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. 1999 Jun 15;517(Pt 3):879–888. doi: 10.1111/j.1469-7793.1999.0879s.x

Noradrenaline synchronizes evoked quantal release at frog neuromuscular junctions

Ella A Bukcharaeva *,†, Kira C Kim †, J Moravec ‡, E E Nikolsky *,†, F Vyskočil ‡,§
PMCID: PMC2269380  PMID: 10358126

Abstract

  1. Noradrenaline (NA) increases synaptic efficacy at the frog neuromuscular junction. To test the hypothesis that one of the actions of NA is to shorten the period over which evoked quanta are released, we measured the latencies of focally recorded uniquantal endplate currents (EPCs).

  2. NA shortened the release period for evoked quantal release. The interval between the time when responses with minimal delay appeared and the point at which 90 % of all latencies had occurred was shortened in the presence of 1 × 10−5 M NA by about 35 % at 20 °C and by about 45 % at 8 °C. Inhibitor and agonist experiments showed that NA acts on a β-adrenoreceptor.

  3. The better synchronization of release significantly increased the size of reconstructed multi- quantal EPCs. This suggests that NA facilitates synaptic transmission by making the release of quanta more synchronous.

  4. The synchronizing action of NA might potentiate neuromuscular transmission during nerve regeneration, transmitter exhaustion and other extreme physiological states where the quantal content is reduced, such as survival in cold and hibernation.

It has long been known that catecholamines facilitate neuromuscular transmission and muscle contraction (Orbeli, 1923; Burn, 1945). The mechanism underlying the facilitation by different sympathomimetics may vary. Thus, noradrenaline (NA), phenylephrine and adrenaline increase the number of quanta released by nerve stimulation (Jenkinson et al. 1968; Hidaka & Kuriyama, 1969; Kuba, 1970; Kuba & Tomita, 1971; Wessler & Anschuetz, 1988; Vizi, 1991). In chick ciliary ganglion, NA appears to elevate transmitter release by enhancing the Ca2+ sensitivity of exocytosis (Yawo, 1996). Isoproterenol (isoprenaline) increases the input resistance of muscle fibres, thereby increasing the amplitude of endplate potentials. Adrenaline can have both pre- and postsynaptic actions (Kuba, 1970; Chen et al. 1991). NA stimulates the electrogenic sodium pump and increases the resting membrane potential (Zemkováet al. 1985). The facilitating action of NA is particularly interesting, since it may underlie a variety of important physiological processes, such as the phenomena of neuromuscular facilitation and muscle contraction and the recovery of animals from hibernation (Hidaka & Kuriyama, 1969; Kuba & Tomita, 1971; Melichar et al. 1973; Moravec et al. 1973; Anderson & Harvey, 1988; Banos et al. 1988; Wessler et al. 1990b; Yawo, 1996).

In recent years, various agents have been shown to alter the time course of the evoked release of neurotransmitter quanta from nerve terminals (Matzer et al. 1988; Van der Kloot, 1988a,b; Molgóet al. 1989). The time course of quantal release can be experimentally estimated by lowering the number of quanta released per stimulus and then measuring the synaptic delays for evoked uniquantal endplate currents (EPCs) (Katz & Miledi, 1965a,b; Barrett & Stevens, 1972a,b). Our preliminary data showed that NA can shorten the time course for quantal release when the number of quanta released per stimulus is low (Bukcharaeva et al. 1998). The aim of the present study was to characterize the action of NA and several other adrenergic compounds on the time course of evoked quantal secretion at the frog neuromuscular junction and to estimate the impact this action might have on the amplitude of multiquantal EPCs. The impact is assessed from mathematical models of EPC summation (Soucek, 1971; Giniatullin et al. 1995).

METHODS

Experiments were performed on isolated m. cutaneus pectoris neuromuscular preparations from the frog Rana ridibunda during the winter period (October-March). Animals were anaesthetized with ether before being stunned and pithed. The isolated preparations were pinned on the bottom of a 1.5 ml translucent chamber with several compartments, superfused with the following solution (mM): NaCl, 113.0; KCl, 2.5; CaCl2, 0.2; NaHCO3, 3.0; MgCl2, 4.0. pH was adjusted to 7.3. The solution flowed through the muscle chamber at a rate of 3 ml min−1. Monitoring of the bath solution throughout the experiment did not reveal any changes in pH after passing through the muscle chamber. The measurements were carried out at either 20.0 ± 0.3°C or 8.0 ± 0.3°C and temperature was controlled by a Peltier semiconductor device. The protocol and the experiments were approved by the Animal Care and Use Committee of The Institute of Physiology, Czech Academy of Sciences.

