 McLachlan & Martin (1981). Binomial analysis of EPPs requires the use of a compound binomial model which incoporates the size and variance of the individual quantal amplitudes into the distribution (Miyamoto, 1975, for derivations and estimates of errors, see Bennett et al. 1975; Robinson 1976; McLachlan, 1978). The value of p was determined from the following equation: S 2 EPP s 2 EPP p=1 _ +, (4) EPP w MEPP MEPP 2 where EPP is the mean EPP amplitude, S 2 EPP is the variance of EPP amplitudes, s 2 MEPPis the variance of MEPP amplitudes and MEPP is the mean amplitude of the MEPPs (Miyamoto, 1975; McLachlan, 1978). Note that Searl & Silinsky (2002, eqn (12)) had an errant power for the first MEPP term in the final printed version of this equation; this has been corrected in eqn (4) above, with apologies to the reader. Finally, n was determined by rearranging eqn (2): n = m/p, (5) where: m = (EPP)/(MEPP). (6) Error estimates of m, n and p were calculated using eqn (18) in McLachlan, 1978 and eqns (9) and (10) in Robinson (1976). MEPP amplitudes for analysis were obtained from spontaneous events occurring between the EPPs and fitted a normal distribution (see Miyamoto, 1975; McLachlan, 1978; Christian & Weinreich, 1992). Sources of error in the binomial analysis of evoked neurotransmitter release The simple binomial may not give truly accurate estimates of n and p when evoked neurotransmitter release is studied (see McLachlan, 1978; Redman, 1990). Three possible sources of error in the estimates of p derived by the simple binomial have been suggested: non-uniformity of p (spatial variance in p); nonstationarity of p (temporal variance of p), and non-stationarity of n (temporal variance of n) see Searl & Silinsky (2002) for a more complete discussion. (For the derivations of the equations for the complex binomial distributions see Stuart et al ) For the convenience of the reader, the specific equations associated with these different sources of error are presented in the Supplementary material (see also McLachlan, 1978; Searl & Silinsky, 2002), together with a more detailed description of how the errors influence the estimate of p. Interpretation of the binomial parameters of neurotransmitter release The exact meaning of n and p derived from the simple binomial distribution remains controversial (for an alternative statistical model see e.g. Clements & Silver, 2000). Values for n are much smaller than those predicted by the size of the readily releasable pool, and they are calcium dependent (Bennett et al. 1975, 1976). Based on our observations (Searl & Silinsky, 2002), and those of others (Bennett et al. 1975, 1976), it appears that the parameters n and p, derived from application of the simple binomial to neurotransmitter release, reflect two independent calcium binding steps in the exocytosis of the synaptic vesicle. According to this hypothesis, n represents the number of primed vesicles with three calcium ions bound to low affinity calcium binding sites of the secretory apparatus, and p represents the probability of a fourth calcium ion binding to the secretory complex, and triggering exocytosis. Thus, changes in the binomial parameter n may result from either changes in the total number of primed vesicles (i.e. those within the readily releasable pool) or changes in the efficacy/affinity of the calcium binding sites corresponding to n or even to changes in calcium entry. The parameter p can also be affected either by changing the affinity/efficacy of calcium binding or by changing the amount of calcium entering the nerve terminal (The calcium dependence of n and p are shown in Fig. 1, Results). It should be stressed that the estimate of p given by the binomial distribution does not represent the proportion of vesicles released from the readily releasable pool (sometimes referred to as the fractional release). Here the proportionate release is given by the ratio of the single evoked EPP amplitude to the total store size (see e.g. Elmqvist & Quastel, 1965). The readily releasable pool of neurotransmitter We shall define primed vesicles as those vesicles which are fusioncompetent (given adequate calcium). We use the term readily releasable pool to refer to the total number of primed, fusioncompetent, vesicles of the nerve terminal in a resting state. In order to determine changes in the size of the readily releasable pool we used short high-frequency stimulation (100 Hz) trains in high (5.4 mm) extracellular calcium concentrations (where release is close to the maximum, Silinsky, 1981; Redman et al. 1997). We assume that the first phase of rapid rundown in transmitter release is due to the depletion