The following drugs were used (all from Sigma): noradrenaline (NA), dobutamine, clonidine, phenylephrine, phentolamine, propranolol, isoproterenol and atenolol. The drugs were added to the superfusing solution. Unless otherwise stated, the measurements were started 20 min after drug application. In most cases, drugs were washed out for another 30 min and EPCs were recorded again.

Electrophysiology

Square suprathreshold stimuli of 0.1 ms duration were delivered to the nerve at 2 s intervals via a pair of platinum electrodes located in a small adjacent moist chamber (Beránek & Vyskocil, 1967). This arrangement minimized the stimulus artifact. Nerve action potentials and extracellular endplate currents were recorded using focal extracellular pipettes filled with low-Ca2+ (0.2 mM) Ringer solution. Extracellular pipettes with tip diameter of 2–3 μm and 1–3 MΩ resistance were positioned under visual control in the proximal endplate region of a large nerve terminal, 3–5 μm from the beginning of the myelination of the axon. In this region, three-component nerve spikes, EPCs and miniature EPCs (mEPCs) were recorded (Mallart, 1984; Shakiryanova et al. 1994). The zero (reference) baseline for each EPC or mEPC amplitude estimation was continuously counted from the 333 noise amplitudes measured randomly during the preceding interstimulus period.

The recorded signals were filtered between 0.03 Hz and 10 kHz, digitized at 3 μs intervals by an analog-digital 9-bit converter, fed into the computer and processed by our application program package. Amplitudes of extracellular responses are expressed in millivolts.

To estimate the time course of individual quantal release from the dispersion of synaptic delay values, uniquantal endplate currents are required (Katz & Miledi, 1965a,b; Barrett & Stevens, 1972a). Experiments were therefore carried out in the presence of 0.2 mM Ca2+ and 4.0 mM Mg2+. The quantal content (mo) of the low-quanta EPCs was determined from five or six stimulation periods (256 stimuli each) in the presence and absence of NA or other drugs. The number of failures in a series of 250–400 uniquantal responses were measured and mo calculated as equal to lnN/no, where N is the total number of stimuli and no is the number of failures (del Castillo & Katz, 1954; Martin, 1955). The number of stimuli was therefore 1280–1536 in each series of stimulation periods. Unless mo is quite low, at least some of the responses in a series of 250–400 responses will be larger than uniquantal.

Latency measurements

Latency was measured as the time interval between the peak of the inward presynaptic Na+ current and the time at which the rising phase of the quantal event reached 20 % of maximum. The latency limits of the early release period were 6 ms for experiments at 20°C and 22 ms for those performed at 8°C. The mean value of the shortest 5 % of latencies in each series was taken as the minimum synaptic delay. The stability of the recording electrode positioning next to the membrane region of interest was crucial during long-lasting extracellular recordings. We therefore monitored the amplitude of the nerve terminal action potentials and the rise and decay of mEPCs throughout each data set. Only data sets in which the terminal spike and time course of mEPCs changed by less than 10 % during drug application and washout were analysed further. Statistical analyses of pre- and postsynaptic events were performed using Student's t test for paired data (Origin; Microcal, Northhampton, MA, USA).

The quantitative characteristics of the change in the time course of evoked secretion produced by the action of NA were obtained by one of the following two methods:

Cumulative curve analysis

Cumulative curves were built from latency histograms of the selected uniquantal EPCs (corresponding to the first peak in the amplitude histograms; see Fig. 1B). The interval between the minimum synaptic delay and the time at which 90 % of all measured uniquantal EPCs had occurred was designated as P90. The statistical significance of the difference between two cumulative curves was assessed by the Kolmogorov- Smirnov test; p < 0.05 was taken as significant (Bronstein & Semendjaev, 1986; Van der Kloot, 1991).

Figure 1. Effect of 1 × 10−5 M NA on the latency of quantal release at 20 °C.

Figure 1

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Extracellularly recorded presynaptic nerve spikes and endplate currents (A), distribution of endplate current amplitude (B) and release latencies (C) before (a), after noradrenaline (NA) application (b) and after washout of NA (c) at 20 °C. A, 12 superimposed recordings of stimulus artifacts (SA), presynaptic spikes (PS) and individual endplate currents (EPC). The stimulus artifact is followed by a 3-phase action potential, which is typically seen in the proximal region of the nerve terminal (Mallart, 1984; Shakiryanova et al. 1994). Insets, 50 averaged presynaptic spikes and 50 averaged EPCs from the same endplate. There was little change in the amplitude of the presynaptic spikes, but Ac (NA washout) shows a marked slowing of the time course of this action potential, which does not, however, seem to be important for the time course of release. The arrow indicates the Na+-inward part of the presynaptic spike. The latency of each EPC was measured from the peak deflection of the spike (see Methods). B, amplitude distribution of all quantal responses. The left bar denotes the incidence of failures. The first peak of the histogram, considered as the uniquantal response, is terminated by the dashed line. C, normalized histograms of 252 latencies of putative uniquantal EPCs from the same endplate. Quantal content (mo) was calculated by the method of failures from 5 stimulation periods (256 stimuli each) in the presence and absence of NA. mo was 0.22 ± 0.08 (n = 5) before, 0.24 ± 0.09 (n = 5) after 1 × 10−5 M NA application and 0.20 ± 0.07 (n = 5) after washout of NA.

Release probability function, α(t)

This method (Barrett & Stevens, 1972b) avoids possible problems of identifying uniquantal EPCs and the overlapping of several quanta released simultaneously by a single stimulus. Only the latency to the first quantal release following each stimulus is measured. The function α(t) is defined as α(t) =S(t)/[1 - S(t)], where S(t) represents the probability that the first quantal latency occurred in an interval of about time t and S(t) is the probability of a first quantal latency occurring between the beginning of the early release period and t. Then [1 - S(t)] denotes the probability that the first latency is longer than t. Both S(t) and S(t) were obtained from the histograms of first quantal latencies. The rising phase of the resulting curves could be fitted with an S-shaped function and the decaying phase by two exponential functions (e.g. Fig. 3B). The statistical significance of differences between the control and experimental curves was estimated by the χ2 test; a probability level (p) of 0.05 was considered significant.

Figure 3. Effect of 1 × 10−5 M NA on the release probability function α(t) at 20 °C.

Figure 3

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Linear (A) and semilogarithmic (B) plots of release probability α(t) against time before (open symbols, continuous lines) and after NA application (filled symbols, dashed lines) are shown. Pooled data from 9 endplates were used. The rise time of α(t) was measured as the time at which the rising phase reached 80 % from 20 % of maximum. In B, the decays of the curves can be approximated by 2 exponentials.

Statistics

Other statistical tests were performed with SigmaStat 0.1 (Jandel Corporation). Analyses of variance (ANOVAs) of the experimental group versus the control group were performed by multiple comparison using the ANOVA Bonferroni t test. The amplitudes, rise times of EPCs and mEPCs (from 20–80 % of amplitude) and exponential decay constants τEPC and τmEPC of the responses are presented throughout this paper as means ±s.e.m. Differences between two groups were considered statistically significant at the probability level p≤ 0.05. The symbol n indicates the number of endplates measured in each group.

RESULTS

Time course of evoked quantal release in the presence of NA at 20°C

Superimposed nerve action potentials and EPCs in response to stimulation under control conditions at 20.0 ± 0.3°C are shown in Fig. 1Aa. The EPCs appear after the synaptic delay or latency (Katz & Miledi, 1965a,b). In the low-Ca2+, high-Mg2+ solution mean mo was 0.33 ± 0.09 (n = 9). One experiment is shown in Fig. 1. The histograms of the EPC amplitudes displayed two peaks (Fig. 1Ba); the mean value of the EPC amplitude for the dominant first peak (uniquantal EPCs) was 0.151 ± 0.02 mV (n = 252) in this particular experiment, which closely matched the mean amplitude of the mEPCs (0.153 ± 0.01 mV, n = 128, not shown) recorded from the same synapse. The number of responses that formed the first peak was 252 (Fig. 1Ba, terminated by the dashed line) while the number of responses forming the second peak was 30; these numbers were similar to those predicted for uni- and biquantal responses by the Poisson distribution (255 and 28 for 1437 stimuli and 1151 failures).