of readily releasable pool of neurotransmitter. The second phase, namely the steadystate plateau level of release at the latter part of the train, presumably represents mobilisation or the rate at which vesicles can dock, prime and release their contents. Statistical comparisons Full details are provided in Searl & Silinsky (2002). Comparisons were made by either parametric statistics (e.g. Student s paired t test) or non-parametric statistics (Mann-Whitney rank sum test, see Glantz, 1992). When more than two groups were compared, an analysis of variance for the normally distributed data was followed by multiple comparisons using the Bonferroni inequality (see Glantz, 1992, p. 93). Data are presented as mean + 1 S.E.M. with P < 0.05 being used as a measure of statistical significance. For detailed statistical analysis of a typical experiment, see the text describing the data of Fig. 4. RESULTS General observations on the binomial parameters of evoked ACh release It has been demonstrated previously that spontaneous ACh release is well-described by a simple binomial distribution and suggested that evoked release might also behave in accordance with binomial statistics (Searl & Silinsky, 2002). To evaluate this possibility further, we constructed calcium concentration response curves for m, n and p in order to confirm that calcium affects both n and p (Bennett et al. 1975, 1976; Miyamoto, 1975) and to choose an appropriate concentration of calcium for the binomial analysis of neurotransmitter release. Figure 1 demonstrates that both n and p depend upon extracellular calcium, with p having a higher affinity and saturating at lower calcium concentrations than n. Based upon the experiments depicted in Fig. 1, we chose a moderate calcium concentration (0.3 mm) where p was

4 448 T. J. Searl and E. M. Silinsky J Physiol estimated to be close to 0.5 for the binomial analysis. The motor nerve was stimulated at a moderate frequency (0.5 Hz), allowing several thousand EPPs to be collected, and then detailed histograms of corrected EPP amplitudes were constructed. Figure 2A shows a typical experimental result (the continuous line represents the theoretical binomial distribution obtained from the experimentally determined values of m, n and p). We then generated Monte Carlo simulations (Fig. 2B) using the binomial parameters estimated in Fig. 2A. The x 2 statistic was then employed to compare actual experimental data and the data from Monte Carlo simulations with the corresponding ideal distributions of EPP amplitudes. As shown here, the experimentally obtained results and the Monte Carlo simulations based on the experimentally obtained values of n and p are indistinguishable, both by visual inspection and according to the x 2 values. Specifically, a comparison between the theoretical distribution of EPP amplitudes and the actual EPP amplitude histogram gave a x 2 value of for the data in Fig. 2A whilst comparison between the theoretical distribution and the Monte Carlo distribution (Fig. 2B) gave a x 2 value of Figure 1. The calcium dependence of the binomial parameters of ACh release This figure shows the effects of calcium concentration on the binomial distribution of EPP amplitudes The graph shows the averages of five individual experiments ± S.E.M. on the parameters m, n and p. For each experimental point, binomial estimates were obtained from EPPs. All experiments were carried out in the absence of Mg 2+ ions. Note the inflection in m occurs close to the point at which p approaches unity. Similar observations were previously made by Bennett et al. 1975, 1976 see text). Figure 2. Comparison between EPP amplitude distributions and a Monte Carlo simulation A, a typical histogram of nerve-evoked EPP amplitudes (2449 events; 0.5 Hz; 0.3 mm calcium). Binomial analysis of EPP amplitude fluctuations gave values of n = 12.4 available quanta/stimuli and p = The continous line, superimposed over the distribution, represents the theoretical distribution of the compound binomial as estimated from the size and distribution of MEPP amplitudes (quantal size) and a binomial distribution where n = 13 available quanta, and p = Comparison between the theoretical distribution of EPP amplitudes and the actual EPP amplitude histogram gave a x 2 value of B, a compound Monte Carlo distribution using the same parameters as for the theoretical distribution. Comparison between the theoretical distribution and the Monte Carlo distribution gave a x 2 value of (A more detailed analysis of the EPP amplitude distribution, in this experiment, indicates a lack of temporal variance in p see the Supplemental material for further details).