The synaptic latency histograms for uniquantal EPCs were asymmetric due to a large number of long synaptic delays (Fig. 1Ca). NA was then applied at a concentration of 1 × 10−5 M. This concentration was selected after preliminary tests with concentrations ranging from 1 × 10−6 to 2 × 10−4 M. NA did not change the presynaptic action potential, the EPC amplitude (see insets in Fig. 1Aa and B and Table 1) or the mean quantal content (Table 1). The mean frequency of mEPCs in the presence of NA (1.73 ± 0.45 s−1, n = 9; p > 0.05) was very similar to that recorded before NA (1.64 ± 0.50 s−1). NA did not change the amplitude and decay time of mEPCs, suggesting that it had no effect on postsynaptic receptor sensitivity.

Table 1.

Parameters of putative uniquantal EPCs and miniature EPCs (mEPCs) in control solution and in the presence of 1 × 10−5 m noradrenaline (NA) at 20 °C and 8 °C

Temperature Amplitude (mV) Rise time (ms) τdec (ms) Quantal content n
EPC, control 20 °C 0.156 ± 0.010 0.162 ± 0.024 1.96 ± 0.27 0.33 ± 0.09 9
EPC, NA 20 °C 0.144 ± 0.030 0.123 ± 0.034 2.02 ± 0.42 0.39 ± 0.08 9
EPC, NA washout 20 °C 0.134 ± 0.041 0.145 ± 0.028 2.12 ± 0.33 0.36 ± 0.06 9
mEPC, control 20 °C 0.162 ± 0.010 0.129 ± 0.029 1.58 ± 0.29 – 9
mEPC, NA, 20 °C 0.159 ± 0.020 0.118 ± 0.023 1.43 ± 0.20 – 9
mEPC, NA washout 20 °C 0.148 ± 0.031 0.127 ± 0.031 1.64 ± 0.14 – 9
EPC, control 8 °C 0.103 ± 0.042 0.349 ± 0.154 5.65 ± 0.21 0.23 ± 0.23 5
EPC, NA 8 °C 0.110 ± 0.050 0.370 ± 0.160 4.99 ± 0.47 0.25 ± 0.28 5
EPC, NA washout 8 °C 0.105 ± 0.043 0.418 ± 0.151 5.40 ± 0.41 0.20 ± 0.43 5
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τdec, decay time constant. n, number of endplates measured in each group. Statistical tests (1-way ANOVA) showed no significant differences among control and test groups for p > 0.05.

NA did, however, change the latency distribution significantly (Fig. 1C b): the number of EPCs with longer synaptic latencies was decreased, as was the modal value (from 0.95 to 0.70 ms). Interestingly, the minimal synaptic delay was unchanged (0.48 ± 0.05 ms in 9 control experiments vs. 0.52 ± 0.04 ms in the presence of NA, p > 0.05). NA washout restored the distribution of synaptic latencies towards the initial pattern (Fig. 1C c).

To quantify the effects of NA on the synaptic latency, normalized distributions of synaptic delay (Fig. 2A) and cumulative curves (Fig. 2B) for uniquantal EPCs before and after addition of NA were constructed. The control value of P90, 2.16 ± 0.23 ms, was significantly reduced (by the Kolmogorov-Smirnov criteria) to 1.41 ± 0.10 ms (n = 9, p < 0.05) in the presence of NA. The ratio between the P90 value in NA versus that in the control solution was thus 0.65, indicating that NA shortened the early release phase by 35 %.

Figure 2. Effect of 1 × 10−5 M NA on the synaptic latency of 9 endplates at 20 °C.

Figure 2

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A, normalized latency histogram of 2268 uniquantal EPCs from 9 experiments. The size of the bin was 0.1 ms. ▪, NA; □, control. B, cumulative plot of latencies for the same data. •, NA; ○, control. The vertical dotted lines indicate the times when 90 % of the quanta have been released (P90). The dotted lines parallel to the curves are confidence limits at p = 0.05. According to the Kolmogorov-Smirnov criteria the difference between the curves is statistically significant if the confidence limits of the 2 curves do not overlap.

The synchronizing effect of NA was also measured by means of the release probability function α(t), which avoids the necessity of deciding whether each EPC is uniquantal or not (Barrett & Stevens, 1972a,b; Baldo et al. 1986). The α(t) functions derived from the synaptic delay distribution histograms for all the EPCs recorded (including multiquantal ones) had a sigmoid rising phase; the rise times were 0.24 ms in the control solution and 0.22 ms in the presence of NA. The decay phases of the functions showed two exponentials (Fig. 3A); the time constant of the fast decay component was 0.075 ms after subtracting the extrapolated slow contribution, whilst that of the slow component was 0.86 ms (Fig. 3B). The addition of NA decreased the time constants by 25 % and 42 %; the fast component was 0.055 ms and the slow one 0.50 ms. Thus by the χ2 test criterion, NA significantly changed the release probability function, in particular the slow component.

In summary, both methods of analysis of the time course of evoked release revealed that NA increased the number of postsynaptic responses with shorter synaptic delays.

Time course of evoked quantal secretion in the presence of NA at 8°C

Lowering the temperature increases the asynchrony of transmitter secretion (Katz & Miledi, 1965b), mostly due to a rise in the number of EPCs with larger synaptic delays. This broadens the histogram of the delay distribution. If the NA effect described above were indeed due to changes in the synchrony of evoked release, then this action of the drug would be expected to be more pronounced at lower temperatures. In experiments carried out at 8°C, the minimal synaptic delays were longer (2.62 ± 0.21 ms, n = 7) than those at 20°C. Rise time and decay time constants of EPCs were also prolonged (Table 1). Synapses selected for analysis of the time course of evoked release had quantal contents of 0.2 ± 0.1 or higher, to ensure obtaining at least 250 EPCs. All EPCs obtained under these conditions were uniquantal according to amplitude analysis (not shown). The time interval during which evoked EPCs appeared increased from ∼6 ms at 20°C to ∼22 ms at 8°C.

The addition of NA markedly shifted the decaying phase of the distribution histogram towards a lower synaptic delay (Fig. 4A). The modal value of the distribution histogram also changed significantly from 3.94 ± 0.17 ms in control solution to 2.84 ± 0.22 ms (n = 5; p < 0.05) in NA. NA reduced the minimal value of the synaptic delay to 2.16 ± 0.24 ms from a control value of 2.62 ± 0.2 ms, although the difference was not significant.

Figure 4. Effect of 1 × 10−5 M NA on synaptic latencies at 8 °C.

Figure 4

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A, normalized latency histograms of 1260 uniquantal EPCs from 5 experiments. The size of the bin was 0.5 ms. ▪, NA (n = 5 endplates); □, controls (n = 5). B, same data as A shown as cumulative plots of latencies in control solution (□), in the presence of NA (▪) and after washout of the NA (+) (n = 5). The dotted vertical lines indicate the times when 90 % of the quanta have been released (P90).

Quantitative analysis of cumulative curves derived from the synaptic delay histograms revealed that NA decreased the P90 from 9.98 ± 1.34 to 5.51 ± 0.55 ms (n = 7; p < 0.05) (Fig. 4B, vertical dotted lines). On washout of NA, P90 recovered to 8.89 ± 1.20 ms.

The release probability function α(t) derived from the synaptic delay histogram at the lower temperature (8°C) also showed a more pronounced NA effect than at 20°C (Fig. 5).Lowering the temperature slowed the decay of the α(t) function: the fast decay time constant was 1.67, while the slow one was 6.62 ms. Addition of NA changed the decay phase so that it followed a single exponential with a time constant of 1.37 ms, which is in fact even significantly smaller (18 % by the Kolmogorov-Smirnov criteria) than the time constant of fast decay in control solutions. The ratio of the P90 in NA to the control P90 was 0.55, so the effect was larger than at 20°C. Thus, NA affected the time course of transmitter release at low temperature even more than at room temperature, as shown by the 45 % shortening of the P90 release period.

Figure 5. Effect of 1 × 10−5 M NA on the release probability function α(t) at 8 °C.