5 J Physiol Changes in ACh pools produced by phorbol esters and adenosine 449 The actions and interactions of PDBu and adenosine as studied by binomial analysis The above results suggest that the binomial analysis of EPPs can be used successfully to describe the ACh release process. Binomial analysis was used to evaulate potential mechanisms of action of PDBu and adenosine. A battery of PKC antagonists do not affect the ability of PDBu to increase in evoked ACh release (Searl & Silinsky, 1998), suggesting that PKC does not contribute to the increases in ACh release produced by PDBu at the frog neuromuscular junction. Here we performed these experiments in the presence of the PKC inhibitor Ro (1 mm) to preclude any possible involvement of PKC. This concentration of Ro is sufficient to inhibit all phorbol ester-sensitive PKCs (Johnson et al. 1993) without producing the non-selective and toxic effects associated with higher concentrations of this agent (see Lingameneni et al. 2002; Brose & Rosenmund, 2002). Neither this PKC inhibitor nor a number of other PKC inhibitors had any effect on nerve evoked ACh release. Application of PDBu (100 nm, in the presence of PKC inhibitors, Fig. 3B) caused a statistically-significant increase in m over control levels (Fig. 3A) and this was due to a selective increase in n. Adenosine (50 mm, Fig. 3D) produced a statistically ignificant reduction in the value of m from the control level (C); this effect was associated with an equivalent Figure 3. The effects of adenosine and PDBu on the binomial parameters m, n and p A and B show the effects of PDBu on EPP amplitude distributions (in the presence of the PKC inhibitor Ro ) obtained from a single endplate. A is the control and B is in the presence of 100 nm PDBu. In the example shown here, the mean values (± 1 S.E.M.) of the binomial parameters for control were m = 5.4 ± 0.1, n = 12.1 ± 1.4, p = 0.45 ± 0.05, and in PDBu they were m = 8.36 ± 0.2, n = 18.1 ± 2.0, p = 0.46 ± C and D show the effects of adenosine on the EPP amplitude distributions. C is control and D is in the presence of 25 mm adenosine. In the example shown here, the binomial parameters in control were m = 12.1 ± 0.1, n = 27.6 ± 3.5, p = 0.44 ± 0.05; in adenosine m = 6.8 ± 0.14, n = 16.4 ± 1.9, p = 0.41 ± The continuous lines show the theoretical distributions of EPP amplitudes with the estimate of n rounded to the closest whole number and the quantal size and variance estimated from MEPPs. Each histogram was constructed from 500 events.

6 450 T. J. Searl and E. M. Silinsky J Physiol reduction in the binomial parameter n with no statistically significant effect on p. Figure 4. The effects of adenosine and PDBu, both separately and combined, on the binomial parameters of release The figure shows the effects of adenosine, PDBu and PDBu with adenosine on the binomial pareameters m, n and p. Experiments were carried out in the presence of either the PKC inhibiter Ro (0.5 mm) or GF X (1 mm). The nerve was stimulated at a frequency of 0.2 Hz. Graphs show the mean values ± S.E.M. from n = 7 experiments. In each experiment, results were obtained from a single endplate, with 300 EPPs recorded in each condition; control, adenosine (50 mm), PDBu (100 nm), and adenosine (50 mm) with PDBu (100 nm). To determine whether phorbol esters and adenosine mutually interact, we examined whether the presence of one agent affected the action of the other (see Fig. 4). These experiments were carried out in 0.35 mm calcium in the presence of a PKC inhibitor (either Ro (1 mm) or GFX X (1 mm) (Searl & Silinsky, 1998), thereby restricting the actions of PDBu to effects independent of PKC (i.e. to Munc-13). Following the recording of control EPPs, we first applied adenosine (50 mm) and, after allowing sufficient time for the solutions to equilibrate (5 10 min), we recorded EPPs. Adenosine reduced the mean level of quantal release m from control (m = ± 2.6 to m = 5.9 ± 1.3; P = 0.02, n = 7 experiments). This decrease in m was associated with an equivalent reduction in n (in control, n = 20.8 ± 3.8 vs. in adenosine n = 10.4 ± 1.9; P = 0.02, n = 7 experiments) and no statistically significant change in p (p = 0.53 ± 0.04 in control, p = 0.56 ± 0.04 in adenosine, P = 0.26, n =7 experiments). We then exchanged the superfusing solution to one containing PDBu (100 nm) without adenosine. As before, PDBu