Figure 5

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Time course of α(t) expressed as linear (A) and semilogarithmic (B) plots. In B, the decay of α(t) can be approximated by 2 exponentials in control solutions (□, continuous lines) and 1 exponential (▪, dashed line) in the presence of NA.

Which receptor type mediates the synchronizing effect of NA on evoked secretion?

To determine the type of adrenergic receptor responsible for the synchronizing action of NA on evoked release, the effects of selective α- and β-adrenergic agonists on secretion kinetics, as well as the ability of α- and β-adrenergic antagonists to depress the effects of NA were studied. The selective α1- and α2-adrenergic agonists phenylephrine (1 × 10−5 M) and clonidine (1 × 10−5 M) did not change the distribution of synaptic delays; the ratio of the P90 values was 0.94 ± 0.04 (n = 6; p > 0.05) for phenylephrine and 1.03 ± 0.03 (n = 6; p > 0.05) for clonidine. The α1- and α2-adrenergic antagonist phentolamine (1 × 10−5 M) and the α2-adrenergic antagonist yohimbine (1 × 10−5 M) did not block the synchronizing action of NA (P90 in the presence of NA during continuous superfusion with phentolamine was 0.79 ± 0.03 (n = 5) and 0.88 ± 0.03 with yohimbine (n = 5); p < 0.05 for both drugs). The selective β-adrenergic agonists dobutamine and isoproterenol (both at 1 × 10−5 M) caused effects similar to those of NA. P90 values in the presence of isoproterenol and dobutamine were 0.59 ± 0.02 (n = 5; p < 0.05) and 0.70 ± 0.04 (n = 5; p < 0.05), respectively.

Addition of NA to a superfusion solution containing 1 × 10−5 M atenolol or 1 × 10−5 M propranolol (inhibitors of β1- and β1,2-adrenoreceptors, respectively) failed to synchronize the secretion process, judging by the lack of significant change in P90 in the direction of synchronization; P90 was 1.01 ± 0.05 in propranolol (n = 5; p > 0.05) and 1.13 ± 0.03 in atenolol (n = 5; p < 0.05) (Fig. 6).

Figure 6. Effects of α- and β-adrenergic agonists and antagonists on P90 values in the presence of the drug (dP90) vs. in control solution (cP90).

Figure 6

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The drugs applied were: PHE, 1 × 10−5 M phenylephrine; CLN, 1 × 10−5 M clonidine; PNT + NA, 1 × 10−5 M phentolamine + 1 × 10−5 M NA; YOH + NA, 1 × 10−5 M yohimbine + 1 × 10−5 M NA; NA, 1 × 10−5 M NA; IPR, 1 × 10−5 M isoprotenerol; DBT, 1 × 10−5 M dobutamine; AT + NA, 1 × 10−5 M atenolol + 1 × 10−5 M NA; and PRO + NA, 1 × 10−5 M propranolol plus 1 × 10−5 M NA. AT or PRO alone were without significant effect (data not given). The number of experiments done with each tested drug was 5 or 6. Significant differences against 100 % dP90: cP90 (shown by the dotted line) are indicated by asterisks. Temperature, 20 °C.

These results suggest that NA speeds up the time course of release via presynaptic β1-adrenoreceptors.

Multiquantal EPC reconstruction using measured values of synaptic delays

The synchronization of transmitter release by NA increased the EPC amplitude (Fig. 7A-C). To quantify the impact of synchronization on the EPC peak amplitude, multiquantal EPCs were constructed from the experimentally measured parameters of the uniquantal EPCs (Table 1) and the values of the synaptic delay by a convolution method (Fig. 7D). The ‘synchronous’ curve represents the EPC obtained during ideally synchronous transmitter release (dispersion of synaptic delays = 0). The amplitude of the EPC constructed from delays and EPC parameters under low-Ca2+ conditions (Fig. 7D, control) at 20°C was lower than that of the synchronous EPC by as much as 26 %. The amplitude of the reconstructed EPC in the presence of NA (NA curve in Fig. 7D) was 17 % larger than in control, evidently due to increased synchronization of release. When the values of the synaptic delay obtained at 8°C were used for reconstruction (Fig. 7E), the amplitude of the resulting multiquantal EPC amplitude (Fig. 7E, control) was only 53 % of the fully synchronous one (Fig. 7E, synchronous), presumably due to the greater asynchrony observed under these conditions. The NA-induced increase in EPC amplitude was also greater than at 20°C (26 % more than in the control). Thus the effect of secretion synchronization by NA is evidently greater at low temperature than at room temperature.