increased evoked release over control, with statistically significant increases in m (19.0 ± 3.3) and n = 31.6 ± 4.4, P < 0.01, n = 7 experiments) but not p (0.58 ± 0.04, P = 0.27). Lastly, adenosine added to the PDBu containing solution ( Both in Fig. 4) reduced m to approximately 50 % of the level observed in PDBu alone, a reduction similar to that seen in the absence of PDBu (m in adenosine + PDBu = 10.2 ± 1.6, P = when compared to PDBu alone); n in adenosine + PDBu = 17.9 ± 2.6; P = when compared to PDBu alone; p in adenosine + PDBu = 0.58 ± 0.04, P = 0.32, when compared to PDBu alone). The results thus suggest that both PDBu and adenosine acted exclusively on the binomial parameter n (see Fig. 4) and do not interact in the presence of PKC inhibitors. The effects of PDBu on the adenosine concentration response relationship Although no signs of an interaction between the effects of PDBu and adenosine were observed in the presence of a PKC inhibitor, it remained possible that there was a PKCdependent interaction between adenosine and PDBu. Also, given that both PDBu and adenosine act on the binomial parameter n it seemed possible that a more subtle interaction between the effects of PDBu and adenosine might have been missed. We tested both of these possibilities by determining the effects of a supra-maximal concentration of PDBu (200 nm) on the adenosine dose response curve. A PKC inhibitor was not used initially, to allow direct comparisons of our results with those of Sebastiao & Ribeiro (1990). A typical experiment shown in Fig. 5A and B. Note the lack of effect of PDBu (200 nm) (B) on the normal sensitivity of EPPs to adenosine (A).

7 J Physiol Changes in ACh pools produced by phorbol esters and adenosine 451 Figure 5C shows the averaged concentration response curves for adenosine in control conditions and in the presence of PDBu (open and filled circles respectively, n = 5 experiments). PDBu had no measurable effect on either the relative potency or the maximal inhibitory effect of adenosine (5 50 mm). Also as depicted in Fig. 5C (square symbols), similar results were observed when the PKC antagonist staurosporine was included at a concentration (1 mm, n = 4 experiments) that fully blocks PKC at the skeletal neuromuscular junction (Considine et al. 1992; see Searl & Silinsky, 1998 for discussion) and also blocks PKCs that are not activated by phorbol esters or calcium (Gschwendt et al. 1992). In order to allow a more direct visual comparison of the adenosine concentration response curves in control and in PDBu, the data of Fig. 5C were normalised in Fig. 5D. These results indicate no Figure 5. The effect of PDBu on the concentration response curve for the inhibition of ACh release by adenosine. A and B, the effects of adenosine on averaged EPPs from a single experiment recorded in the absence (A) and presence (B) of PDBu (200 nm). EPP amplitudes were increased by PDBu, but, as shown here, there was no significant difference in the degree of inhibition produced by adenosine on the neurotransmitter release process. C, the effect of PDBu on the adenosine concentration response inhibition of ACh release both in the absence of any PKC inhibitor and in the presence of the PKC inhibitor staurosporine. In the absence of any PKC inhibitor, control EPPs in the absence of PDBu are shown by 1 and the presence of PDBu (200 nm) (0). Each point represents the average ± 1 S.E.M. of EPP amplitudes as a percentage of the control EPP (recorded in the absence of adenosine or PDBu). Each individual experiment was carried out on a single endplate (n = 5 experiments). Also shown is the concentration response curve for inhibition of ACh release by adenosine in the presence of staurosporine (1 mm) (ª) and the presence of staurosporine (1 mm) and PDBu (200 nm) ( ). Each individual experiment was carried out on a single endplate (n = 4 experiments) and plotted with each point representing the average ± 1 S.E.M. of the EPP amplitudes as a percentage of the control EPP (recorded in the absence of adenosine or PDBu). The data from the dose response curve plotted in the absence of any PKC inhibitor was replotted in D with the EPPs recorded in the absence of adenosine in the presence and absence of PDBu normalised to the same scale. This allows a more direct visualisation of the lack of effect of PDBu on the inhibition of release by adenosine.

8 452 T. J. Searl and E. M. Silinsky J Physiol change in the sensitivity to adenosine and further suggest that these two agents are acting independently. The effects of PDBu and adenosine on the readily releasable pool of neurotransmitter as studied by high frequency trains of stimuli While the above experiments suggest that the opposing effects of phorbol esters and adenosine are mediated by changes in the same statistical parameter, n, these effects on n are likely to occur through different sites of action. Observations that phorbol esters increase ACh release at all calcium concentrations (Redman et al. 1997) suggest that phorbol esters increase the total number of primed vesicles (those within the readily releasable pool). In contrast, adenosine produces a shift to the right of the [Ca 2+ ] ACh release curve without a change in maximum, suggesting that adenosine reduces the apparent calcium affinity of a component of the secretory apparatus (Silinsky, 1981, 1984, 1985). To define more clearly the sites of action of PDBu and adenosine we used EPPs generated by high frequency trains of stimuli to examine the readily releasable pool of ACh quanta. For these experiments we stimulated the motor nerve with short (400 ms) high-frequency (100 Hz) trains every 2 min, in high extracellular calcium concentrations (5.4 mm). This is an approach similar to that employed by Elmqvist & Quastel (1965) and is highly suitable for use at the skeletal neuromuscular junctions, because these short high-frequency trains of EPPs are very stable and reproducible. From Fig. 6, two phases in the EPP trains can be discerned. The first phase of rapid rundown in transmitter release is thought to represent the depletion of the readily releasable pool of quanta. The steady-state plateau of release seen at the latter part of the train is thought to be determined by the rate of mobilisation or the rate at which vesicles can dock at the active zones of secretion. If a change in n is due to a change in the number of synaptic vesicles in the readily releasable pool, then such an effect should be reflected in the early part of the train but absent at the latter part of the train (where release is primarily dependent on the mobilisation of vesicles). Indeed as shown in Fig. 6A, application of PDBu resulted in an increase in the size of the readily pool of ACh without affecting the rate of mobilisation. Making the assumption that the rate of mobilisation (i.e. the plateau phase) is constant throughout the train (from the second stimuli), it was found that PDBu increased the readily releasable pool of neurotransmitter by 141 ± 9 %, an effect that Figure 6. The effects of PDBu and adenosine on high-frequency (100 Hz; 5.4 mm Ca 2+ ) EPP trains Short (400 ms) trains of EPPs were evoked by highfrequency (100 Hz) stimulation of the motor nerve. Each train of stimuli was separated by 2 min of rest. The experiments were performed in 5.4 mm calcium, which is close to the maximal level of the [Ca 2+ ] EPP amplitude curve (see e.g. Silinsky, 1981; Redman et al. 1997). Each bar represents mean of each EPP amplitude (n = 5 experiments). EPP amplitudes were normalised as a percentage of the first EPP in the control train (± S.E.M.). As shown in A, PDBu (100 nm) in the presence of the PKC inhibitor Ro increases EPP amplitudes over the first few stimuli of the train (1 7 stimuli), but had no effect on the latter part of the train (compared with control). B shows the effects of adenosine (50 mm). Note that adenosine reduced EPP amplitudes throughout the train (n = 5; P < 0.05). The difference between adenosine and control EPPs was maximal at the initial part of the train. However, a much smaller, but significant, difference between adenosine and control EPPs remained during the late plateau phase. Conditions were chosen such that endogenous adenosine derived from ATP would saturate the 5 -ectonucleotidase (Cunha, 2001), eliminating the involvement of endogenous adenosine derived from ATP (Redman & Silinsky, 1994) in this study.