Figure 7. Effect of 1 × 10−5 M NA on summed multiquantal (A-C) and reconstructed (D and E) multiquantal EPCs.

Figure 7

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A-C, summations of 40 measured uniquantal EPCs from a single endplate. The summations were normalized according to the downward deflection of the stimulus artifact (SA) before (A), during application of NA (B) and after washout of NA (C) at 20 °C. D and E, computer reconstruction of amplitude (AEPC) and time course of the multiquantal EPCs. For reconstruction, 100 uniquantal EPCs were used with mean parameters (see Table 1) obtained at 20 °C (D) and 8 °C (E). Dashed curve, synchronous or ‘ideal’ EPCs with maximal amplitude (ordinate) considered as 100 %, when dispersion of latencies was zero. Dotted and continuous curves, maximum amplitudes expressed as a percentage of the reference curve peak; EPCs were constructed with measured latency dispersion before (Control) and after NA application, respectively.

DISCUSSION

According to the conventional model of synaptic transmission, the EPC results from the almost simultaneous release of several hundred quanta of acetylcholine in response to a single action potential in the motor nerve. In 1965, Katz & Miledi (1965a,b) demonstrated that the evoked release of neurotransmitter quanta is asynchronous, as shown by the variability in the values of the synaptic delays for uniquantal postsynaptic responses. Mathematical modelling of the uniquantal EPC summation process has been used to show that alterations in the degree of synchrony of the release evoked by a single action potential can modulate synaptic transmission even when other parameters of postsynaptic responses are unchanged (Soućek, 1971; Giniatullin et al. 1995; Zefirov & Gafurov, 1997).

In the present study we addressed the hypothesis (Bukcharaeva et al. 1998) that NA - which is well known to facilitate synaptic transmission, reportedly via a presynaptic mechanism - can not only change EPC quantal content (Kuba, 1970; Kuba & Tomita, 1971; Banos et al. 1988) but also synchronize the release of single quanta. NA (1 × 10−5 M) changed the variability of the synaptic delays but not the minimal delay. This phenomenon of shortening of the delay histogram is not associated with a significant change in quantal content. The modal values of the synaptic delays were reduced, since the numbers of responses with longer synaptic delays were reduced. The latter effect was demonstrated by the reduction in the value of the P90 parameter observed when the cumulative curves were compared (Fig. 2). The EPC amplitude, mean quantal content and mean frequency and amplitude of the spontaneous mEPCs were not changed. These observations (suggesting also the lack of quantum size change after NA) are in agreement with previously published results showing that NA action on both the frequency of spontaneous mEPCs and the quantal content of stimulation-evoked secretion is absent at lower extracellular Ca2+ concentrations (Kuba, 1970; Kuba & Tomita, 1971). The fluctuations of synaptic delays observed in our experiments were not likely to be due to the recording of evoked events together with spontaneous ones. The mean frequency of spontaneous random mEPCs was 1.6 ± 0.5 s−1 in our experiments, and so the probability that an mEPC occurred during the 6 ms time window allocated once every 2 s for evoked EPC recording is as low as 0.0034. From the methodological point of view, the lack of change in the parameters of spontaneous quantal secretion in the presence of NA in successful experiments, as well as the return of synaptic delay histograms towards their initial levels on washout of the drug, also suggest that the effect of NA was not related to any erroneous recording of mEPCs.