9 J Physiol Changes in ACh pools produced by phorbol esters and adenosine 453 is statistically indistinguishable from the increase in n observed in the binomial analysis (147 %, Fig. 3). The same type of analysis indicated that adenosine reduced the readily releasable pool of transmitter to 53 ± 6 % of control (Fig. 6B). The reduction in the readily releasable pool by adenosine was statistically indistinguishable from the inhibitory effect of adenosine in the binomial parameter n (50 % of control, Fig. 3). However, in contrast to the effects of phorbol, the inhibitory effects of adenosine also occurred over the entire train. If adenosine reduces the apparent affinity of a calcium-dependent step in the release process (see Silinsky, 1981, 1984), then the effects of adenosine would be expected to occur throughout the train, with reduced effectiveness during the plateau phase. This is because during the plateau phase the intracellular calcium concentration become elevated when compared to that at the beginning of the stimulation train (Wu & Betz, 1996; Suzuki et al. 2000). This higher calcium concentration would be predicted to reduce the degree of inhibition produced by adenosine. (For a competitive relationship, the degree of inhibition would be greater at the lower concentration of calcium ions see e.g. Segel, 1975.) This is consistent with the experimental results (see Fig. 6 legend for further details). The different effects of PDBu and adenosine on the pattern of release during these trains of stimuli demonstrate that while both agents have opposing effects on the parameter n, these actions are likely to be mediated through actions on independent presynaptic mechanisms. DISCUSSION The first goal of this work was to test the applicability of the binomial distribution to the process of evoked neurotransmitter release at the skeletal neuromuscular junction. In this regard, a large number of EPPs were analysed in an attempt to detect deviations from the simple binomial distribution. No detectable deviations from the simple binomial distribution were observed using the x 2 test and Monte Carlo simulations. In addition, when EPP amplitudes were summed and binomial analysis performed, the estimate of p was found to be independent of n, suggesting a lack of significant temporal variance in p (see Supplemental material, eqn (7)). The actions of PDBu and adenosine as studied by binomial analysis and high-frequency trains of stimuli Our next goal was to use binomial analysis, in consort with studies of high-frequency stimulus trains to determine the possible sites of action of phorbol esters and adenosine. In both the binomial analysis of EPP amplitudes and the analysis of the pattern of ACh release evoked during highfrequency trains, the effects of PDBu were studied in the presence of a PKC inhibitor. This allowed us to restrict our interpretations of the actions of PDBu to those involving Munc-13 or another phorbol ester receptor other than PKC. These results demonstrate that both the increases in evoked ACh release produced by phorbol esters and the reductions in ACh release produced by adenosine were accompanied by directly proportionate changes in the binomial parameter n. No measurable effect on p was produced by either agent. Because these effects were exclusively on n, they must be independent of any error due to variance of either p or n (for a full discussion, see Supplemental material). In addition, these results suggest that both agents act downstream of calcium entry. If calcium ion entry were affected then we would expect changes in both binomial parameters n and p (see Fig. 1 and Bennett et al. 1975, 1976; Miyamoto, 1975). Indeed, measurements of perineural calcium currents demonstrated that the effects of neither phorbol esters nor adenosine on ACh release can be attributed to effects on calcium currents in the frog (Silinsky & Solsona, 1992; Redman et al. 1997). It thus seems likely that these changes in n must reflect real changes in the readily releasable pool of calcium-charged vesicles (even if n does not represent the actual size of the readily releasable pool of vesicles). Given the opposing actions of adenosine and PDBu on n, we also tested the effects of these agents on neurotransmitter release during high-frequency trains of stimuli. This method provided an alternative measure of the readily releasable pool of neurotransmitter as well as the mobilisation process. The results demonstrated that the increase in ACh release by PDBu was accompanied by an increase in the size of the readily releasable store of ACh, without an increase in the rate of mobilisation of neurotransmitter for release. In addition, the increase in the size of the readily releasable pool produced by phorbol esters was similar to that produced on the binomial parameter n. The increases in the binomial parameter n and the readily releasable pool of ACh by PDBu thus entirely support the proposed role of Munc-13 to act in consort with syntaxin to promote vesicular priming (Brose et al. 2000). Our results are also consistent with the effects of overexpression of Munc-13 in amphibian motor nerve endings (Betz et al. 1998). Indeed, PDBu causes a similar increase in the readily releasable pool of ACh at the mouse neuromuscular junction (Silinsky & Searl, 2003; Fig. 5). Adenosine also decreases n and the immediately available store but this is likely to be due to an effect on the release properties of pre-primed vesicles (see below). Studies of the effects of phorbols on CNS neurons (e.g. Stevens & Sullivan, 1998; Rosenmund et al. 2002) are hindered by the inherent lack of experimental accessibility to the presynaptic element and the paucity of presynaptic release sites and postsynaptic receptors. Because of this, estimates of the readily releasable store at central synapses rely upon more indirect methods than those we have used