To characterize changes in the kinetics of evoked secretion in more detail, the release probability function α(t) was calculated (Barrett & Stevens, 1972a,b; Baldo et al. 1986). Decreases in both the fast and slow components of the decay time constant of this function indicate that not only the early release period but also the late phase found in low-Ca2+ solutions (Barrett & Stevens, 1972a,b), or the period controlled by a high-affinity Ca2+ sensor as suggested by Goda & Stevens (1994), were shortened in the presence of NA. Taken together these data suggest that NA facilitates synaptic transmission by synchronizing the release of neurotransmitter. Two approaches were used to examine the effect of NA-induced synchronization of transmitter release on the amplitude of the multiquantal EPC. First, experimentally measured uniquantal EPCs were summed in the control condition, in the presence of NA and after washout of the drug. The results thus obtained (Fig. 7A-C) suggest that NA can produce a considerable increase in the amplitude of the resulting EPC without affecting quantal content or postsynaptic receptor sensitivity. Second, computer reconstructions of the EPCs were carried out from 100 values of the amplitude-temporal parameters of uniquantal responses on the basis of averaged variations of EPC synaptic delays obtained in nine experiments at 20°C. The increase in EPC amplitude of 17 % by NA observed in the computer model (Fig. 7D) also suggests that the synchronizing effect of NA on release can have a strong impact on the amplitude of the multiquantal EPC even in the absence of a change in the number of quanta. When the bath temperature was lowered to 8°C an expected increase in the asynchrony of release was observed (Fig. 4). At this temperature NA also decreased the dispersion of synaptic delays. Comparison of the numeric parameters of NA action at 8°C with those at 20°C indicated an increase in drug efficacy at the lower temperature. This seems especially interesting when the predictions from mathematical modelling (Van der Kloot, 1988a; Zefirov & Gafurov, 1997) are taken into account. These studies suggest that the increase in mEPC duration observed at lower temperatures (or after cholinesterase inhibition; Giniatullin et al. 1995) renders the effects of secretion synchrony on multiquantal EPC amplitude less prominent. Despite this, the asynchrony leads to a more prominent decrease in amplitude at low temperatures, which to a large extent can be reversed by NA.

Pharmacological tools were applied to identify the types of adrenoreceptor mediating the synchronizing action of NA. β1-Adrenergic agonists such as isoproterenol and dobutamine, but not α-adrenergic agonists, were shown to affect release in a similar way to NA. An α1,2-adrenergic antagonist, phentolamine, also failed to prevent the synchronizing NA action. Since the β1-adrenergic antagonist atenolol or the β1,2-adrenergic antagonist propranolol blocked the effects of NA, it can be concluded that NA acts through presynaptic β1-adrenoreceptors. This action of NA might be coupled via β1-adrenoreceptors to the N-type calcium channel, as suggested for ACh release from the rat phrenic nerve by Wessler's group (Wessler et al. 1990a), and NA might serve as a substitute for the synchronizing role of external Ca2+ in low-Ca2+ medium. This mechanism is apparently separate from the facilitatory effect of NA on ACh exocytosis (Yawo, 1996) but requires further analysis, including the role of cyclic nucleotides and modulators of phosphorylation- dephosphorylation cascades.

Concerning the possible physiological role of the observed phenomena, it is an important finding that the synchronizing action of NA is expressed in the absence of any change in EPC quantal content or quantal size when the initial probability of neurotransmitter release is low. This suggests that the above mechanism might play an important role in those situations where quantal content is considerably decreased, for example during transmitter exhaustion (Ceccarelli et al. 1973; Ruzzier & Scuka, 1979), release by regenerating axons (Dennis & Miledi, 1974; Di Gregorio et al. 1989) or during extreme physiological states such as survival in cold and hibernation (South, 1961; Moravec et al. 1973). Hibernation is of particular interest since an increase in the concentration of NA in the blood is known to precede recovery from hibernation and the potentiating effects of NA on quantal release are virtually absent (Melichar et al. 1973; Moravec et al. 1973).

From a theoretical point of view the present data, which demonstrate that the synchrony of release of transmitter quanta has substantial effects on multiquantal EPC parameters, suggest that caution should be used when calculating the quantal content of evoked synaptic currents by the direct method (i.e. dividing the mean EPC amplitude by the mean mEPC amplitude) because drugs that can affect the synchrony of release might make the summation of uniquantal responses non-linear even when the subsynaptic membrane is voltage clamped.

Acknowledgments

We thank Dr Ch. Edwards, Dr W. Van der Kloot and Dr L. Kheerough for their help during preparation of the manuscript. This research was supported by grants VS-97099, RFBR 99–04-48286, The Physiological Society Foreign Programme 1998 and EU grant Nesting 1997–99. F. V. was also supported by the Grant Agency of the Academy of Sciences, A7011902 (1999–01).

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