10 454 T. J. Searl and E. M. Silinsky J Physiol here. The method generally employed to estimate the readily releasable store in the CNS is the sucrose method in which the size of the pool of transmitter is estimated by quantifying the large increases in spontaneous quantal secretion evoked by hypertonic sucrose superfusion (Stevens & Sullivan, 1998). This method is not applicable to evoked release at the frog neuromuscular junction (see Kashani et al. 2001; Silinsky & Searl, 2003) where the effects of sucrose on ACh release are related to the effects of tonicity on integrins (see Kashani et al. 2001). Interestingly, as reported by Rhee et al. (2002), deletion of the phorbol ester binding site on Munc13-1 decreased refilling of the primed pool of vesicles associated with neurally evoked release but had no effect on refilling of the pool evoked by hypertonic sucrose solutions. One possibility is that sucrose can release both primed and docked (but unprimed) vesicles. Such a mechanism would explain the reduced effect of phorbol esters on the readily releaseable store (as assessed by the sucrose method) as priming by phorbols would not be required for release from this store, and hence a significant fraction of the store would be phorbol-independent. Alternatively, these results could be due to real differences between central and peripheral synapses, to different isoforms of Munc-13 or to effects on another, as yet undiscovered non-pkc target for phorbol esters in motor nerve endings. Do adenosine and PDBu act to modulate neurotransmitter release via a common mechanism? The final goal of this paper was to determine if there is any interaction between the effects of phorbol esters and those of adenosine. In the experiments employing binomial analysis experiments, no evidence was found for any interactions between PDBu and adenosine. Thus, the effects of adenosine and PDBu on m and n were simply additive (in the presence of a PKC inhibitor), However, these experiments did not rule out a possible interaction between PDBu and adenosine through a PKC-dependent mechanism. Therefore, in order to further test for a possible interaction between PDBu and adenosine, simple adenosine concentration response curves in the absence and presence of PDBu (200 nm) were constructed. These experiments were performed both in the absence of a PKC inhibitor (Fig. 5C) to allow more direct comparison with the experiments of Sebastiao & Ribeiro (1990), and in the presence of a PKC inhibitor (Fig. 5D). Again, no evidence was found for an occlusive effect of PDBu on the action of adenosine, suggesting that the two agents exert their effects on the readily releasable pool of ACh through separate mechanisms. In particular, the effects of adenosine in this preparation are independent of either Munc-13 or PKC. The reason for the disparity between the present results and those of Sebastiao & Ribeiro (1990) is unknown. Sebastiao & Ribeiro (1990) found that relationship between adenosine concentration and percentage inhibition of ACh release was decreased slightly by the presence of the phorbol ester phorbol diacetate (the effect of the phorbol ester on the maximum response to adenosine was not determined). The differences between our results and theirs could be related either to differences in the preparations used (the frog proprialis nerve cutaneous muscle preparation in our studies, the sciatic nerve sartorius muscle preparation in the study of Ribeiro & Sebastiao) or to differences in the phorbol ester used. Indeed, the increase in ACh release produced by phorbol diacetate was very modest (Sebastiao & Ribeiro, 1990) when compared to the published results using other phorbol esters at the frog neuromuscular junction (Shapira et al. 1987; Redman et al. 1997; Searl & Silinsky, 1998). Another possibility is that the high levels of basal endogenous adenosine present in the sartorius preparation (Ribeiro & Sebastiao, 1987; see Redman & Silinsky, 1994 for discussion) in combination with the adenosine released in response to nerve stimulation under the conditions of these experiments helped reduce the increase in ACh released by the phorbol ester. In addition to limiting the apparent effectiveness of the phorbol ester, basal levels of adenosine might also reduce the effects of exogenous adenosine under the stimulation conditions used by Sebastiao & Ribeiro (1990). Differences between the mechanisms of action of phorbol esters and adenosine While the effects of both phorbol esters and adenosine are exerted exclusively on the binomial parameter n, the mechanisms of action differ. Specifically, these present results, when taken with the results of others suggest that phorbol esters increase n by increasing the conversion of docked but unprimed vesicles to primed vesicles (Betz et al. 1998; Brose & Rosenmund, 2002), without a change in the calcium dependence of the ACh release process (Redman et al. 1997). Indeed, studies using high-frequency trains (Fig. 6) demonstrate that the effect of phorbol esters occurs early on in the train when priming is still the rate limiting step, but not during the plateau phase when mobilisation is rate limiting. In contrast, the decrease in n by adenosine receptor agonists is associated with a change in the concentration response curve for Ca 2+, shifting the potency for extracellular Ca 2+ (Silinsky, 1981) and intracellular Ca 2+ or Sr 2+ (Silinsky et al. 1999) to lower apparent affinity. The effect of adenosine also occurs with an extremely short latency as assessed by fast-flow superfusion with 25 mm 2-chloroadenosine (E. M. 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